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Multi-omics analysis of Aspergillus niger glucoamylase producing strains
and exploration of NADPH metabolic engineering
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vorgelegt von
B. Sc.
Yufei SUI
ORCID: 0000 -0002-1504-5717

von der Fakultät III –Prozesswissenschaften
der Technischen Un iversität Berli n
zur Erlangung des akademischen Grades
Doktor der In genieurwissenschaften
- Dr.-Ing. –
genehmigte Dissertation

Promotionsausschuss:
Vorsitzende r: Prof. Dr. Yong WANG
Gutachterin: Prof. Dr. Vera ME YER
Gutachterin: Prof. Dr. Yingping ZHUANG

Tag der wissenschaftlichen Aussprache: 23. June 2020

Berlin 20 20

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Appreciate for all the efforts and gains, to make me be a better self.

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ACKNOWLEDGEMEN TS
A t f i r s t , I w o u l d l i k e t o e x p r e s s m y s i n c e r e g r a t i t u d e t o m y s u pervi sor Prof. Vera Meyer for providing
me such a valuable opportunity t o study in this en ergetic lab a nd work with these lovely colleagues.
Vera profoundly influences me not only on sci ence but also on t he attitude towards life. I am deeply
grateful for her constructive g uidance on my r esearc h, as a bea con to light up my direction.
I would l ike to gi ve particular thanks to my colleague Dr. Tabe a Sch ütze fo r her gre at hel p i n my
project and my life in Berlin. W i thout her patient and professi onal direction, I canno t learn so much
experiment skills in a short time . Most importantly, she always gives me reliable support to help m e
overcome di fficulties.
Moreover, I want to thank Dr. Mi nJin Kwon for her ki nd experim e ntal suggestions to help me
accelera te my research. She is always an excellent example for me.
Billions of thanks to all other co lleagues for their w arm help and professional d irections on
experiments.
Deeply gratitude to my friends in Berl in, Jiaying Gong, Zui Xu, Yifan Zhang, Shenzi Tan, Zheng
Wang, my life would not be such fruitful without their company in Ber lin.
And specia l than ks to the gran t sponsored by China Sch olarship Co uncil.
I n t h e e n d , I w o u l d l i k e t o e x t e n d a l l m y g r a t i t u d e t o m y w a r m famil y for their support and
encouragement. They are always my most substantial backing to l et me pu rsue and reali ze al l my
dreams.

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I
Zusammenfassung
Der Fadenpilz Aspergillus niger ist aufgrund seiner außergewöhnlichen Fähi gkeit zur
Proteinexpression und -sekretion eine der wi chtigsten Z ellfabri ken, die heutzutage in der Industrie
für die homologe oder heterologe Protei nproduktion verwendet we rden. Das systematische
Verständnis seiner Stoffwechselre gulationseigenschaften während des Fermentationsprozesses is t
von erheblicher Bedeutung, um di e Leistung dieser vielseitigen Zellfabrik durch metabolisches
Engineering zu verbessern. Aus der Sicht der Genomik, der Zei tv erl auf s-Tr an skr ipt om ik- Anal ys e
und des Cofaktor-Engin eerings entschlüsselte diese Arbeit den h ocheffizienten
Enzymproduktionsmechanismus in A. ni ger und opti mierte erfolgreic h seine Enzymfähi gkeit
durch metabolisches Engineering.
Da frühere Studien berichteten, dass eine v er ringerte Sporulati on die Protein sekretion in A. nig er
begünstigt, führten wir in dieser Arbeit e ine vergleichende Gen omanalyse des nicht sporuliere nden
industriell genutzten A. niger Stammes LDM3 in Chin a und des Re ferenzprotein sekretionsst amms
CBS 513.88 durch, um den S chlüssel vorherzusagen Gene, die gene t i s c h e B a s i s d e s h o h e n
Proteinprodukti onspotentials von LDM3 in silico definieren könn ten. Nach d em Zusammenbau
enthält LDM3 11.209 offene Leserahmen (ORFs) und weist im Vergl eich zu CB S 51 3.88 große
chromosomale Umlagerungen auf. E in Alignment der beiden Genomse quenzen erg ab, dass die
Mehrheit der in LD M3 ein deutig vorhandenen 457 ORFs vorhergesag te Funktio nen in
Redoxwegen, Proteintransport- u nd Protei nmodifikationsprozessen besaß. Darüb er hinaus zeigten
bioinformatische Analysen das Vorhandensein von 656 ORFs in LDM 3 mit nicht synonymen
Mutationen, die für Proteine kodieren, die mit Proteintranslati on, Proteinmodifi kation,
Proteinsekretio n, Metaboli smus und Energieproduktion zusammenhä n g en. Basierend auf der
verfügbaren Literatur und der An alyse des Koexpressionsnetzwerk s wurde tupA a l s
Schlüsselfaktor für die asexuelle Sporulation v on A. nige r vorgeschlagen. Durch Knockout-
Experimente zeig ten wir, dass die Δ tupA -Mutante eine verringerte Sporulation (3 5%) zeigte,
begleitet von einer höheren Gesa mtprot einsekretion (65%) im Ver gleich zu ihrem Elternstamm.
Ein solcher Effek t wurde jedoch bei der Δ prpA -Mutante nicht beobachtet.
Die Sauerstoffbegrenzung ist ei ne rentable Strategie für die Üb erproduktion von i ndustrieller
Glucoamylase (GlaA) durch A. niger . Um das Verständnis darüber zu vertiefen, wie das
Transkriptionsnetzwer k auf die S auerstoffbegrenzung während der indust riell en Glucoa mylas e-
Fermentation reagierte, wurden Zeitverlaufstran skriptomproben a us den Fed-B atch-Kulturen eines
GlaA-produzierenden A. nige r-Stamms DS03043 mit hoher Ausbeute sequenziert. Die Analyse
des Genex pressionsmusters von 515 iden tifizierten differentiell exprimie rten Genen (DEGs)
deutete auf einen hochflexibl en Metabolismus hin, wenn die Zell e mit dem hypoxischen Status
f e r t i g w u r d e . Z u s a m m e n m i t d e m b e g r e n z t e n W a c h s t u m d e r B i o m a s s e , das durch die geringe
Sauerstoffverfügbarkeit verursacht wurde, führte dies zu einer geschwächten Bi osynthese des
Zellpro teins, aber der Fetts äure kata bolism us, die GlaA-Bi osynt h ese und die Bereitstellung von

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II
Vorläuferamin osäuren wurden in sbesonder e a ktiviert. Die o bigen Beweise unterstreichen, dass
zelluläre Ressou rcen wie Energiesubstrate und Vorläufer-Metab ol iten de r hoch effiz ienten GlaA -
Akkumulation unter Bedingungen ni edriger Energ ieversorgung Vorr ang einräumen.
Bemerkenswert ist, dass der definierte Transkriptionsfaktor Srb B für sterolregulatorische
elementbind ende Proteine (SREB P) während der gesa mten Kulturen kontinuierlich und
d r a m a t i s c h h o c h r e g u l i e r t w i r d , w a s a u f e i n e w e s e n t l i c h e r e g u l a t orische Rolle von Srb B be i der
Transkriptionsanpassung an die Hyp oxie hinweist.
Unsere kürzlich durchgeführten Mu lti-Omics-Analysen d er Biosyn these von Glucoamylase
(GlaA) in der Fabrik für filamentös e Pilzzellen A. niger zeigten, dass eine geringe Verfügbarkeit
von NADPH ein begrenzender Fakt or f ür die Überpr oduktion von Gl aA sein kön nte. Angesichts
der Vorhersage von GSMM un d der verfügbaren Literatur h aben wi r insgesamt neun
vorhergesa gte NADPH-Generati onsenzym e in A. niger ausgewählt, um ihre individuell en
Auswirkungen auf d ie Glucoamyla se und d ie Gesamtproteinproduk ti on im im Labor etabliert en A.
niger - S t a mm A B 4 . 1 ( m i t n u r e i n e K o p i e d e r f ü r d a s G e n glaA kodierenden Glucoamylase). Dabei
haben wir ein e zusätzli che Kopie dieser Gene unter der Kontr oll e des starken und abstimmbaren
Tet-on- Schalters des Gens in den pyrG -Locus von A. niger integriert. Ob wohl die intrazelluläre
NADPH-Versorg ung nach genetischer Störung in AB4.1 leicht verbe s s e r t w a r , w u r d e k e i n e
signifikante Veränderung der Proteinbiosynthese beobachtet. Dah er könnt e die NADPH-
Versorgung aufgrund des geringen NADPH-Bedarfs in AB4.1 nicht d er Engp ass für die
Proteinpr oduktion sei n.
Das I n-vitro-CRISPR/Cas9- System hat aufgrund seiner hohen Effiz ienz bei d er Geneditierung,
jedoch einer Schwäch ung der Mö glichkeit außerhalb des Ziels, um fangrei che akademische
Aufmerksamkeit erregt. In dieser Arbeit wurde das in vitro opti mi erte A. niger C R I S P R / C a s 9
unter Verwendung des Ribonu kleopr otein (RNP) -Ansat zes übernommen, um die auxotrophe
Uridinmutante ei nes GlaA-produzierenden Stamms A. ni ger B36 mi t hoher Ausbeute effizient zu
konstruieren und so eine hervorra gende Pl attform für weiteres N ADPH bereitzustellen
Engineering unter einem Hi ntergru nd mit hoher Proteinsekretion.
Der kl assische DBTL-Zy klus (Desi gn-Build-Test -Learn) des M etabolic Engineeri ng wird
zunehmend genutzt, um di e rati onale Stammentwicklung v oranzutre iben . Um zu verste hen, ob
eine starke Tendenz zur GlaA -Bio synthese (sieben Genkopien) ein e höhere proteinogene
NADPH-Versorg ung im Ver gleich zum nativen Zustand erfordert, be richteten wir über di e
Implementierung von DBTL-Zyklen , um effek tive Co-Fak tor-Enginee ring-Strategien in einem
GlaA mit hoher Ausbeute zu i dentifizieren und zu priorisieren P roduzent B36 mit sieben glaA -
Genkopien. Eine detaillierte Ana ly se aller sieben identischen N ADPH-Generat ionssysteme wie in
AB4. 1 in Schüttelkolben kulturen er gab, d ass eine Tet-on-g esteue rte Überexpression des gsd A -
Gens (Glucose-6-phosph at-Dehydrogenase), des gndA -Gens (6-Phosphogluconat -Dehydrogenase)
und des maeA -Gens (NADP) a uftrat -abhängiges Äpfelsäur eenzym) unterst ützte die GlaA-

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Produktion auf einem su btilen, aber signifikanten Niveau (10%) im Hintergrundstamm, der sieben
glaA-Genkopien trägt. Wir führten daher für diese drei Isolate Maltose-limitierte Chemostat-
Kultur en durch, um den intrazellulä ren NADPH-Pool unt er station ären Bedingung en zu
bestimmen. In diesen Kulturen erhöhte die Überexpression des gnd A - und des maeA -Gens den
intraze llulären NADPH-Pool um 45% und 66% und die Ausbeut e an G laA um 65% bzw. 30%.
Trotzdem hatt e die Überexpression des gsdA -Gens einen negativen Einfluss auf die
Gesamtprotein- und Glucoamylase-Produktion. Diese Daten legen z um erste n Mal nahe, dass eine
erhöhte Verfügb arkeit von NADPH tatsächlich die Protein - und insbesondere die GlaA-
Produktion in St ämmen untermauern kann, i n denen ei n starker Zug zur GlaA-Biosynthese besteht .
Wir schla gen daher vor, dass das NADPH-Cofakto r-Engineering tat sächlich eine gültige St rategie
für das metabol ische Engineering von A. niger zur Verbesserung der GlaA-Produktion i st, ei ne
Strategie, die sicherlich auch a uf das rat ionale Design anderer mikrobieller Zellfabriken
anwendbar ist.
Schlüsselwörter: Aspergillus niger ; NADPH; Cofakt or-Engineering; Glucoamylase; Multi-
Omics-Analyse; CRISPR/Cas9

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IV
Summary
The filamentous fungus Aspergi llus niger is one of the main cell fact ories used nowadays in the
industry for homologous or heterol ogo us protein production due to its extraordi nary ability for
protein expression and secr etion. Systematic comprehension of i ts metabolic regul ation
characteristi cs during the fe rmentati on process is of cons idera b le significance to improve the
performance of thi s versatile cell factory through metaboli c engineeri ng. Fro m the perspective of
genomics, time-course transcripto me analysis an d cofactor engin eering, this thesis d eciphered the
high efficient enzyme production mechanism in A. niger and successfull y opti mized its enzyme
capability through metabolic engineering.
As previous stud ies reported that reduced s porulation favors pr otein secretion in A. niger , in t his
thesis we conducted a comparative g enomic analysis of th e non-s porulating industrially exploited
A. niger strain LDM3 in Chi na and the ref erence protei n secretion strai n CBS 513.88 to predict
the key genes that might define t h e g e n e t i c b a s i s o f L D M 3 ’ s h i g h protein produci ng pot ential in
silico. After assembly, LDM3 harbors 11,20 9 open reading frames (ORFs) and exhi bits large
chromosomal rearrangements i n comp arison to CBS 513.88. An ali g nment of the two genome
sequences revealed that the majo rity of t he 457 ORFs uniq uely p resent in LDM3 pos sessed
predicted functions in redox path ways, p rotein tr ansport, a nd p rotei n modificati on pr ocesses. In
addition, bioinfo rmatic a nalyses rev ealed the presence of 656 ORFs in LDM3 with non-
synonymous mutati ons encodi ng f or p roteins related to protein t ranslati on, protein modification,
protein secretion, m etabolism, and energy production. Based on available literature and co-
expression network analysis, tupA was proposed as the key facto r involved in asexual sporul ation
of A. niger. By knockout experiments, we sh owed that the Δ tupA mut ant displayed reduced
sporulation (35%) accompanied by higher total prot ein secreti on (65%) compared to i ts parental
strain. Such an ef fect was, however, not observed in the Δ prpA mutant.
Oxygen limitation is a profitabl e strategy for industrial gluco amylase ( GlaA) overproduction by A.
niger. To deepen t he understan ding of how the transcri ptional networ k responded to t he oxygen
limitation during th e industrial glucoamylase fermentation, ti m e-course transcriptome samples
from the fed-batch cultures of a high-yield GlaA producing A. niger strain DS03043 were
sequenced. Gene expression patt e rn a nalysis of 515 identified d ifferentially expressed genes
(DEGs) s uggested a highly flexible metabolism when the cell cop ed with the hypoxic status. Along
with the l imited biomass growth caused by the low oxygen availa bility, it led to a weakened
biosynthesis of cell protein, but fatty aci d cat abolism, GlaA b i osynthesis, and the provisi on of
precursor amino acids were notably act ivated. Above proofs unde rline th at cellular resources, such
as e nergy subst rates a nd precursors met abolites, were prioritiz ed to the highly efficient GlaA
accumulation under the low energy s upply condition . No teworthy, the defined sterol-regulatory
element-binding proteins (SREBP) tran scription factor SrbB cont inuously and dramaticall y up-

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V
regulated during the entire cultures, indicating an essential r egulatory role of SrbB in the
transcripti onal adapt ion to th e hypoxia .
Our recent multi-omics analyses o f glucoamylase (GlaA) biosynth esis in the filamentous f ungal
cell factory A. niger indicated that low availabil ity of NADPH might be a limiting f actor for GlaA
overproduction. Given the pred iction of GSMM and availab le l iterature, we selec t e d i n a t o t a l o f
nine predicted NADPH g enerati on e nzymes in A. niger to test their individual effects on
glucoamylase and total protein pr oduction in the lab e stablishe d A. niger strain AB4.1 (only
carrying one copy of glucoamylase encodi ng gene glaA ). In doing so, we integrated an additional
copy of these genes under the control of the strong and tun able gene Tet-on swit ch into t he pyrG
locus of A. niger . Ho wever, albeit intracellular NADPH suppl y was slightly improved after genetic
perturbation in AB4.1, no significant alt eration was o bserved o n protein bio synthesis. Hence,
NADPH s upply could not be the bott leneck for prot ein producti on beca use of the low NA DPH
requirement in AB4.1.
I n v i t r o C R I S P R / C a s 9 s y s t e m h a s a r o u s e d e x t e n s i v e a c a d e m i c a t t e ntion due to its high gene editing
efficiency but weakened off-target possibility. This t hesis ado pted the optimized in vitro A. niger
CRISPR/C as9 system u sing the ribonucle oprot ein (RN P) approach t o efficientl y construct the
uridine auxotrophic mutant of a high-yield GlaA p roducing strai n A. niger B36, so as t o provi de
an excelle nt platform for fu rther NADPH engineer ing under a hig h protein secretion backgrou nd.
The classic Design-Build-Test-Learn (DBTL) cycle of metabolic e ngineering is increasin gly
leveraged to advance rational st rain development. To understand if a strong pull towards glaA
biosynthesis (seven gene co pies) mandat es a h igher proteinogeni c NADPH suppl y compared to
the native condition, we report ed on t he impleme ntation of DBTL cycles to i dentify and prioritize
effective co-factor engineering strategies in a high-yield GlaA producer B36 carrying seven glaA
gene copie s. Det aile d analy sis of all seve n i dentical NADPH ge n eration systems as in AB4.1 in
shake flask cult ures uncovered that Tet-on dr iven overexpr essio n of the gsdA gene (glucose 6-
phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA g e n e
(NADP-depend ent m alic e nzyme) suppor ted GlaA product ion on a su bt le but signifi ca nt level
(10%) in the background strain which carries seven glaA gene copies. We thus performed malt ose-
limited chemostat cult ures for these three isolates to determin e the intracellular NADPH pool
during steady-state conditions. In these cultures, overexpressi on o f g ndA a n d maeA gene increased
the intracellular NADPH pool by 45% and 66%, and t he y ield of G l a A b y 6 5 % a n d 3 0 % ,
respectively. Nevert heless, ove rexpression of gsdA gen e had a negative effect on both total protein
and gl ucoamylase producti on. This data suggests for the first t ime that increased NADPH
availabili ty can indeed underpin protein an d especially Gl aA pr od uction in strain s where a strong
pull t owards GlaA biosynthesis exists. We thus propose that NAD PH cofactor engineering is
indeed a valid strategy for metabolic engineering of A. niger to i mprove GlaA product ion, a
strategy which is certainly al so applicable to the rational des ign of other microbial cell factories.
Keywords: Aspe rgillus nig er ; N ADPH; cofactors e ngineeri ng; gl ucoamylase; multi-omi cs
analys is; CRISPR /Cas9

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VI
Index
Zusammenfassung .... ........ ........... ................ ........ ................ ........ ........ ........ ........ ........ .......... ........... I 
Summary ............... ................ ........ ........ ........ ........ ........ ........ ........... ................ ................ ........ ..... IV 
Chapter 1 Introduc tion .......... ........ ........ ........ ...... ........ ........ ........ ........ ........ .......... ................ ........ .. 1 
1.1 Aspergillus nige r ........... ................ ........ ........ ........ ........ ........... ................ ................ ........ .... 1 
1.2 Progress in t he genomics of A. niger ............. ................ ........ ........ ........ ........ ........ ........ ....... 1 
1.3 Progress in the transcri ptomics of A. niger ........... ................ ................ ........ ........ ........ ....... 3 
1.3.1 Comparative transcriptome analysis to uncover the high efficient production
mechanism in A. niger ...... ........ ........ ........ ........ ........ ........ ........... ................ ........ ........ ........ .... 3 
1.3.2 Transcriptome analysis to revea l the regu latory mecha nis m of seco ndary metabolites
biosynthesis i n A. nige r ....... ........ ........ ........ .......... ................ ........ ........ ........ ........ ........ ........ .. 4 
1.3.3 Transcription p r ofiles t o describ e cellular re sponse on d iverse carbon sour ce ............. 4 
1.4 The metabolomics of A. niger .......... ........ ........ ........ ........ ........ ........ .......... ................ .......... 5 
1.5 Multi-omics integrated analysis ....... ........ .......... . .......... ........ ........ ........ ........ ........ ........ ....... 6 
1.6 Genetic modification platforms in A. niger ................ ........ ........ ........ ........ ........ ........ .......... 7 
1.6.1 The applicati on of Tet-on inducible gene expression switc h ..... ................ ........ ........ .... 7 
1.6.2 Applicati on of the CRISPR /Cas9 gene-editing in filamento us fungi ........ ........ .......... .. 8 
1.7 Optimization of the produc tion capability of microbial cell factory via metabolic
engineering .............. ........ ........ ........ .......... ... ........... ........ ........ ........ ........ ........ ........ .......... ........ 13 
1.7.1 Construction of efficient smart biomanufacturing cell f actory driven by DBTL cycl e
.......... ........ ........ ........ ........ ........ ........ ........ ........... ................ ........ ........ ........ ........ ........ ........ .. 13 
1.7.2 Recent adva nces of ce ll f actory A. niger .. ................ ........ ........ ........ ........ ........ .......... 15 
1.7.3 Optimization of cell fa ctory productivi ty by cofactor e ngineering .................. .......... 16 
1.8 Objecti ve of this pr oject ........ ........ ........ ........ ............. ........... ................ ................ ........ ..... 19 
Chapter 2 Comparative geno mics of the aconidial Aspergill us niger strain LDM3 predicts genes
associated w ith its high protein sec retion capacity ........... ............... ................ ........ ........ ........ ..... 22 
2.1 Introduction ................ ................ ........... .. .............. ................ ........ ........ ........ ........ ........ ..... 22 
2.2 Materia ls and methods .......... ........ ........... ....... ...... ................ ........ ........ ........ ........ ........ ..... 23 
2.2.1 Strain s and culture .......... ........... ............... ...... ........ ........ ........ ........ ........ ........... .......... 23 
2.2.2 Genome DNA ex traction and seque ncing ................... ........... ........ ........ ........ ........ ..... 24 
2.2.3 Hybrid assembly of the A. niger LDM3 genome seq uence usi ng Ill umina and Pa cBio
sequencing ..... ........... ................ ........ ........... . ............ ........... ................ ................ ........ ........ .. 24 
2.2.4 Bioinformatic an alyses ................ ........... ...... ....... ................ ........ ........ ........ ........ ........ 2 4 
2.2.5 SNP, INDEL and SV analysis ............. ........... ..... ... ................ ........ ........ ........ ........ ..... 24 

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VII
2.2.6 Synteny analysis .......... ........... .................. ...... ........ ........ ........ ........ ........ ........ ........ ..... 25 
2.2.7 Strain-spe cific genes in the LDM3 genome comp ared to CBS 513.88 .......... ............. 25 
2.2.8 Co-expression netwo rk of TupA and PrpA encoding gene ... ...... ........ ........ ........... ..... 25 
2.2.9 Determination of biomass, t otal secreted protein, residu al glucose and enzyme activity
of glucoamy lase ... ........ .......... ................ .......... .............. ........ ........ ........ ........ ........ ........ ........ 2 5 
2.2.10 Generation of Δ tup A and Δ prpA strains .... ........ ........ ........... ................ ....... .... .......... 26 
2.2.11 Data access ........... ........... ........ ........ ..... ... ........ ........ ........ ........ ........ ........ ........ .......... 28 
2.3 Result and discuss ion ......... ........ ........ ........... ............. ................ ........ ........ ........ ........ ........ 28 
2.3.1 Strai n cultiva tion and q ualit y detect ion of genome DNA . ........ ........ ........ ........ ........ .. 28 
2.3.2 Characteristics of A. niger LDM3 genome ........ .......... ................ ........... ..... ........ ........ 29 
2.3.3 Genome structu re variation analysis.......... .......... . ............. ........ .......... ................ ........ 32 
2.3.4 LDM3 strain-specifi c genes analysis .............. ...... ............... ................ ........ ........ ........ 33 
2.3.5 SNP and INDEL analys is ............ ........... .......... ... ........ ........ ........ ........ ........ ........ ........ 34 
2.3.6 In vivo analysis of two selected genes putati vely involv ed in sporulation ............... .. 39 
2.4 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ ........... .. 43 
Chapter 3 Gl obal transcriptional response of Aspe rgillu s niger in t he process of glucoamylase
fermentation ............. ........ ........ ........ ........ ........ ................ ................ ........ ........ ........ ........ ........... .. 44 
3.1 Introduction ................ ................ ........... .. .............. ................ ........ ........ ........ ........ ........ ..... 44 
3.2 Materia ls and Methods .......... ................ ........ .. ... ........ ........ ........ ........ ........ ........ ........... ..... 45 
3.2.1 Strain ............ ................ ........ ........ ..... ... ................ ........ ........ ........ ........ ........ ........... ..... 45 
3.2.2 Flask-level cultures ......... ................ ........ .. ... ........ ........ ........ ........ ........... ................ . .... 45 
3.2.3 Bioreactor fed-b atch fermentat ion .................. .... .... ................ ........ ........ ........ ........ ..... 45 
3.2.4 Sampling for R NA-Seq ....... ........... ................. ............ ........ ........ ........ ........... ............. 45 
3.2.5 Determination of biomass a nd enzyme activity of glucoamy lase .. ................ ........ ..... 45 
3.2.6 RNA Extraction ........... ................... ........... .. ................ ........ ........ ........ ........ ........ ..... ... 4 6 
3.2.7 Strand -specific RNA-Seq .......... ........ .......... .... .. ........ ........ ........ ........ ........ ........ ........ .. 46 
3.2.8 Alignment of reads to the reference genome and normaliza tion of gene expression .. 46 
3.2.9 Differ ently expres sed genes............ ................ . ....... ................ ........ ........ ........ ........ ..... 46 
3.2.10 Functional gene a nnotation ........ ........... ......... ....... ........ ........... ................ ........ ........ .. 47 
3.3 Result and Discuss ion ................ ........... ........ . .... ........ ........... ................ ................ ........ ..... 47 
3.3.1 Growth physiology of gl ucoamylase overproduction by A. nige r ............. ................ .. 47 
3.3.2 Overview of RNA-Seq data ............ ........ ........... ............. ................ ........ ........ ........ ..... 48 
3.3.3 Function al and gene expression pattern analyses of DEGs ............ ........ ........... .......... 49 
3.3.4 Key transcriptional respo nse during glucoamylase overpro duct ion ....... ........ ........ ..... 53 


VIII
3.3.5 Transcriptional changes re levant to the secretion pathwa y ............... ........ .......... ........ 54 
3.3.6 Transcriptional changes re levant to transcription factor ............. ........ ........ ........ ........ 5 5 
3.4 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ ........... .. 56 
Chapter 4 Ex ploring the e ffect of NADPH supply on enzyme biopr oductio n in low-yi eld GlaA
producing Aspergillus niger ............. ........ ........ ........ ........ ........ ........ ........... ................ ........ ........ .. 58 
4.1 Introduction ................ ................ ........... .. .............. ................ ........ ........ ........ ........ ........ ..... 58 
4.2 Materia ls and methods .......... ........ ........... ....... ...... ................ ........ ........ ........ ........ ........ ..... 60 
4.2.1 Strain and medium ............... ........ ........ ....... . ................ ........ ........ ........ ........ ........ ........ 60 
4.2.2 Soluti ons ......... ........ ........ ........ ........ ..... ... ........ ........ ........ ........ ........ ........ ........ ........... .. 61 
4.2.3 Plasmids construc tion ................ .......... ........ ........ ........ ........ ........ ........ ........ ........ ....... . 61 
4.2.4 Gibson cloning and AQUA cloning ............... ......... ....... ........ ........ ........ ........ ........ ..... 62 
4.2.5 E.coli c olony PCR ....... ........... ................ ........ ......... .. ................ ........ ........ ........ ........ .. 63 
4.2.6 Construction o f ΔAn14g004 3 and ΔAn16g02510 disruptants . ...... ........ ........... .......... 63 
4.2.7 Preparation of A. niger spore suspension .......... .......... ................ ........ . ....... ........ ........ 64 
4.2.8 A. niger transform ation ................ ........ ........... ............ .... ........ ........ ........ ........ ........ ..... 64 
4.2.9 Southern ana lysis ................. ................... .. ........... ........ ........ ........ ........ ........ ........... ..... 64 
4.2.10 Analysis of broth ....... ........... ........ ........... ............. ........ ........ ........ ........ ........ ........ .. ... 65 
4.2.11 NBT staini ng procedures ....... ........... .............. .. ........ ........ .......... ................ ................ 65 
4.2.12 qRT-PCR ... ........... ................ ........... ...... ....... ........ ........ ........ ........ ........ ........ ........... .. 65 
4.3 Result and discuss ion ......... ........ ........ ........... ............. ................ ........ ........ ........ ........ ........ 66 
4.3.1 Construction o f integr ati ve plasmids used for g ene overe xpression ........... ........ ........ 66 
4.3.2 Characte rizing the effect of NADPH e ngineeri ng on enzyme production in the low-
yield GlaA producer ...... ........... ................ ........ .. ... ........ ........ ........ ........ ........ ........ ........ ........ 6 7 
4.3.3 Exploration of the functi on of NADPH oxidase regulator R iaA on protein formation
in A. niger ......... ........ ........ ........ ........ ........ ........ ........... ................ ................ ........ ........ ........ . . 7 0 
4.3.4 Exploration of the func ti on of t he NADP+ depend ent trans hyd rogenase oxidase in A.
niger ............. ........ ........ ........ ........ ........ ........ ........ ........ .......... ................ ................ ........ ........ 73 
4.4 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ ........... .. 75 
Chapter 5 Co nstruction of a uridi ne aux otrophic mutant of a hi gh-yield GlaA producing
Aspergil lus niger s train B36 via CR ISPR/Cas9 gene edi ting .. ........... ......... ............ ........ ........ ..... 76 
5.1 Introduction ................ ................ ........... .. .............. ................ ........ ........ ........ ........ ........ ..... 76 
5.2 Materia ls and methods .......... ........ ........... ....... ...... ................ ........ ........ ........ ........ ........ ..... 77 
5.2.1 Strain s and media....... ........ .......... ........ ...... ................ ........ ........ ........ ........ ........ ....... ... 7 7 
5.2.2 Homologo us recombination s trate gy to constr uct the urid i ne auxotroph of B36........ 77 
5.2.3 A. niger PEG-mediated protop last transformation ............ ............ .... ........ ........ ........ .. 78 


IX
5.2.4 Biosynthesis and in vitro purificati on of Cas9 protein . ............... ........ ........ ........ ........ 78 
5.2.5 In vit ro synthesized sgRNA ......... ........... ......... .... ........ ........ ........ ........ ........ ........... ..... 80 
5.2.6 Gene editing strategy for pyrG via CRISPR /Cas9 .............. ........ ........ ........ ........ ........ 80 
5.3 Result and discuss ion ......... ........ ........ ........... ............. ................ ........ ........ ........ ........ ........ 80 
5.3.1 Homologo us recombination s trategies t o const ruct the B36 uridi ne auxotroph ......... 80 
5.3.2 In vitro puri fication of Cas9 protein ............... ... ............. ................ ........ ........ ........ ..... 83 
5.3.3 In vitro CRISPR/Cas9-mediated pyrG gene editing....... ............................. .............. .. 84
5.4 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ ........... .. 87 
C h a p t e r 6 E n g i n e e r i n g c o f a c t o r metabolism for impr oved protein a nd glucoamylase p roduction
in Aspergillus niger ............. ........ ........ ........ ........ ........ ........ .......... ................ ........ ........ ........ ........ 88 
6.1 Introduction ................ ................ ........... .. .............. ................ ........ ........ ........ ........ ........ ..... 88 
6.2 Materia ls and methods .......... ........ ........... ....... ...... ................ ........ ........ ........ ........ ........ ..... 89 
6.2.1 Strain and media .......... ................ ........... .. ...... ........ ........ ........ ........ ........ ........ ........ ..... 89 
6.2.2 A. niger colony PC R ............... ........ ........... ................ . ....... ........ ........ ........ .......... ........ 90 
6.2.3 Batch cultu res ......... ........ ........... ............. ........ ........ ........ ........ ........ ........ ........ ........... .. 90 
6.2.4 Chemostat cultures .............. ........... ............ . ........ ........ ........ ........ ........ ........... ............. 90 
6.2.5 Analysis of cu lture broth ........... .................. . ..... ........ ........ ........ ........ ........ ........ ........ .. 9 1 
6.2.6 Analysis of GlaA by dot blot ........... ........... ...... ....... ........ ........ ........... ................ .......... 91 
6.2.7 Quantification of extr acellul ar organic acid ........... ................ ................ ........ ........ ..... 91 
6.2.8 Fast ex tractio n of intracel lular metabolite s ....... .... ...... ........... ................ ................ ..... 91 
6.2.9 GC-MS.... ................ ........... ................ .... .... ........ ........ ........ ........ ........ ........ .......... ........ 91 
6.2.10 LC-MS .......... ........... ................ .......... .. ............ ........ ........ ........ ........ ........ ........ .......... 92 
6.2.11 Quantification of intrace llular NADPH ............. .... ....... ........ ........ ........ ........ ........... .. 92 
6.2.12 Quantitative real-time PCR (qPCR) .. ........... ........ ... .......... ................ ................ ........ 92 
6.2.13 Multivariat e statistical analysis of intracellular meta bolites ........... ................ .......... 92 
6.3 Result and discuss ion ......... ........ ........ ........... ............. ................ ........ ........ ........ ........ ........ 93 
6.3.1 Transformation efficien cy of B36 uri dine auxotroph ..... ........ ........ ........ ........ ........ ..... 93 
6.3.2 Optimization of the prot o plast transformation effi ciency of B36 ....... ........... ............. 93 
6.3.3 The impac t of NADPH eng ineering on GlaA produc tion is st rain-dependent ............ 9 4 
6.3.4 Effect of diverse NADPH gen eration systems i n batch cult ures ............ ........ ........... .. 96 
6.3.5 Impact of NADPH gene rati on on physi ologi cal chang es and metabolic adaptio n ..... 98 
6.3.6 Learning new biological insig h ts ............... ........ ........ ........ ........ ........ ........ ........ ........ 110
6.4 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ . ....... ... 111 
Chapter 7 C onclusion and outlook ............. ................ .......... ........ ........ ........... ................ ........ .. . 11 2 


X
7.1 Conclusion .......... ........ ........... ................ .. ...... ........ ........ ........ ........ ........ ........ ........ . ....... ... 112 
7.2 Future work ........ ................ ........... ........... .. ........ ........ ........ ........ ........ ........ ........ ..... ... ...... 113 
Appendix A . .......... ................ ........ ........... ...... ....... ........ ........ ........ ........ ........ ........ ........ ........ ...... 115 
Appendix B .............. ................ ........... ........... ..... ................ ........ ........ ........ ........ ........ .. ...... ........ 120 
Appendix C .............. ................ ........... ........... ..... ................ ........ ........ ........ ........ ........ .. ...... ........ 132 
Appendix D . .......... ................ ........ ........... ...... ....... ........ ........ ........ ........ ........ ........ ........ ........ ...... 142 
Reference ......... ........ ........... ................ ........ . ....... ........ ........ ........ ........ ........ ........ ........ ........ ........ 143 



XI
List of Abbrevia tions
2PG 2-phosp ho-D-glycerate
3PG 3-phosp hoglycerate
6PGDH phosphogluconat e dehydrogenase
AC acetat e
AcCoA acetyl-CoA
AKG α-Ketogl utaric acid
Ala alanine
Arg arginine
Asn asparagine
Asp aspartate
ATP Adenosine Tri Phosphate
CIT citrate
CER Carbon-dioxide Escape Rate
DHAP dihydroxy acetone-ph osphate
DO Dissolv ed Oxygen
E4P erythrose-4- phosphate
EMP Embden-Meyerhof-Parnas pathway
F6P fr uct ose -6-ph os pha te
FBP 1,6 Fructose Diphosphate
FUM f uma rate
G6P glucose-6-phosphate
GlaA glucoamylase
GAP glyceraldehyde-3-phosphate
Gln glutami ne
Glu glutamate
G6PDH Glucose 6-phosphate dehydrogenase
His histidine
ICT i so cit rat e
Ile isoleucine
Leu leuc ine
Lys lysine
Mal malate
Met methioni ne
NADP-ICDH NADP+ depende nt i socitrate de hydrogenase
NADP-ME NADP+ depe ndent malic enzyme
OA oxala te
OAA cytosoli c oxaloacetate
Orn ornithine
OUR Oxygen Uptake Rate
PEP phosphoen olpyruvate
Phe phen ylalanine
Pro p roline
PYR py ruvate


XII
PPP Pentose Phosphate Pathway
PLS-DA Partial Least Square s Discrimination Analysis
R5P ribose-5-phosph ate
Ru5P ribulose 5- phosphate
S7P sedohept ulose-7-phosphate
Ser serine
SUC succinate
Th r threon ine
Trp tryptophan
Tyr ty rosi ne
TCA Tricarboxylic Acid Cy cle
Val vali ne
X5P D-Xylu lose 5-phosphate




C h a p t e r 1 1 
Chapter 1 Introd uction
1.1 Aspergillus niger
A. niger is a filamentou s ascomycete f ungus commonly found in the envir onment, which has been
a widel y used cell factory in bulk manu facturing of indust rial enzymes and or ganic acids (Sui et
al. , 2017 ; Cairns et al . , 2018). To date, fun gi have pr ovi ded a profound source for t he majority of
antibiotics (Liu et al. , 2010; Liu et al. , 2012). As it has recently been reprogrammed to produce
secondary metabolites and pharmaceutical ingredients at a hi gh level, A. niger i s o f g e n e r a l i n t e r e s t
as a multipurpose cell factory (Boec ker et al. , 2018). In the past decade, the market for ab undant
products derived from Aspergilli , especially e nzymes a nd o rgani c acids, ha s been w itnessed a
continuous and rapid increment. Their estim ated ma rket volum e i n 2014 was $ 2.6 bill ion, which
was expected to reach up to $ 3.6 bil lion until 2020, displayin g huge economi c prospects (Cairns
et al. , 2019).
1.2 Pr ogr ess in the genom ics of A. niger 
With the rapid development of diverse o mics technologies, a sea o f d a t a f r o m m u l t i p l e l a y e r s o f
omics including genome, transcrip tome , proteo me, and metabolom e are accumulating
exponentially, d eclaring the coming of big da ta era for t he versatile cell factory A. niger . In
partic ular, ev olvemen t from single omics to the integra tions of multi-omics centred on the genome-
scale me tabolic network model (GSMM) l argely extends t he intens ive and systematic
understan ding of the complex regulatory mechanisms in A. niger .
In the last decade, with the development and maturation of NGS (next -generation sequencing), it
has played a c rucial role in genome informatio n mi ning and brin gs transformati ve breakthroughs
for genome and t ranscriptome seq uencing. Particularly after a s ig nificant fall of sequencing costs,
a sea of strains has been performed genome resequencing or de n ovo sequenci ng, which
progressively accelerates the develop ment of genomi cs and provi des new insights into genome
assembly, methylati on, transcription, and metagenomic. To date, a t o t a l o f 1 7 A. niger genomes
have been sequenced (Baker, 2006; Pel et al . , 2007; Andersen et al. , 2011; Yin et al. , 2 014a; Paul
et al. , 2017; Yin et al. , 201 7). In 2000, DSM ( Netherland) s t a r t e d t h e w h o l e g e n o m e s e q uencing
of a glucoamylase producing strain CBS 513.88. Based on the gen ome seq uence and the annotation
information, a genome-scale metabolism model of A. niger with 1069 specific rea ctions was built ,
which well explained the mechanism of the high effi cient GlaA p rodu ction i n A. niger (Pel et al. ,
2007). In light of the comparati ve genome analysis, a 0.8 M new sequence was identified in citric
acid prod ucer ATCC 1 015 co mpared to CBS 513.88, accompanied by frequent genomic
rearrangement, deletion, a nd strain-specific horizont al gene tr ansfer (Andersen et al. , 2011). An
alternative example also verified the genome diversity of the c ell factory A. niger . Genome
sequenci ng o f an aconidial, high glucoamylase yield s train SH2 proposed that a large f ragment
deletion in prpA may be respo nsible for the aconidial phenotyp e (Yin et al. , 2014a). I ncreasing


C h a p t e r 1 2 
genome sequencing of Aspergilli has laid a solid foundation for t he establishment of comparati ve
genomics studies so as to decipher spec ie s p hyl oge ny .
Compared to Sanger sequencing, NGS platform conf ers high throug hput sequencing. However, its
drawbacks, such as short reads and GC bias (Gong et al. , 2016), still limit its app lication and impair
the ge nome assembly accuracy. To overcome t hese defec ts, the third-generation sequencing also
named as single molecule sequenc ing h as been developed, such as Si ngle-molecule real-time
(SMRT) released from Pacific Bioscien ces (PacBio), the single-m olecule nanopore DNA
sequencing from Oxford Nanopore c omp any, and t SMSTM (th e from c ompany and True single-
molecule sequencing) f rom Helicos (Braslavsky et al. , 2003). Neve rtheless, on l y PacB io RS
sequenci ng pl atform from Pacfic Biosciences has been succ essful ly commercialized by now. Zero-
mode w aveguides (ZMWs) is one of the core technol ogies of SMRT seq uencing (Eid et al. , 2009)
(Figure 1.1 A), where only a single DNA molecule c an b e combined w ith a DNA polymerase
immobilized at the bottom of ZMW (Rhoads and Au, 2015), and the n DNA is synthesize d by
polymerases from a template using f our fl uorescently-labelled nucleotides within the ZMWs. This
method does not require any prior amplification of the DNA temp late (Ferr arini et a l. , 2013), thus
avoid sequencing errors c aused by GC preferences.  Additionally, t he width of ZMWs ( 10-50 nm)
provides an optical observati on volume confinement (Eid et al. , 2009; Ferrarini et al. , 2013), which
ensures the f luorophore ex cited fr om each ZMW is only related t o the growin g DNA stra nd but
not propagate through t he wavegu ide. This detecti on method thus confers a dramatic r eduction of
background inter ference, enabling simultan eous detection of tho usands of single-molecule
sequencing reacti ons on the arr ay alo ng with t he extension of t he DNA strand. Another
breakthrough of SMRT is the c onjugation of fluorophores to the terminal phosphate moiety o f the
dNTPs, which is cleaved from the nucleotide with the gr owing DN A strand (Rhoads and Au, 2015) .
Thus, it all ows the continuous synthesis of the DNA strand with reduced steric hindrance, resul ting
in lon g reads wi th over thousands of bases (Figure 1.1B). Albei t the prominen t advantages of
SMRT, its high error-prone ( up to 15 %) are always argued. These errors are randomly distributed.
While th e accuracy can be improved up to 99.3% b y enhanc ing t he sequenci ng depth (> 1 5) a nd
the coverage (Eid et al. , 2009).
At present, it has been hi ghlighted t hat the hybrid sequencing method incorporating NGS a nd
PacB io platf orms enable to co mplemen t the stren gths and weaknes ses of these t wo platforms,
which g reatly reduce the sequenc ing cost, but surprisin gly impr oves the coverage and the accuracy
of genome assembly (Rhoads and Au, 2015). Since 2 017, a growing number of researc h group s
have employed hy brid sequencing to reveal the genetic character istics of A. niger with diverse
phenotypes. For example, to explore th e mole cular mechanism of efficie nt productivit y of
industrial A. nige r , Yin et a l. (2017) highl ighted the genetic factors of citrate accumulation i n A.
niger through co mparative genomics and t ranscriptome analysis. Lao th an achareon et al. (2018)
first published the whole genome sequence of a master lab-estab lished strain N402, which is nearly
identical to the typical citrate producing strain A. niger ATCC 1015. Besides, they also uncover


C h a p t e r 1 3 
the molecular rol e of the goxB in gl ucose oxidase produc tion t hrough comparative genomics
analysis.

Figure 1.1 Sequencing principles of Pac Bio RS plat form (Rhoads and Au, 2 0 15). (A) SMRTbell (grey)
diffuses into a ZMW, and the adap tor binds to a polymerase immo bilized at the b ot tom. ( B) A
fluorescently-labelled nucleotid e associates with the template in the active site of the polymerase. The
fluorescence outpu t of the color correspon ding to the incorpora ted base is elevated. The dye-linker-
pyrophosphate product is cleaved from th e nucleotide and diffus es out of the ZMW, endin g the
fluorescence pulse. The polymer ase translocates to the next pos ition to initiate the next fluorescence pulse
1.3 Pr ogr ess in the transcriptomi cs of A. nige r
The whole-transcri ptome level co mparative analysis prov ides res earchers with a more pr ecise
estimation of the c orrelation between cel lular phenotype and ge ne expression, t hereby deepening
our understanding of the cellular regulatory network. With the development of sequencing
technology, the new generation high- throughput transcriptome se quencin g (RNA sequencin g,
RNA-seq) has been t he mai nstream technol ogy adopted b y comparat ive transcriptomic studies in
recent years, due to its advantages in identifying new genome s tructure variations compared to t he
microarray, such as new tran scripts and alterna tive splicing.
1.3.1 Comparative transcriptome analysis to uncover the high e fficient pr oduction
mechanism in A. nige r
At present, transcriptome analysis has been widely u sed to i den t i f y k e y f a c t o r s u n d e r l y i n g t h e
optimize d enzyme capacity resul ting f rom diff erent environm enta l variabl es or d iverse gene tic
characteristi cs.
In 2 011, Andersen et al. ( 2 0 1 1 ) c a r r i e d o u t t h e c o m p a r a t i v e t r a n s c r i p t o m e a n a l y s i s b e t w een a
citrate-producing strain A TCC 1015 and an enzyme-producing strain CBS 513.88 under the same
culture conditions. Transcriptome analysis suppo rted the up-reg ulation of the glyco lysis pathway,
TCA cycl e, and amino acid metabolism i n CBS 513.88. Part icularl y, the exp ression of genes
involved i n the Thr , Ser, and Try biosynt hesis were highl y acti vated. These three amino a cids are
dominatin g in the c omposition of GlaA, tRNA synthet ase, and p ro tein transporte rs. Moreover, it
was obser ved higher expression in el ectron transpo rt, carbohydr ate transp ort, and or ganic acid


C h a p t e r 1 4 
transport in ATCC 1015 . Genome-level transcriptome analyses tel l u s t h e d e t a i l e d g e n e t i c b a s i s
associated with these two indu strial phen otypes. To ident ify th e transcriptional regulatory network
of the protein secretory pathways in A. niger , Kwon et al. (2012) defined the core gene set
including 40 genes which generally respond in several high-thr o ughput protein secretion
conditions. These core genes may p lay a key role in regulating high p rotein traf fic through the
secretory pathway . Alazi et al . (2018) confir med t hat overexpression o f the transcripti on fact or
GaaR activates the extracellula r accumulation of pectinases in A. niger through transcriptome and
exoproteome analysi s. The above proofs gleaned from comparative transcript ome analysis provide
us wi th a wealth of valuable references fo r optimizing t he prod uction of target protein thr ough
genetic engineering.
Bio-based organic acid productio n gradually substitut es pet rochemical as building-block
c h e m i c a l s , t o b e a p r o m i s i n g a l t e r n a t i v e s t r a t e g y f o r t h e c h e m i c a l s i n d u s t r y ( H o s s a i n et al. , 20 19c).
Transcriptome analysis provid es more drivin g forces t o optimize itaconic acid (IA) heterologous
production in A. nig er systematically . Hossain et a l. (2019b) i nitiated with i dentifying two novel
IA biodegradation pathways in A. niger, and t hey found that the deletion of critical genes ictA a n d
ichA on one bioconversion pathway enab led to improve IA production. However, disrupting tmtA
o n t h e o t h e r d e f i n e d w a y n e a r l y e n t i r e l y i n h i b i t e d I A f o r m a t i o n . M oreover, they also identified
that acetyl-CoA c ould be the bottleneck for cytosolic IA produc tion i n t heir previous study
(Hossain et al. , 2019a). Thereby, t o en hance th e cyto solic acety l-CoA abundanc e, they furt her
performed the metabolic engineering of ACL (ATP-citrate lyase) combining the optimization of
medium composition and cu lture conditions, l eading to improved IA accumulatio n.
1.3.2 T ranscriptome analysis t o r eveal the regulatory mechanis m of secondary met abolites
biosynthesis in A. niger
The r ichness of data from varied layers of omics (genomics, proteomics, transcriptomics, and
metabolom ics) in A. nige r accelerat e the identificat ion and characteriza tion of a sea of g e n e s o r
gene clust ers putati vely involved in natur al products b iosynthe s i s , s u g g e s t i n g t h e p o t e n t i a l o f A.
niger to be a promising bio-based production platform for nat ural dr ug discovery a nd biosynthesis.
Wang et al. (2018) characterized the global transcriptional regulator LaeA relevant to the
activation of 281 putativ e seco ndary metabolite genes, providin g potential resources for new drug
exploration. Based on the accurat e prediction of co-expressi on sub-networks, Schäpe et al. (2019)
identif ied two unreported novel global regu lators (MjkA and Mjk B). Wet lab experiments
confirmed their surprising regulatory roles on dozens o f natura l p roducts at the transcript a nd
metabolite level. Noteworthy, to induce ascomycete secondary me tabolism throu gh the conditional
expression of MjkA coul d be an attractive strat egy in drug deve lopment.
1.3.3 T ranscription profiles to describe cellular r esponse on div erse carbon source
Bioconversion of the compl ex cellu lose and hemicellul ose to fer mentable carb on sou rces is sti ll
challenging in the second generation biofuels production (Delma s et al . , 2012). Fungi are


C h a p t e r 1 5 
outstanding hosts for the product ion of plant cell wall polysac charid es–degrading enzymes, such
as cellulo se and hemicellulose, which is playing an i ncreasingl y importan t role in biofuels
biomanufacturing. More import an tly , to optimize the productivi t y of these enzy mes mandates to
control the cost of in dustrial b iofuel production. T ranscrip tio nal evidence indicates that the sorts
or concentration of carbon sources remarkably influence the pro duction of polysaccharide-
degrading enzymes i n A. niger . Jorgensen et al. (2 009) compared the transcriptome of A. niger
grown on xyl ose o r maltose at the same specific growth rate in carbon-limi ted chemostat cultures.
They found that when grown on maltose, the yield of the extrace llul ar en zyme was about three
times hi gher than that on x ylose, accompa nied by the up-regula t ion of genes related t o protein
secretion. de Souza et al. (201 1) recognized that 58% of cellulases and hemicell ulose predicted in
A. niger were highly expressed at the transcriptional level when grown o n sugarcane bagasse
compared t o glucose. Subsequently , de Souza et al. (20 13) r eported the gene sets regulated by two
transcription activ ators XlnR and AraR respo nsible for plant po lysaccharide degradati on, which
are mainly involved in the degrad ation of cell ulose and hemicel lulose and pr otein t ransport ation.
Diverse carbon sour ces used in bi ofuel prod uction perform diffe rently in inducing t he expression
of plant biomass-degrading enzymes. Therefore, to identify avai lable carbon sources may not only
improve t he yield or ef ficiency o f e nzymatic cocktails but also e f f e c t i v e l y r e d u c e t h e c o s t i n t h e
industrial fermentation of renewable fuels.
1.4 The metabolom ics of A. niger
Metabolomics a nalysis mandates to identify biomarkers and key m etabolic nodes at the metabolic
level un der specific environment a l or genetic perturbat ions. Co mbining with the reliable extr action
and quantification methods of intracellular metabolites and met aboli c f lux anal ysis, i t reinforces
the possib ility to ref lect the actua l intrac ellular meta bol ic s tatus that is pivotal in precise l y dir ecting
the industrial and b iotechnological bio-based productio n.
In light of the multi-omics analysis incorporating 13 C isotope labeling, metabolomics, and
metabolic flux a nalysis, our lab recognized that two high-yi eld GlaA produci ng A. nige r s t r a i n s
GAM15 and DS03043 displayed similar cellular metabolic respo nse in comparison to the
reference strain CBS 513.88 (Chen et al. , 2014; Lu et al. , 2015). When forced to overexpress GlaA,
GAM15 and DS03043 channeled more carbon flux t owards PPP accomp anied by a weakened
carbon input into the TCA cycle compared to the reference, l ead ing to a dramatic reduction of by-
products (oxalic acid a nd citric acid). Moreover, the activated PPP accelerated the biomass growth,
and it also reflected an extra d emand for NADPH for the biosynt hesis of amino acid as bui lding
blocks for proteins. However, CBS 513.88 showed a higher r edox state due to the imbalance of
NADH generation and consumption, r esulting in the accumulation of oxalic acid. Meanwhile , the
accu mulated p ool size of int erme diates OAA and PEP on t he lower glycolysi s pathway in CBS
513.88 decreased the glucose uptake rate. Overall, we learn fro m the autonomous adap tion of

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C h a p t e r 1 6 
cellular m etabolism in high GlaA producers that th e enhanced NA DPH provision and the i nhibited
by-products formatio n could be key modification strategies for the subseque nt strain dev elopment.
1.5 Multi-omics integrated ana lysis
Cellular metabo lism is the result of the interaction of multi-d imensi onal network s so t hat single-
level omics could not ent irely reflect complex metabolic activi ties. Varied layers of omics data
closely interact, but e ach omi c c arries its strengths a nd we akn esses. For example, the transcriptome
is easier to degrade, which may a f f e c t t h e a c c u r acy of subseque n t a n a l y s i s w i t h l o w q u a l i t y o f
transcriptome samples. With r egard to pro teomics, the isolati on and accurate quantification of
many low- abundance proteins still face technical c hallenges. Be sides, the reliable met abolites
extraction and accu rate quantifi cation for complex small molecular compounds still have a long
way to go. Therefore, integration analysis combining multi-omic s data is advan tageous to r einforce
the reli ability of data analysis, providing a more detailed and comprehensive profile of intracellular
metabolic activities and the interaction b etween diverse omics.
C u r r e n t l y , t h e i n t e g r a t e d a n a l y s i s o f t r a n s c r i p t o m i c s a n d p r o t e omics is commonly applied i n A.
niger study. A. n iger adopts a delicate qualit y control m echanism for p rotein secreti on, which is
closely related to the highly fl exible ER-associated pr otein de gradation (ERAD). The secretory
system in A. nige r s h a r e s a p a r t i a l s i m i l a r i t y w i t h t h a t o f m a m m a l i a n c e l l s a n d Saccharomyces
cerevisiae (Pel et al. , 2007). Aft er compared the trans criptome and proteome data bet ween the
wild type and th ree overexpression strains (heterologous lipase , homologous pr otease, and
hydrolase), Jacobs et al . (2009) identified that proteins invol ved in carbon and nitroge n met abolism,
(oxidation) stress response, protein folding, and ERAD wer e up- regulated at both transcriptional
and protein level. Howev er, only pro teins on the protein degrad ation pathways showed higher up-
regulation in all three engineered strains, suggesting that it coul d be selected as l eads f or genetic
strain improvement. Based on this result , they deleted t he ERAD f a c t o r doaA to weaken protein
degradation an d overex pressed oligosaccharyl transferase st tC to facilitate prot ein folding in a gus
(β-glucuronidase) overproducing strain, leading to a slight i nc r e m e n t o f G U S . S i m i l a r l y , C a r v a l h o
et al. (2011) disrupted derA gene associated with ERAD in a multi-copies GlaGus overproducing
A. niger , resulting i n 6 fo lds higher of GlaGus. In sum, the above proo fs suggest that c ombining
the dele tio n of certa in genes on the ERAD pathway and the intro duction of multi-copies
heterologous protein is an excellent strategy for optimizing he ter ologous pr otein product ion in
Aspergil li .
W i t h t h e s u r g e o f s t r a i n a n d d a t a b a s e i n f o r m a t i o n , t h e G S M M m o d el has be en con struct ed and
upgraded in increasing organism s. The i ntegrate d multi-omics an alysis cen tred on GSMM enables
to provide new insights int o revealing the glob al cellular meta bolic regul ation. Vongsangnak et al.
(2011)  integ rated multi ple laye rs of omics data, that is, genom es, tra nscr iptomes, interactome s
(protein-protein interacti ons), metabolic networks, and fluxome , to determin ing key cellular
components i nvolved in α-amylase synthesis and secretion, nucle otide metabolism and amino acid

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C h a p t e r 1 7 
metabo lism in Aspergillus oryzae . To shed light on the co rrelation betwee n env ironmental pH a nd
organic acid pro duction, Andersen et al. (2009 ) systematically uncovered t he high efficient acid
production in A. niger through int egrated analysis of GSMM, genome and transcriptome d ata. The
updated GSMM with optimized extracellular p roton-production cou l d better predict the preferred
pH for citrate an d oxalate p roduction. Comparative transcri ptom e a nalysis al lowed the
identif ication of pH-depend ent cis-acting promoter elements and four secondary metabolite gene
clusters regulated by pH. I n addition , t hey also found t he o rth olog of pal/ pacC pH signall ing
pathway of Aspergillus nidulans in A. ni ger . This integrated analysis provides abundant theoretical
basis for g uiding commercial acid producti on by A. niger and for studyi ng the transcriptional
adaption mechanism arouse by pH per turbation in industrial prod uction processes.
1.6 Genetic modific ation platforms in A. niger
1.6.1 The a pplication of T et-o n inducible gene expression switc h
In a typical molecular biology st udy, the characterization of g ene function or regulator y networks
is generally based on gene knoc kout or overexpression. However, i n S. cerevisiae and other higher
eukaryotes, the majority of gene disruption does not lead to vi sible phenotype alteration, whi ch is
covered by the genome robust ness or redundancy (Gi aever et al. , 2002; Kamath et al. , 2003; Meyer
et al. , 2011) , thus imposing barriers to disti nguish the gene functio n. A t p resent, the m ajority gene
overexpression studies still adop t metabolism-dependent promote rs, while this may limit the
choice of medium and inhibit the signif icance of gene overexpre ssion due to the pleiotropic effects
aroused by metabolic alteration (Meyer et al. , 2011). Therefore, it is of considerable significance
to find a strong, inducible, and metabolic-independent promoter for academic app lication. With in
the three published induci ble gene expr ession system (P thiA , hER, a nd Tet), Tet (tetracycline
resistance operon) system sh owed the highest potential to be pr of oundly appli ed in f ilamentous
fungi (Meyer et al. , 2011; Dichtl et al. , 2012).
Meyer et al. (2011) fo r the first time appl ied the Tet-on/tet-off system in A. niger and successfu l ly
established a tunable gene expression system, which is silent w ithout inducer and carbon or
nitrogen s ource independent as well. The Tet-on swi tch is c ompo sed of two expression modules,
one cont aining a const itutively expressed tetracycline-dependen t transa ctivat or rtTA2 S -M2, the
other comprisi ng a the rtTA2 S -M2-dependen t promoter fused with the int erested gene. Based on
the previous stu dy, Wanka et al. (2016) up graded the Tet-on system to improve its genetic stabi lit y
in A. niger by substitu ting the PgpdA promoter with t he PfraA promote r. To date, in light of it s
outstanding induction intensity, Tet-on swi tch has been widely used i n a variety of filamentou s
fungi, such as A. n iger , A. f umigatus , A. nidulan s and A. terreus ( M e y e r et al. , 20 11; Ouedraogo
et al. , 2011; Di chtl et al. , 2012; Warten berg et al. , 2 012; Samantaray et al. , 201 3; Kwon et al. ,
2014; Richter et al . , 2014; Kalb et al. , 2015; Macheleidt et al. , 2015; Paun et al. , 2016; Sun et al. ,
2016; Schütze and Meyer, 2017).

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C h a p t e r 1 8 
Tet-on switch is rega rded as an e xtraordinary tool for function al genomics and systems b iology
studies in filamentous fungi, such as acti vating natural metabo lites or explori ng unknown gene
functions. Through mining genomic informatio n, a wealth of gene clust ers of natural met abolites
has been predi cted in th e seque nced fi lamentous fungal genomes. However, gene clusters of the
majority of natural pr oducts keep silent in th eir natu ral ho sts, inducing difficulti es for t he novel
secondary metaboli tes discovery. There are five non-ribosomal p olypeptide synthase (NRPS)
encodin g gene s pgnA, at melA, atqA, apvA an d btyA i n A. terreus (Guo and Wan g, 2014), while
only pgnA has not been well characte rized. Sun et al. (2016) adop ted the Tet-o n t o act ivate t he
silent pgnA g e n e , r e v e a l i n g t h e g e n e p r o d u c t o f t h i s c r y p t i c N R P S - l i k e g e n e. Through the
heterologous expression in A. nidulans , pgnA is verified as the essential gene for the biosynt hesis
of phenguignardic acid. M acheleidt et al. (2015) ov erexpressed a t ranscriptional regulator of cyclic
AMP-dependent protein kinase A (PKA) pat hway recognized by tran scriptomics analysis , fmpR ,
through Tet-on switch. This initi ated the specific expression o f the other six genes of the fmp
cluster, enab ling the prod uction of a novel natural product 5-b enzyl-1H-pyrrole-2-carboxylic acid
(also named as fumipyrrole).
Conditi onal g ene expression is a powerful molecular biological tool for characterizing unknown
intracellular p roteins, allowing th e disco very of specific phen otypes relevant to gene dosage effects
(Helmschrott et al. , 2013). The adven t of Tet-on s ystem realizes the ti trat able a nd conditional
control of genes in filamentous fungi. Therefore i t has been wi de ly u sed in th e p re di cti on o f ge ne
biological function. Rho GTPase rho1 is an essential regulator for cell wal l i ntegrity (Kwon et al. ,
2011), thus the disruption o f which may lead to letha l phen otyp e . B y s u b s t i t u t i n g t h e n a t i v e
promot er of the Rho GTPase rho1 with the Tet-on switch in A. fumigat us , it a llows defining the
gene fun ction of the e ssential gen e rh o1 (Dich tl et al. , 2012). Schalén et al. (2016) individ ually
overexpressed 14 g enes relev ant t o protein secretion via the Te t-on promoter in an A. nidulans
secreting mRFP as a fluorescent reporter. The overproduction of Rab G TPase RabD n otably
improved the prot ein secretion by 40%. However, several constru cts reduced t he secreti on of
mRFP, suggesting that the up-regulation of par tial genes on sec retory pathways aims to relieve the
cellular burden resulted from the high protein t raffic.
1.6.2 Applicati on of t he CRISPR/Cas9 gene- editing in fi lamento us fungi
In recent ye ars, with the de velo p m ent of genome sequencin g tech nology and the drop in financial
cost, it provides us with considerable genome knowl edge of fila mentous fungi. Nevertheless, the
molecular biology and biochemical mechanisms of only several mo del fungi have been well
characterized, and t remendous unknown knowl edge remains to be e xplored. However, due to the
complex g enome structure of filamentous fungi, t he existing gen e-editi ng tools not allow efficient
genetic modification, which s everely hinders the molecular biol ogy research in these industrially
important platforms. To date, a variety of gene-editing too ls h ave been de velo ped for f ilamentou s
fungi (Meyer, 20 08). The non-homologous end-joi ning (NHEJ) medi ated repair is t he dominating

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C h a p t e r 1 9 
DNA repa ir m echanism in filame ntous fungi , leadi ng to t he very low efficiency of homol ogous
directed repair (HDR) based gene manipul ation (<5%) (Meyer et a l. , 2 007; Ful ler et al. , 2015).
The disruption of Ku70 or Ku80 encodi ng gene on the NHEJ-pathwa y is the most represent ative
method t o elevat e t he homologous recombination (HR) efficiency (up to 65%) in filamentous fungi
(Weld et al . , 200 6; Liu et al. , 2015 ). However, th e loss of this i mportant DNA repai r mechani sm
may make cells more se nsitive whe n expose d to ɣ radia tion and t he culture environment containing
specific chemicals, such as phleo my cin, bleomycin, and methylme sy late (Meyer et al. , 2 007; van
Attikum et al. , 2014; Weyda et al. , 2017). In addition, Δ ku is not available for the agrobacteriu m-
mediated fu ngal transfo rmation that requires th e NHEJ proteins for the integ ration of T-DNA into
the host genome (van Atti kum et al. , 2 014). Ind ependent on th e NHEJ, Niel sen et al. (2 006)
established a split marker metho d employing a bipart ite gene-ta rgeting substr ate by PCR, leading
to three fol ds higher of HR efficiency (Weyda et al. , 2017). However, this str ategy still calls for a
huge workload to achi eve a high concentration of biparti te subs trates used for fungal
transformation, and sometimes the positive rate of transformant s is not such attractive wi thout
adopting Δ ku nul l mutant. Over all, because of the weakn ess of t he existing genet ic edi ting tools,
it provides a br oad platform fo r the applicati on of CRISPR/Cas9 gene- editing system in
filamentous fungi.
1.6.2.1 Discovery of CRISPR/Cas system in the bacterial system
In 1987, Ishino et a l. (1987) for the first time reported the clustered regu larly int erspaced
palindromic repeats (CRISPR) in E. c ol i an d a similar str ucture was ex t ensively identified in a lot
of bacterial genomes as well. The hypothesis that CRISPR-Cas sy stem provides the acquired
resistance against viruses or pha ge invasion was init ially conf irmed in Streptococcus thermophilus
in 2007 (Barrangou et al. , 200 7). To date, three CRISPR sys tems have been identified (Ma karov a
et al. , 2006), of which the type II system has attracted t he most ext ensive attention. Si nce Cas9
protein i s regarded as the onl y protein gui ded by a two-RNA str ucture combi ning CRI SPR-derived
(crRNA) and trans- activating crR NA (tracr RNA) to direct cleav ag e in t arget DNA (Ji nek et al. ,
2012). Type I I system is more dir ect, simple and easier to be a ppl ied than the ty pe I and type III
systems, which has brought huge breakthr oughs for modern biolog y in such a short time (Doudna
and Charpentier, 2014).
1.6.2.2 Applicati on of CRISPR /Cas in filamentous fungi
CRISPR/Cas9, as an efficient and versatile gene manipulation to ol, has been wildly used in
mammalian cells, yeast and plant c ells. Since 2015 , with the su ccessful applicat ion of CRISPR
system in increasing filament ous f ungi, for instance, N. crassa ( M a t s u u r a et al. , 2015), A. nidulans
(Nodvig et al. , 2015), T. reesei ( L i u et al. , 2015), Candida albicans (Vyas et al. , 2015) Peni cillium
chrysogenum ( Pohl et al. , 2016), op portunistic pathogens A. fumigatus (Krappmann, 2007; Fuller
et al. , 2015; Mil es et al . , 2016 ), A. n iger (Nodvig et al. , 2018; Zheng et al. , 2 018) and A. oryzae
(Katayama et al. , 2016), makes it possible to achieve the high eff icient genet i c modification in

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C h a p t e r 1 10 
these economic important microbe s. Compared t o traditional HDR based gene disruption,
CRISPR/Cas9 contributed to an increase of 6 folds for knockout e fficiency (Pohl et al. , 2016).
Moreover, with the support of abundant genome sequence i nformat ion, CRISPR/Cs s9 g ene-
editing gets access to accelerate the constructi on of a genome- wide single-gene knockout library
of filamentous fungi (Liu et al. , 2015).
With respect t o CRISPR/ Cas9 tech nology, t he Cas9 end onuclease is guided by a single-chimeric
sgRNA to the PAM si te (protospacer adjacent motif ) to introd uce a d o u b l e - s t r a n d b r e a k ( D S B )
within the target DNA at the 3-4 bp upstream of the PAM. Two ce ll ular DNA re pair mechanism s,
NHEJ or HDR, are a dopted to repair t he DSB, lead ing to prec ise gene manipulatio n (Jinek et al. ,
2012). sgRNA i s co mposed of two par ts, including a 2 0 nt gRNA/P rotospacer de fines the target
locus adjace nt a 5’-NGG DNA motif (PAM) and a 77 bp sgRNA tail (Ran et al. , 2013; Poh l et al. ,
2016) (Figure 1.2) .

Figure 1.2 CRISPR /Cas9 gene-ed iting platform (Pankowicz et al. , 2017) . (A) A single-c himeric sgRNA
combi nes w ith Cas9 prote in to gene rate C RISPR -Cas9 ribonu cleopr oteins (RNPs) to ca talyze a DSBs within
target DNA . (B) Two types of D NA repair mec hanism, the no nhomo lo gous e nd-joinin g (N HEJ) as well a s
homolo gous repai r (HR), are used to fi x the genome
At the initial application stage of the CRISPR/Cas9 platform in filamentou s fungi, the majority of
studies focused on o ptimizing i ts gene-editing e fficiency in di verse fungal hosts. Therefore,
pigment biosynth esis genes ya or wa (Z h en g et al. , 2017 ) were generally targeted to evaluate t he
gene manipulation efficiency from phenot ype. With t he rapid dev elopment of this master tool in
filamentous fungi , it has been gra dually employed to r ewire spe cific metabolic pathways for the
purpose of optimizing the productio n capacit y of fungal bi ologi ca l fa ct o ri es , in pa rti cu lar , fo r th e

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C h a p t e r 1 11 
development of bioactive pha rmaceut ical products. Kuivanen et al. (2016) f irst e mployed
C R I S P R / C a s 9 i n t h e m e t a b o l i c e ngineeri ng studies of f ilamentous fungi. Through di srupt ing
galactaric and D ‑ galacturonic acid cata b o l i s m v i a C R I S P R / C a s 9, i n parallel wi th expressing a
heterologous uronate dehydrogen ase produced galactari c acid fro m D ‑ galactur onic acid , they
successfully achieved an efficient galactaric acid pro ducing en gineered A. n iger stra in. S imila rly,
Weber et al . (2017) activated t he polyketide synthase encoding gene tynC involved in the
trypacidin biosynthetic pathway by CRISPR/Cas9-based tool, allo wing i ts pr oduction i n a
nonproduci ng A. fumigatus strain.
Both in vitro or in vivo transcription of s gRNA are att ractive strategi es, but with their streng ths
and weaknesses. In vitro synthesized sgRNA is e asy in molecular manipu lation without any
cloning efforts, but its stabilit y i s always argued. Kuivanen e t al. (2016) and Sar kari et a l. (2017)
reported that th e absorptivit y and stabil ity of i n vitro synthe sized sgRNA might affect gene editing
efficiency. In vivo transcribed sgRNA is primarily initiated by t w o t y p e s o f p r o m o t e r s , R N A
polyphase II or RNA polyphase III. Compare d to the type II prom oter, RNA polyphase III allows
the direct regulation of sgRNA transcr iption. However, only lim ited RNA polyphase III promoters
have been identified i n filamentous f ungi, becoming an urgent n eed to rea lize eff icient in vivo
sgRNA biosynthesis. The RNA polyphase type III U6 promoter is a per fectly suit able tool , which
could directly initiate the transcri ption of sgRNA wit hout i ntr oducing the cap structure and po ly-
A t ail. Thus it has been widely used i n t he in vivo biosyn thesi s of sgRNA (Arazoe et al. , 20 15;
Nodvig et al. , 201 5; Katayama et al. , 2016; Pohl et al. , 20 16; Schuster et al. , 2016; Zhang et al. ,
2016; Liu et al. , 2017; Zheng et al. , 2017; Nodvig et al. , 2 018). Zheng et al. (2018) are the first to
identif y and validate a n endogenous U6 promoter in A. niger, estab lishing a novel CRISPR/Cas9
system which coul d o btain a high efficient in-frame gene insert ion using donor DNAs with o nly
40 bp length of homologous arms. Noteworthy, recognition of the endogenous U6 promoter in A.
niger e nables to s implify genetic modifi cation, r ed uce cloning effor ts for the i n vivo expressi on of
sgRNA, and enrich the CRISPR/Cas9 tool box as well.
It has been val idated t hat providing donor DNA is conduciv e for the hig hly efficient CRISPR/Cas9
gene edit ing, where cell tends to repair the DSB through HDR (P ohl et al. , 2016). In particular,
supplying circular plasmid type donor DNA allowed for higher HR efficiency than that o f
lineari zed donor DNA (Nodvi g et al. , 2015). To date, several resear ch groups h ave exploited donor
DNA wi th sho rt fl anking re gions, or even s ingle-st raine d oligo n ucleotides to achieve effi cient gene
disruption or gene i nsertion i n T. reesei (W eyda et al. , 2017), A. fumigatus ( N i e l s e n et al. , 2006 )
and P. chrysogenum (Nodvig et al. , 2015). This strategy does not require to construct complicate d
gene deletion cassettes, thus greatl y reduce the w orkload and t ime require d for clonin g, paving the
road for the high-throughput and st raightforward genetic manipu lation in filamentous fun gi. Pohl
et al. (2016) d emonstrated a large gene fragment deletion (>18 kb) an d al so an e ntire SM gene
cluster deletion using donor DNA wit h f lanks as sho rt as 6 0 bp. Nodvig et al. (201 8) e nabled to
induce up t o 100% specific point m utations as well as gene dele tions by adopting 90 bp single-

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C h a p t e r 1 12 
stranded oligonucleotid es for C RISPR-Cas9 medi ated gene editing . Zhang et al. (2016)
successfully achieved the marker-free gene tagging of GFP prot e in through a highl y efficient
C R I S P R m u t a g e n e s i s s y s t e m i n a ku -80 proficient A. fumig atus at accuracy approxima tely 95–
100% via on ly 39 bp homology arms . They named this NHEJ-inde pen dent system as
microhomology-mediated end j oining (MMEJ).
Optimization of ind ustrial-scale commercial production from cel l facto ries generally requires
combinational genetic engineeri ng, but it is always time- and l abour-intensive when using limited
selection markers for tradit ionally sequent ial strain developme nt, cau sing high demand for
molecular manipulati on (Katayama et al. , 2019). Therefore, efficient marker-fre e mu ltiple gene
editing has become one of the re search focuses on the current C RISPR/Cas9 technology (Li u et
al. , 2015) . The auton omously replicating plasmids based on AMA1 se quence allows the reuse of
selective marker for multi rounds of genetic manipulation, whic h h a s b e e n a p r o m i s i n g s t r a t e g y
for seam less genomic editing (Nodvig et al. , 2015; Po hl et al. , 2016). Add itionally, the long gr owth
cycle of filamentous f ungi typically causes several months of c ostl y development time for multiple
rounds of genetic modificat ion, whic h severely reduces the effi ci ency of metaboli c engineeri ng in
filamentous f ungi. Th e emergence of CRISPR/Cas9 platform open a new d oor for simultaneous
multiplex g ene-editing in fi lament ous fungi, but there is still a long way to go to achieve a
frequency for mult iple recombination as high as singl e-gene mod ification. Liu et al. (2 0 1 5) p u t a n
effort t o disrupt multi ple gen es simultaneousl y v ia c o-transfor ming multip le in vitr o synthesized
sgRNA and donor DNA in T. reesei , but the current editing s trategy only resulted in a vi sible l ower
frequency than single-gene d isrup tion. In this syste m, the freq uency of double r ecombination was
only 16%, while dropping to 4.2% for t riple-gene deletion. The majority of cur r en t establis hed
fungal CRISPR/Cas9 systems are not available f or in vivo multip lex sgRNA expression.
Noteworthy, tRNA-spacer based system provides a promising so lut ion for this issue, which has
been widely harnessed in various organisms for multiple sgRNAs expression, including pla nts,
yeast, human c ells and fruit cells (Schwartz et al. , 20 15; Xie et al . , 2 015; Do ng et al. , 201 6; Port
and Bullock, 2016; Qi et al. , 2016). Nodvi g et al. (2 018) are the first to report its application in
Aspergil li . In t his syste m, each sgRNA sequenc e is fl anked by identical g lycine tRNA sequen ce
on two sides, and then multiple sgRNA tran scribes are liberated f rom a sin gle pr e-RNA transcript
spliced by RNase P and RNase Z at the 5' and 3' splice sites of tRNA (Fig ure 1.3).
The advent o f CRISPR/ Cas9 has induced a break through rev olution for the g enetic engineering o f
filamentous fun gi. Although CRISPR / Cas9 technologies are m ain ly exploi ted for gene disruption
at present, with i ts extensive a nd in-depth application in prec is e gene modif ication, such as gene
silencing, secondary metabolite development, gene overexpressio n, gene mu tations, gene
regulation, and gene tags, i t will provi de great possi bilities for rati onal str ain devel opment of
filamentous fungi in t he near fu ture (Wang and Co leman, 2 019) ( Fi gure 1.4).

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C h a p t e r 1 13 

Figure 1.3 tR NA cleaving r eleases mu ltiple sgRN A specie s from a single pr e-RNA transcr ipt. Individ ual
sgRNA spec ies are fla nked by tRNA sequences . Dotted lines and “ P acmen” indica te cle avage sit es fo r RNa se
P and RNase Z in the pre- RNA tra nscrip t. Di fferent colors of pr otospacer present different se q uences (Nodvig
et al. , 201 8)

Figure 1.4 Application of CRISPR /Cas9 technology in filamentou s fu ngi (Wang an d Coleman, 2 0 19)
1.7 Optimization of the production capability of microbial cel l factory via met abolic
engineering
1.7.1 Construction of efficien t smart biomanufacturi ng cell f actory driven by DBTL cycl e
It is commonly accepted that traditional strain modification is n o t a t a s y s t e m l e v e l b u t i s t e s t e d o n
a trial-and-error basis to m odulate the strength of specific me tabol ic pa thways to spur the
maximum potential of production chassis (Opg enorth et al. , 2019 ). How ever, t his typical research
route unable t o provide a systematic v iew of cellular regulatio n and also leads to higher time and
labor costs. For example, the activation of the entir e artemisi nin biosynthesis gene clusters carrying
16 genes cost 150 person-years of eff ort and mil lions of dol lar s (Opgenorth et al. , 2019 ).

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C h a p t e r 1 14 
Noteworthy, t argeting regulation sometimes may ex hibit a hi gher eff iciency in optimi zing the
desired output than t uning speci fic metabolic pathways (Lawson et al. , 2019). With the r apid
advance of DNA s ynthesis, gene m odifi cation tool box, high-t hrou ghput screening and t he
popularity of machine learning, it paves a new way to speed up rational engin eering thr ough
systematic metaboli c engineering (Opgenort h et al. , 2019). Metabolic e ngineering invol ves
rewiring the native metab olic pathways and reconstructing heter ologous product biosynt hesis
pathways (Niel sen and Keasling, 2016). Ho wever, enormous challe nges lie ahead for
reprogramming proof-of-concept cells i nto high e fficient cell f actories. D ue to the ev olved robu st
cellular metabolic networks attempting to keep homeostasis, our engineer ing eff orts to enh ance
flux towards desired products are always r esisted (Nielsen and Keasling, 2 016). Theref ore, the
iterated application of design-bu ild-test-learn (DBTL) loops is exp loited to engineer cell factories
to achi eve the desired design that mee ts specifi cations. DBTL c ycl e initiat es wit h designing (D)
biological systems to set clear engi neering goals (Lawson et al. , 2019). Biol ogical systems are then
built (B) by adopting system biology tool boxes such as DNA part s or appropriate microbial c hassis.
The n ext phase incorporates the use of a variety o f assays (for example, physiological data or omics
analysis) to test (T) wh ether the performance of the biosystem meets the original desired design.
The la ck of compre hensive datab a ses for non-model strains sever ely hinders the success rate of
DBTL cycle . Thus, data capture, data analysis, and data inte rpr etation are urgent issues to be
improved (Nielsen and Keasling, 2016). The l earning (L) phase o f the DBTL cycle is currently the
most ch allenging step d ue to limited biological experience (And o and Gar cia Martin, 2018). This
phase interprets the data obt ained from the test stage t o acqu i re new biological insights, for instance,
novel regulatory structures or pr imary metabolic activities, to drive t he design of the next round of
DBTL cycle (Nielsen and Keaslin g, 2016). Moreover, recent advan ces in model prediction, mult i-
omics integration analysis, hi gh-throughput strain s assembly, a nd screening have provided more
driving forces for speeding up t he DBTL c ycle (Liu a nd Nie lsen, 2019).
The systematic engineering centred on DBTL framework has long b een a core e lement in
traditional engineering practice s t o s a t i s f y t h e d e s i r e d s p e c i f ication. This systematic and rational
metabolic engineering framework has led to notable advances f or cellulase production in T. reesei
(Zou et al. , 2012) and fatty acid formation in E. coli (Chen et al. , 2018). Surpr isingly, aft er t wo
rounds of DBTL c ycles, an increase of 40-fold yield of pinocemb rin was ac hieve d (Carbonel l et
al. , 2018). More i mportantly , through aut omated DBTL c ycles to acc elerate the constitution of
smart biofoundries has stirred up extensiv e discussions in rece nt years. Automated recursive
DBTL cycle starts wi th gleaning candidate enzymes in silico. Su bsequent ly, it direct s rat ional
redesi gn through au tomated DNA d esign, robot-assisted pathway a ssembly, fast assay , and
autonomous learning, expecting to r ealize the entirely automati c and hi gh-throughput strain
development pipeline (Chao et al. , 2017). Therefore, t he automated DBTL pipeli ne dramatically
reduces the turnover rate of each cycle a nd enables to get rid of human errors and bi ases resulted
from man ual interferen ce, thus maximizing exp erimental obj ectiv ity (Chao et al. , 2 017). Carbonell


C h a p t e r 1 15 
et al. (2 018) leveraged two round s of automated DBTL loop to rapidly and succ essfully obtain a
500-fold h igher titer of flavon oid (2S)-pinocembrin i n E. co li , reaching up to 88 mg/L.
Additionall y, with the advance of novel technologies a nd the st imu latio n of large comme rcial
investment, t he estab li shment of automated and versatile e ngine eri ng pl atf orm s will not only
introduc e a m ore rapid, predic table and sustaina ble production pipeline for high val ue-added
biologi cal products but al so a cceler ate the p roof-of-concept st udies.
1.7.2 Recent advances of cell factory A. niger
A. niger has been developed in to one of the promising industri al cell f actories for the
biomanufacturing of a vari ety of organic acids (citric acid, c h loric acid, malic acid, itaconic acid,
etc.), enzyme preparation (GlaA, cellulase, pectinase, etc.) a n d natural products (Meyer et al. ,
2015). With the rapid development of systems biology and synthetic biology, improving a proof-
of-principle strain t o a superior cell factory meeting indu stri al T RY （ titer, rate, and yield ）
requirements t hrough rati onal engineering has become a signific ant g oa l for t he subsequent A.
niger studies (T ong et al. , 2019).
In t he last two decades, heterologous and h omologous e nzyme pro duction has achieved remarkable
progress in A. niger  accel erated by the availability of synthetic biology tools, for inst ance, codon
usage optimi zation for heterologou s expressing protein, promote r optimization (Liu et al. , 2003 ),
the increment of gene copy, fusion protein expression (Xu et al . , 2018), inhibition of extracellular
protease (Punt et al. , 2008), or optimization for protei n structure (Lubertozzi and Keasling, 2009).
However, due to the lack o f syst ematic and comprehensive unders tanding of t he secretion system
in filament ous fungi , it introd uces obstacles for maximizing pr otein secr etion and rewiring
secretory pathways by genetic e ngineering. Therefore, a system a tic s tudy for t he regulatory
mechanisms that profits fungal se cretion i s speculated as a pri mary research focus for the next 20
years (Cairns et al. , 2018).
A. niger -derived citric acid accoun ts for pr obably 80% of the global ci tri c aci d market (Tong et al. ,
2019). Systemic metabolic engi neering is a powerful strategy to unco ver the underlying molecular
regulation mechani sm for citr ic acid biosynthesi s, so as to rew ire A. niger to s at is fy th e indus trial
specification for large-scale ci tric acid pr oduction. Enormous biotech nology endeavours have also
been paid in optim izing the production of this commercially imp ortant organic acid in A. niger
(Tong et al. , 2019). For instance, identifying ke y genes associated with ci tric acid biosynthesi s
through multi-omics analysis (Andersen et al. , 2011; Xi an et al. , 2017) , i mproving the carbon
source utilization, modulatin g citri c acid biosynthesis pathway s, en hancing precursor suppl y,
regulat ing the respira tory chai n to boost NADH generatio n (Wang et al. , 2015), reducing f eedback
suppression or inhibiti ng by-produ ct formati on. Based on the ab ove biological efforts, the titer of
bio-based citric acid production by A. ni ger reaches up to 184 g/ L total sugar.


C h a p t e r 1 16 
With respect to ind ustrial applicatio n, the co ntrol of micro or macromorphology a nd the biomass
growth t hrough regulating the cu lture conditions t o optimize th e desired producti on have also
attracted extensi ve attention. F or instance, a hyperbran ching A. niger is used to charact erize t he
morphogenetic network of A. niger. It has b een wildly a ccepte d that proteins ma inly secrete at the
hyphae tips; however, if the increase of hyphal bran ches i n fi l amentous fungi induces secretion
remains argued (Kwon et al. , 2013). Based on the construction of Rho-GTPase RacA del etion
mutant accompanied wi th a hype rbranching phenotype, Fiedler et al . ( 2018a) fi rmly demonstrated
that an increment of post-Golgi secretory vesicles at the h ypha e tips merits th e h igh protein traffic
through secret ory pathway s in Δ ra cA null mutant, confirming t he positive co rrelation between the
amount of hyphal ti ps and secretion. They also revealed that th e additio n of insolub le microt alc
partic les enabl ed disperse d mycelium in smaller clumps with hig her frequent branching in the
submerged A. niger cultures, leading to improved mass transfer. Additi onally, oth er fermentation
parameters, su ch as carbo n source, ion content, pH, spore inocu lation concentration, agitation,
aeration, a nd osmotic pressure, have al so been extensively defi ned to obtai n t he desi red gr owth
phenotyp e (Cairns et al . , 2019 ). The above genetic engineering or pr ocess regul ation ap proaches
lead t o p recisely control the hyphal diameter of A. n ig e r aggregates, to optimize the viscosity of
the fermentation broth, and to d ram ati cally reduce the cell s en sitivity t o shear force, thereby
accumulating valuable lessons for the high cell density ferment ation of A. nig er .
Currently, owing to the growin g demand for anti-pathogen ic drug s and the increasin gly seve re
i s s u e o f a n t i b i o t i c r e s i s t a n c e , s e e k i n g f o r n e w b i o a c t i v i t i e s h as become a research hotspot in recent
years. Since natura l pr oducts derived from microorganisms are a bundant sources for novel
unreported bioa ctivities, exce llent cel l factory A. niger is gradu ally compatible as a promising
platform for drug development and industri al natural products b iomanufacturing (Cairns et al. ,
2018). Schütze and Meyer (2017) i ntroduced an entire biosynthes i s p a t h w a y ( esyn1 , ekivR ) of
secondary metabolite enniatin from Fusarium oxysporum to A. niger under control of a
polycistronic gene e xpression system, resul ting in A. niger as a heterolo gous pro duction host for
enniatin.
O v e r a l l , t h e a b o v e g e n e t i c a p p r o a c h e s h a v e s h o w n g r e a t p o t e n t i a l to optimize the production
capacity of A. n iger i ncluding protein carrier approaches, tunab le Tet-on driven gen e expression
and morphology engineering , to name but a few (Cairns et a l., 2 018; Cairns et al., 2019; Fiedler et
al., 2018a,b). However , in addit ion to the optimization of spec ific metab olite pathw ay flux via
genetic eng ineering, cofactor e ngineering is also believed to b e a profitable strategy to m odulate
efficient productio n through rebalancing intracellular redox st atus (Li et a l. , 2 018; Yao et al. , 2 019).
1.7.3 Optimization of cell fact ory productivity by cofactor e ngineeri ng
Nicoti namide adenine dinucl eoti de phosph ate (NADPH) is a n i mpor tant cofactor ensuring
intracellular red ox bal ance, anabolism and cell growth in all l iving systems. The newly upd ated
GSMM iHL1210 m odel ide ntified the in volveme nt of NADPH in a bout 1 73 intracellular redox


C h a p t e r 1 17

reactions in A. niger (incl uding 49 NADPH generating reactions). Therefo re, NADPH is a critical
cofactor to re gulate cell growt h and t he e fficien cy of product biosynthesis ( Lu et al. , 2016).
However , due to the imbalance of generation and consumption of cofactors, it may lead to the
severe a ccumulati on o f by -products (polyhydric alcohols o r or ga nic acids) during A. niger
submer ged cultivation, causing the loss of a lot of carbon subs trate and inducing dif ficult ies for
downstre am extract ion.
NADPH is a limiting fac tor for the biosynthesis of ami no acids. For in st anc e , 3 m ol o f NA DP H i s
required for pr oducing 1 mol of arginine and 4 mol of NADPH is required for the biosynthesis of
1 mo l of lysi ne (Mo ritz et a l. , 2002). Thus adequ ate cytosolic NADPH supply is in dispensable to
maintain t he intracellular redox balance and serves as a drivin g force for efficien t amino acid
biosynthesi s (Park et al. , 2014).
Figure 1.5 P rimary NADP H bios ynthesi s path ways
T wo comm on strategies have been profoundly employed to optimi ze the availa bility of NADPH.
One is to activate the e nzyme activities of NAD(H) kinases (EC 2.7.1.86, EC 2.7.1.23) to obtain
NADPH o r NADP+ t hrough phosphoryl ati on of NADH an d NAD+, respec tively . The oth er is to
modulate the expr ession stre ngth of typica l NADPH gene rating en z y m e s o f t h e g l y c o l y t i c p a t h w a y ,
the pentose ph osphate pathway or the cit ric acid cycle (Figure 1.5). These include glu cose-6-
phosphat e dehydrogen ase (G6PDH), 6-p hospho-gluconate dehydrogen ase (6PGDH), NADP-
dependen t isocitrate d ehydrogena se (NADP-ICDH ), and NADP-depen d ent malic e nzyme
(NADP-ME) (Mar x et al., 199 9, 2015a, b). No teworthy , het erologo us protein expression in f ungi
can be generally triggered throug h boosted carbon flux to the p entose phosphat e pathway (PPP)
(Driouch et al. , 2012; Nie et al. , 20 14), suggesting that the central carbon m etabolism may have
evolved to ensure th e production of cel lular components under t he balance of ener gy pro duction
and consumpti on (Niel sen and Keasling, 2016). Drio uch et al. (2012) biosynthesized the
exogenous fructofuranosidase u nder the control of a constitutiv e promoter o f the pyruvate kinase
gene in A. n iger , an d
13
C metabolic flux analysis demon st rated that the activated PPP a nd
mitochondrial malic enzyme enabled an elevat ed sup ply of NADPH. L i k e w i s e , t h e m e t a b o l i c f l u x
through the PPP increased by 15-26% compared t o the parental st r a i n s w h e n t h e e n z y m e
glucoamylase was overproduced i n A. niger (Pederse n et al. , 2000b) or the enzy me am ylase was
overproduced in A. or yzae (Peders en et al. , 1999).
NADH NAD P H
NADP + 
NADH ki nase
G6PDH,
6PGD H,
NADP-ME,
NADP-I CDH
NAD+ kinase
NAD+


C h a p t e r 1 18 
In t he past two decades, extensi ve stu dies have focused on engi neering a high flux through the PPP
in E. coli , C. gl utamicum , A. nidulans a n d A. ni ger (Canonaco et al. , 20 01; Li m et al. , 20 02;
Panagiotou et al. , 2 009; Poulsen et al. , 2010; Park et al. , 2 0 1 4 ) ( F i g u r e 1 . 6 ) . A b l o c k o f t h e
glycolytic pathway by down-regulating the pgi gene enco ding a phosphoglucose isomerase was
one succ essful strategy in C. glu tamicum (Park et al. , 2014). In orde r to elevate the NADPH pool
origin ating fro m the P PP in A. niger , Poulsen et al., (2010 ) overexpressed the gsdA gene (gluco se
6-phosphate dehydrogenase), the gndA gene (6 -phosphogl uconate dehydrogenase) and th e tktA
gene (transketolase) indivi dually i n A. niger . Strong overexpression of gndA led up to a nine-f old
increase i n intr acellul ar NADPH c oncen tration, while gsdA and tktA af fected the NAD PH level
only weakl y . Howeve r , a ny correlatio n b etween t he NADPH suppl y and enzyme overproduction
remained unclear .
G6P 6PGL 6PG
RU5 P
PEP
Glucose
PYR
AC-Co A
ICIT
OAA
MA L
AKG
SU CC-Coa
FUM
gsdA gndA
icdA
maeA
NADP+ NADPH
NADP+
NADPH
NADP+
NADPH
Al a Val Leu
As p
As n
Met T hr Ly s
Glu
Gln
Pro
Arg
IIe
PYR
MA L
CIT
Mit ochondri on
HIS
Try
Tyr
Phe
OAA
CIT
IC IT
AKG
CIT
OAA
NADP+ NADPH
Cytop l as m
F6P
pgi
FBP
pfkA
G3P DHAP
G1,3P
NADP+
NADPH
NAD- GapN N ADP-GapN
fbp

Figure 1.6 Mainly published NADP H regulation strategies to ele vate NAD PH avail abili ty . Upr egula ted a nd
knock-down/knock ed-out genes are labelled in r ed or green, r esp ectively
Irrespect ive of t he key r ole of t he PPP on NADPH gene ration, an e fficien t carbon economy is only
guaranteed wh en the carbon flux enters the glycol ytic pathway ( Embden-Meyerhoff-Parnass
pathway, EMP) instead of t he PPP because the PPP releases o ne c arbon as CO 2 when oxidizing 1
mole of hexose. Taken o et al. ( 2010) thus sub stituted the en dogenous NA D-dependent


C h a p t e r 1 19 
glyceraldehyde-3-phosphate dehyd rogenase (GAPDH) in the EMP in C. glutamicum w i t h a
heterol ogous NADP- depend ent GAPDH, leadin g to 2 mol of NADPH ge neration instead of 2 m ol
NADH from 1 mol of hexose dur ing glycolysis. This genetic modif i cation pr ovoked a subst antial
improvement in the yield of L-lysine production by 7 0-120%. Sim ilar strategi es also ha ve been
followed to overprod uce ethanol in the yeast Saccharomyces cerevisiae ( V e r h o et al. , 200 3) o r
lycopen e and ε-caprolactone in the bacterium Clostridium ac etobutylicum (Ma rtí ne z et al. , 2008).
NADPH also provides the main anabolic reducing power for biomas s growth, lipid formation an d
is even indirectl y required by the pr ocess of natu ral product b iosynthesis (Nielsen, 2019). C e n t r e d
on CiED mathematical m odel, Chemler et al. (2010) identified genetically modifi ed t argets to
improve NADPH produ ction in E. coli , and tri ple-gene deletion experiments (Δ pgi , Δ ppc a n d
Δ pldA ) l e d t o a r i s e o f 4 a n d 2 - f o l d w i l d - t y p e l e v e l f o r t h e a c c u m u l ati on of two po lyols,
leucocyanidin and (+)-catechin, respectively. Lee et al. (2010) optimized thymidine production by
increasi ng the ratio of NADP H/NADP+ in E. coli . W asylenko et al. (2015) exploited 13 C metabolic
flux analysis to pr edict the metabo l ic flux distribution of two lipid producing Yarrowia li polytica .
Interestingly, the relative flux through t he oxi dative PPP in m odified strain is ab out two-f old hig her
than that in the parental strain , coupling well with the two-fo ld increment of fatty acid produ ction
and the NADPH-ge neratio n rate o n the PPP is consiste nt with t he consumption rate of NADPH
for fatty acid biosynthesis in bot h str ains. However, the flux through th e malic enzy me did not
display a visible difference between the two strains, which is reverse to the role of cytosolic
NADP-ME as the p rimary NADP H source f or lipid ac cumulatio n in o ther oleaginous fungi (Zhang
et al. , 2007; Hao et al. , 2014). Therefore, the oxidative PPP may pr ovide the primary N ADPH for
fatty acid biosynthesi s in Y. li polytica . Moreover, NADPH engi neering coul d also ind irectly
improve the formation of natural products through op timizing the provision of amino acids (L-α-
aminoa dipic acid, L-cy stein, and L-valine) whi ch are precursor s for intermediates on the peni cillin
biosynthesis pathway (Ni elsen, 2019). Similarly , the deletion o f one of the isomers of
phosphofructoki nase ( pfkA2 ) also enabled to channel more flux from EMP towards PPP to ele vate
the NADPH ava ilabil ity, which significantly improved the accu mu lation of actinomycetes in
Streptomyces coelicolor (Irina et al. , 2 008).
1.8 Objective of this project
A. niger , as an important cell factory, has been widely used as a versa tile and powerful platform
for homologous and heterologo us protein produc tion a t the acade mic or industrial level. This thesis
carried out a systematic study for the purpose of optimizing th e pr oduction capabili ty of cell
factory A. ni ger from the per spective of comparative genom ics and t ranscriptome analysis, and
cofactor engineering (Figure 1.7).


C h a p t e r 1 20


Figure 1.7 Framework of the sys temat ic optim ization of e nzyme performance in A. niger
1) Comparative genomics of the aconidial A. niger strain LDM3 predicts genes associated with
its high protein secretion capacity
This dissertation aims to adopt a comparati ve genomics analysis of an ac oni di al i ndu st rial ly
exploited A. niger s t r a i n L D M 3 i n C h i n a t o u n c o v e r m u t a t i o n s r e l e v a n t t o i t s h i g h protein
producing potential. Moreover, l oss-of- function m utants were ge nerated to investigate the validate
the links betwe en the non-sporul ation and the high yi eld of Gla A.
2) Global tran scriptional response of A. niger i n the process of glucoamylase fermentation
Oxygen limitation is one o f the most effective strategies for i ndustrial GlaA production. H owever,
the t ranscriptional level r esponse duri ng GlaA fermentation und er oxygen-l imited condition has
not been well defi ned. To expl ain the gl obal transcriptio nal level alterations m ay help u s deepen
the comprehension of the m etabolic adapti on and cellular resour ce allocation in the low oxygen
availability condition. Through time-course comparative transcr iptome a nalysis at four- time points
during the fed-batch GlaA fermentatio n, this dissertation aim ed t o r e v e a l t h e m e t a b o l i c
characteristics at di fferent fermentatio n phases, and focused o n the key t ranscriptional response
relevant to transcr iption factor s, protein secretion, fatty aci d metabolism, and amino acid
biosynthesi s, i n or der to present val uable targets for the adva nc e o f ind ustrial-scal e Gl aA
production by A. nig er .
3) Exploration of th e i mpact of NADPH engi neering on enzyme p roduc tion in the low-yield GlaA
producing A. nig er
NADPH generation is regarded as one of the main limiting steps for the enzyme biomanufacturing
in cell fact ory A. niger hypothesized by multi -omics integration analysi s
(Lu et al. , 201 6)
. A wealth


C h a p t e r 1 21 
of metabo lomic and fl uxomic data in A. niger have proved that strain s ad apted to the high
requirement of NADP H und er protein overproduci ng con ditions by channel ing more flux t o t he
PPP. To date, much progress has been harvested f or facilitating the yield of amino acids or fatty
aci ds via NADPH m etabol ic e ngineerin g i n C. gluta micum and oleaginous y east. However,
cofactor engineering has not been considered yet or performed i n A. niger to guide e nzyme
overproduction. On account of t he prediction of GSMM recently u pdated in our lab and available
literature, nine NADPH generatin g genes in A. niger were identified to play a notable role in
intracellular anabolic reductant supply. Driven by the Tet-on g ene expressi on switch, above nine
candidate genes were individually overexpressed in A. ni ger strain AB4.1 carrying one glaA gene
copy, to investigate the impact o f NADPH engineering on protein biosynthesis through metabolic
engineering.
4) To constr uct uridine auxotrophic A. niger via CRISPR/Cas9 gene e diting
In order to directly compare the effect of cofactor engi neering i n a n A. n iger strain carrying one
glaA (AB4.1) or seven glaA (B36) gene copies, it emphasizes the ne c essity of introducing gen etic
modifications under the same genetic control and at the same g e nomic locus in both recipient
strains. To achieve th is goal, we first need to establish a uri dine auxotrophic B3 6 carrying simil ar
unfunctional pyrG genotype as in AB4.1. This dissertation harnessed in vi tro CRIS PR/Cas9 gene-
editing system to build a uridine auxotrophic mutant of a high- yield GlaA producing A. niger B 3 6 ,
so as to provi de a novel platform for gene function studies und er a high protein secretion
background.
5) Engineering cofactor metabolism for imp roved protein and glucoa mylase production in A.
niger
We reported the implementation o f the design-build-test-learn ( DBTL) metabolic engineering
framework to syst ematically stu dy t he impact of NADPH availabil ity on GlaA production in A.
niger . I n o r d e r t o e v a l u a t e i f a s t r o n g p u l l t o w a r d s glaA biosynthesis mandates a higher NADPH
supply, identical NADPH regula tion strategies were appli ed in a high- yield GlaA producing strain
B36 carrying seven glaA gene copies. Combin ed with car bon-limited chemos tat cult ures a nd
multivariate s tatistical analysis, we expected t o shed light on th e physiologi cal altera tions and
global metabolic rearrangement ar oused by genetic perturbation at the same dilution rate.
Furthermore, this dissertation also attemp ted to provide new b i ological insights for the
optimizati on of GlaA or other NADPH-depe ndent pr oducts through recursive D BTL cycles .


Chapter 2 22 
Chapter 2 Com parativ e genomics of the a conidia l Aspergillus niger strain
LDM3 predicts genes a ssocia ted wi th its h igh protein s ecretio n capacity
2.1 Introduction
Presently, a total of 17 A. niger gen omes have been sequence d s ince the firs t A. niger genome
became available in 2007 (Baker, 2006; Pel et al. , 2007; Ander sen et a l. , 2011; Yi n et al. , 2014a;
Gong et al . , 2016; Paul et al. , 2017; Yin et al. , 2017 ; Vest h et al. , 2018). Remarkably, all A. niger
strains have hi gh genome flexibility and share about 7,500 gene s in their core genome but differ
in hundreds up to t housands of gen es, wh ich define the pan-geno me and s pecies-unique genes,
respectively (Ves th et al . , 2018).
A. niger LDM3 is an industrial glucoamylase production strain wi th a ve ry high GlaA product ion
level and is phenot ypic ally characterized by an aconidial pheno type. This is o f speci al interest
because ano ther hi gh GlaA production A. niger strain exploited in China (strai n SH2) is aconidial
as well (Yin et al. , 2014a). Most interesting ly, solid-state fermentations uncover ed that pr oteins
become mainly secreted in t he central and peripheral regions of a n A. niger colony but not in
mycelial re g ions undergoing sporulation, indicating th at sporul ation inhi bits p rotein secretion
(Krijgsheld et al. , 2013). Swift et al . (Swift et al. , 1998) already proved about 20 years a go that an
aconidial phenotype of A. niger is beneficial for protein biosynth esis and/ or s ecretion. Sever al
mutants with reduced sporulati on were isolated f rom maltodextri n-limited chemostat and pH
auxostat cult ivations of strain A. niger B1 (carrying 20 copies of the GlaA encoding gene glaA ) .
Two of these spontaneous m orphological mutants showed almost ye l l o w a n d w h i t e c o l o n i e s w h e n
cultivated on agar plates and had a significantly improved GlaA production when compared to
their parental strain B1, even though fo r one of t he mutants mo re t han half of the glaA c o p i e s w e r e
lost. Similarly, Jørgensen et al. obtained t wo sporulat ion deficient A. nig er s t r a i n s b y U V -
muta genesi s, scl-1 and scl-2 , for which seve ral secon dary metabolites were produced l ess bu t
extracellular prot ein increased (Jorgensen et al . , 2011a). However, the molecular mechanisms
linking protein secretion with a sexual sporulation are not full y understo od so far. In general,
sporulation-deficient Aspergillus strai ns are known to be defecti ve in many regu lators, includin g
the transcription factors (TFs) BrlA and Fl bA. The functions of both p roteins are well-docu mented
for A. niger , A. nidulans , A. fumigatus a n d A. oryzae (Boyl an et al. , 1987; Adams et al. , 1988; Lee
and Adams, 1996; Yamada et al. , 1999; Mah and Yu, 2006; Pavezzi et al . , 2011; K rijgshel d et al. ,
2013; van Munst er et al . , 2015). Br lA is the cen tral regulator of the conid iophore deve lopment
which becomes activated by FlbA (Boyl an et al. , 19 87; Krijgsheld et al. , 2013). Notabl y, the
deletion of the flbA g e n e i n A. niger results in a mutant strain with a fluffy phenotype, it also f o rms
thinner cell walls and displays a more com plex secretome (Krijg sheld et al. , 2 013).
To shed light on the molecul ar me chanis ms beh ind the aconidial and high-secretion p henoty pe of
strain LDM3, we sequenced i ts genome by a hyb rid approach combi ning t he PacBio RS


Chapter 2 23 
sequencing an d Ill umina Hiseq 4000 technol ogies and compared it s genome to the genome of the
GlaA pr oducing model s train CBS 513.88. In addition, gene knock -out experimen ts were
performed with two gen es of our interest, tupA a n d prpA , to investigate their impact on protein
secretion.
2.2 Materials a nd methods
2.2.1 Strains and culture
The A. niger strains used i n this study are listed in Table 2.1. The A. niger s t r a i n L D M 3 w i t h t h e
aconidial phenotype was k indly pro vided by Longda Biotechnology (Shand ong, China). Czapek–
DOX slope a nd submerged m edium were use d to cul tivate LDM3. The composition of Czapek–
DOX slope medium is as fo llows: sucrose 3%, NaNO 3 0 . 2 % , M g S O 4  7H 2 O 0.05 %, KCl 0.05%,
FeSO 4  7H 2 O 0.001%, K 2 HPO 4 0.1%, Agar 1.7%, pH 5.5~6.0. LDM3 was cultivated a t 34  C for 5
days. Czapek–DOX submerged medium is the same as a bove without Agar. Cultivation was done
at 34  C, 180~200 rpm for 72h and pellets were collected by filtration .
Table 2.1 Aspe rgillus ni ger st rains used in t his study
Strai n na me Background
strain Relevant genotype/des cription References
LDM3

Aconidial phenoty pe
Longda
Biotec hnology ,
Shan dong
MA16 9.4 AB4.1 cspA1 - ,kusA::DR-amdS-DR, pyrG − (Carvalho et
al. , 2010)
FW35.1 AB4.1 cspA1 - , pyrG + (W a nka et al. ,
2016
)

YS33.10 MA169.4 kusA::DR-amdS- DR, tupA:: AopyrG, pyrG + ,
sing le co py This study
YS34.16 MA169.4 kusA:: DR-amdS-DR , pr pA:: AopyrG pyrG + ,
sing le co py This study
YS39.1 MA169.4 kusA ::D R- amdS -DR, tupA :: AopyrG pyrG +,
pMA1 71-t upA This stud y
The other A. ni ger strains were cultured by the followi ng method: Strains were gr own at 30 °C
using the complete or minimal medium ( Arentshorst et al. , 2012) s upplemented with 1 mM uridine
w h e r e n e c e s s a r y . T o t e s t t h e y i e l d o f G l a A a m o n g d i f f e r e n t m u t a nts, 10 6 spores/mL of strains
FW35.1 (Wanka et al. , 2016), YS33.10 ( kusA ::DR- amdS -DR, tupA :: AopyrG , pyrG +), and
YS34.16 ( kusA::DR-amdS-DR, prpA::pyrG, pyrG + ) were inoculated into 50 mL CM liquid
medium with 3% w/v gluc ose as the carbon sour ce and culti vated at 30°C and 250 rpm. Samples
w e r e t a k e n a t 2 4 , 4 8 , 7 2 , 9 6 , a n d 1 2 0 h a f t e r i n o c u l a t i o n . P h y siolo gical p arameters (dry weight ,
total secreted protein, r esidual g lucose concentration a nd enzy me activ ity of GlaA in the med ia)
were measured. Experime nts were performed in biological quadrup licate s.


Chapter 2 24 
2.2.2 Genome DNA e xtraction and sequencing
A. niger transformati on, genomic DNA extra ction, and Southern blot were p e r f o r m e d a s p r e v i o u s l y
described (Arentshorst et al. , 2012). Quality analysis o f the genom ic DNA, l ibrary constr uct ion,
and sequencing on PacBio (RS II) and Illumina (HiSeq 4000) inst ruments was carried out by BGI
(Shenzhen, China).
2.2.3 Hybrid assembly of the A. niger LDM3 genome seq uence us ing Illumina and PacBio
sequencing
The development of several hybrid genome assembly algorithms al lows the taking of reads from
multiple read sources (Rhoads and Au, 2015; C hen et al. , 2 0 1 7 ) . R e a d s f r o m t h e I l l u m i n a p l a t f o r m
are short but accurate, w h ile reads from the PacBio are long bu t acco mpanied by a high erro r rate.
Hence, h ybrid sequencing allows the use of long reads for geno m e assemb ly ( PacB io reads) while
Illumina reads can be used for cor rections. A total of 296,149 subreads (2.37 Gb) were generated
on the PacBio RS II platform with an average length of 8,006 bp , and 6.45 Gb of clean data (read
length 150 bp, insert size 300 bp ) were generated on the Ill umi na HiSeq 400 0 platfo rm. The
subreads were self-corrected and then assembled using Falcon v 0.3.0
(https://github.com/Paci ficBiosciences/falco). The resulting as sembly was corrected through
Illumina reads using Proofread v 2.12 (https://github.com/BioIn f-Wuerzburg/proovread).
2.2.4 Bioinformatic ana lyses
Gene m odels were predicted using Augustus v 3.2.1 (http://bioi n f.uni-gr eifswald.de/augustus/),
SNAP v 2 010-07-28 (http://korflab.u cdavis.edu/s oftware.html) an d GeneMark-ES v 4.28
(http://exon.gatech.edu/) with A. niger CBS 513.8 8 as the r eference . Gene st ructure was predicted
using Ge neWise v 2.20 (htt p://www.sange r.ac.uk/Softwa re/Wise2/) . The predi cted gene mod els
were functionally annotated by aligning t heir protein sequences against the KEGG (Minoru et al. ,
2015), SwissProt (ht tp:// www.gpmaw.co m/html/swi ss-prot.htm l), G O (Ashburner et al . , 2000),
COG (Cl usters of Orthologous Groups) ( Galperin et al. , 2014), KOG (E uKaryot ic Orthologou s
Groups) (Jaime et al. , 2015), TrEMBL (https: //www.uniprot .org/) , and No n-Redundan t p rotein
databases (https:// www.ncbi.nlm .nih.gov/) w ith BLASTP (E -value ≦ 1.0e-5). tRNAScan-SE v
1.3.1 (http://gtr nadb.ucsc.edu/ ) was used for tRNA predi ction.
2.2.5 SNP, INDEL and SV anal ysis
High quality filtered short reads from the I llumina pl atform we r e m a p p e d t o t h e r e f e r e n c e g e n o m e
via BWA (Burrows- Wheeler Aligne r) (Li and Durbin, 2010). After filtering for Q < 20, the paired-
end (PE) reads were aligned t o a ll c hromosomes and th e aligned PE reads with a dis tanc e of >
1000 bp were screened for the genome assembly. GATK v1.6-13
(http://www.broadinstitute.or g/gatk/) was used to detect single nucleotide polymorphism (SNP),
insertion and del etion (InDel) between A. niger LDM3 and t he reference CBS 513.8 8 b ased on


Chapter 2 25 
high-quality alignment results. Raw SNPs and I nDels were filter ed under a stringent criterion of
GATK Unified Genotyper (Yin et al . , 2014a).
2.2.6 Synteny analysis
The assembled A. niger LDM3 genome sequence was frag m ented into 1 kb length and was
compared to the genome sequence of A. niger CBS 513.88 by BLASTN usin g the cutoff val ue
1×10 -7 5 . The sequence of t he target fu ngus was sort ed according to tha t of t he reference fungu s
based on MUMmer alignment results (Kurtz et al . , 2004). Synteny analysis was performed as
previously de scribed (A ndersen et al. , 201 1).
2.2.7 Strain-specific genes in the LDM3 genom e compared to CBS 513.88
The imprint al gorithm used to dete rmine strain-specific genes i n L D M 3 w a s p e r f o r m e d a s
previously described (Andersen et a l. , 2011). Using BLASTP (E-val ue ≤ 1.0e-10, identity > 50%,
coverage > 80%) t he CDSs of A. niger LDM3 was compared to the amino acids sequence of A.
niger CBS 513.8 8. The corresponding amino acids sequence from CBS 51 3.88 was named as
‘Imprin t’. For the comparison of CDSs and Imprint, gene variati ons such a s InDel, syn onymous
mutations, frameshift mut ations, and partial hit f or each CDS p air were c ollect ed i nto gene
muta tion lists . To extract th e co rresponding f ull-length nucleo tide sequence of t he genes that were
not aligned to CBS 513.88 un der t he above BLASTP criteri a, gen e s with 100% alignment to the
reference were removed. In addition, the remaining genes were m anu ally a nalyzed for frameshift
mutation, start codon and stop codon loss, partial match (cover age < 5 0%), ea rly termi nation and
no hits in LDM3 compared to CBS 513.88.
2.2.8 Co-expressi on network of TupA and PrpA encoding gene
From 283 microarray experiments of A. nig er hosted by Fungi DB (Basenk o et al. , 2018), the co-
expression networks of TupA and P rpA encodi ng genes were retri e ved accord ing to a p revious
study (Sch äpe et al. , 2019). Gene pai rs passing a Spearman’s correlation coeff icien t of |0.7| were
used t o co nstruct co-expression networks. For the TupA network, 32 gen es were negati vely
c o r r e l a t e d , w h i l e f o r P r p A , t h i s n umber increased to 5 76 genes. Bot h TupA and PrpA networks
were assessed for GO-enrich ed biological process relative to A. niger genome usi ng defau lt
parameters in FungiDB, and the ge nes of interest were m anually filter ed when Benj ami ni–
Hochberg FDR corrected p -values were above 0.05.
2.2.9 Determinati on of biomass, total secrete d protein, r esidual glucose and enzy me
activity of glucoamy lase
4 ml broth was t aken at t he indicat ed tim e points from shake fl ask cultures. Biomas s and cultur e
supernatant were s eparated by vacuum filtration followed by 3 t i m e s w a s h i n g w i t h d e i o n i z e d w a t e r ,
frozen at − 8 0 °C, and f reeze-dr ied overnight for the determina tion of biomass. Total extracellula r
protein in the cult ure supernatant was determined via the Bradf ord assay (BioRad, Califor nia, USA)


Chapter 2 26 
according to the manufacturers’ protocols, and absorbance (60 0 nm) was measured using the
GloMax®-Multi Detection System (Promega, Madison, USA). Quantif i cation of residual glucose
in t he cu ltivation medium was performed using the Glucose GOD/P AP k it ( Human, Wi esbaden,
Germany) according to the manufact urer’s manual. Enzym e activit y is e xpressed in AGI units,
which is relat ed to an officia lly assigned G laA standard. One A G I u n i t i s d e f i n e d a s t h e a m o u n t of
enzyme th at produces 1 µmol gl ucose pe r min at 60 °C and pH 4.3 from the solubl e starch substrate.
20 µl super natant was mix ed with 2 30 µl p-NPG su bstrate (2 g/l 4-nitrophenyl α-D-
glucopyranoside acetate buffer pH 4.3, pre-warmed for 5 min at 37 °C). After incubatio n at 37 °C
f o r 2 0 m i n , 1 0 0 µ l o f 0 . 3 M N a 2 CO 3 was added to stop the reaction an d the absorbance was
immediate ly measured at 405 nm using a plat e reader. The st anda rd GlaA from A. niger ( E . C
3.2.1.2; Sigma Aldr ich, Darmstadt, Germany) was used to buil d a standard c urve with GlaA
enzyme activity= 008 . 0
01 . 0 405  OD
×dilu tion ra te (R 2 > 0.999).
2.2.10 Generation of Δ tu pA and Δ prpA strains
To improve the homologous recomb ination efficiency, the split m arker method and the non-
homologo us end jo ining (NHEJ) defici ent r ecipient st rain MA16 9. 4 (Carvalho et al. , 2010) were
used. For gene deleti ons, deleti on cassettes containing homolog ous 5′ and 3′ flanks (~1.5 kb) for
targeted integration an d the selective ma rker AopyrG ( A. oryzae ) were constructed. These were
co-transformed into the pyrG- recipient s train MA16 9.4 and t r ansformants were scr eened based
o n u r i d i n e p r o t o t r o p h y . A. niger transformations were carrie d out usin g the prot oplast
transfor mation method as descri bed i n Ar entshorst et al. ( 2012). The 5′ and 3′ flan ks were
amplified by PCR with the primers descr ibed in Table A.1 and A. 2 . T h e d e t a i l s o n d e l e t i o n c a s s e t t e
construction were illustrated in Figure 2.1 and 2.2 and positive tupA or prpA deletion strains w e re
confirmed by di agnostic PCR and Southern analysis (Figu re C.1, C.2) . Pr obes used for Southern
blot analysis are listed in Table A.3. T h e a utonomousl y r eplica ting plasm id pMA171 (Carvalho et
al. , 2010b) was exploi ted in the complementation studies. The ORF of tupA including
approximately 0.6 kb promoter and 0.6 kb t erminator regi ons was amplifi ed, tak ing N4 02 geno mic
DNA as a template and cloned into Not I-li nearised pMA171 (Figur e 2 .3). Then the constructed
plasmid pMA171-tupA was t ransformed into the Δ tupA deletion mutant. Primary transformant s
containing the complement ation p l asmid were purified on MM medi um contai ning 100 µg/ml of
hygromycin and further anal yzed by diagnostic PCR.


Chapter 2 27


Figu re 2.1 Plasmid of pMF 22.1. The pla smid pM F22.1 was used a s a template w ith a recy clable functional
AopyrG which can be counterselected under 5-FOA selective pressure

Figure 2.2 tupA and prp A knockout cassettes and homolo gou s recombination str ategy. Prim er pair s
An15g00140fw1 up/An15g00140 revup and p rpA-fw1-up/prpA-fw2-u p wer e used to amplify the upstream
flank of tupA and prpA, res pectively. In addition, prim er pairs An15g00140fwdow/An15g0 0140re2dw and
prpA-f w-down/pr pA-rev1-d own wer e used to ampl ify t he down str eam of tup A and prpA, resp ectively. Primer
pairs Ao pyrG 1_fw/Ao pyrG 2_rev a nd Ao pyrG 3_fw/Ao pyr G 4_r ev we re us ed to a mplify two parts of the
functi onal pyrG , respec tivel y. T hen th e fus ed up stream f lank s were obtained by fusion PCR via primer pairs
An15g00 140fw2u p/Ao pyrG 4_rev and prpA-fw2-u p/Ao pyrG 4_re v, an d the f used d own stream fr agments were
gaine d by pr imer pairs A o pyrG 3_fw/An15g001 40re1dw and Ao pyrG 3_fw/prp A-rev 2-down . The two
lineariz ed cassettes (f u sed upstr eam fragment and fused downstr eam fragm ent) (1µg for each ) were
cotran sformed into A. niger pyrG
—

strain MA169 .4



Chapter 2 28 
1
pAMA-tupA
14997bp
AMA1
tupA
hph
PtupA
TtupA
AmpR
NotI
NotI




Figure. 2.3 Con struct io n of the complem entatio n pl asmi d pMA171 -tupA an d diagnostic P CR of Δ tu pA
complemente d strains. Pr imer pair AMA -F/tupA-R were exploi ted t o verify the poten tial Δ tupA complemented
transformants, where transform ants carrying the p MA171-tupA pla smid w ill give a signal at 3.3 kb (lane 1-9),
while the parental strain will give no signal (lane 10). hph represen ts h ygromyci n
2.2.11 Data access
The complete chromosomal sequen ce of LDM3 is ava ilable at th e G enBank under the assigned
accession number VTFN00000000.
2.3 Result and discussion
2.3.1 Strain cultivation a nd quality detection of genome DNA
A sin gle c olony of aconidial A. nige r LDM3 wa s ino culated on the Czapek-DOX solid plates,
cultured at 34 °C for 5 days. Then, the Czapek-DOX solid medium carrying LDM3 colonies was
transferre d into the Czapek-DOX l iquid medium, and g enome DNA w as ext racted a fter cultured
for three d ays. A s shown in Figure 2.4, g enome DNA is of high q uality without degradation and
r e s i d u a l R N A . G e n o m e D N A s a m p l e s w e r e h a n d e d o v e r t o B G I ( S h e n z hen, China) for l ibrary
preparation.
1 2 3 4 5 6 7 8 9 c o n t r o l
3 kb
5 kb
3.3 kb
AMA -F
t
up

A-R


Chapter 2 29


Figure 2.4 El ectro phore sis a nalys is of A. niger LDM3 genome DNA samples
2.3.2 Characteristics of A. niger LDM3 genome
Table 2.2 Gen ome characte ristics of sele cted A. niger st rains w ith in dustrial rel evance

Strain name
（ NCBI ） ASM285v2 NRRL3 ATCC1015 SH2 LDM3
Name
synonym CBS 513.88 ATCC 9029 NRRL 328 FGSC A1279
Accession
Numbe r
b
GCA_000002855.1 unpublished GCA_000230395.2 GCA_000633045.1 VTFN00000000
Institute
and
coun try

DSM, Netherland
Integrated
Genomics,
USA
DOE/JGI, USA SCUT, China ECUST, China
Project
Chronology 200 0-2 007 200 0 2005-2011 2013-2014 2016-2 017
Genome
length 34.02 Mb 33.7 Mb 34.85 Mb 34.63 Mb 35.28 Mb
Sequencing
technology BAC tiling Shotgun Shotgun Illumina Hiseq Illumina Hiseq
+ PacBio
Coverage ~7.5 × ~6× ~8.9 × ~120 × ~177 ×
Genomic
library
insert size < 150 kb 1-2 kb 3 kb 8 kb 40 kb 500 bp 270 bp
(I llu mina )
Numb er of
cont igs or
scaffolds 19 Scaff olds 9510
Conti gs 24 Contigs 349 Scaffolds 11 Sca f folds
Numb er of
predicted
genes 14,165 14,000 11,200 11,517 11,209
Strain
application
Gluc oamyla se
production
Gluconate
production Citric acid
production
Glucoamylase
production
Gluc oamylas e
production
Refe re nce (Pel et al. , 2007) (Bake r,
2006; Vesth
et al. , 2018)
(Andersen et al. ,
201 1) (Yi n et al. , 2014a) This study
10 kb


Chapter 2 30 
In ord er to identify the genet ic determinants r esponsible for t he unique phenot ype of LDM3, the
entire genome o f L DM3 was sequenced us ing a h ybrid approach t ha t combined Pacific
Biosciences with Illumina sequencin g , obtaining 6,447 Mb and 2, 679 Mb data after filtering from
the Illumina Hi Seq 4,000 and PacBio RS II platfor m, respectivel y. The high- quality reads were
further used to assemble the genome of LDM3 after quality c ontr ol , resulting in a 35.28 Mb
genome sequence with 11 scaffolds and sequenci ng depth of 177× ( T a b l e 2 . 2 ) . T h e a s s e m b l e d
genome ba se c alls were co rrected with Il lumina high-quality PE reads. A t otal of 11,209 ORFs
were identified in LDM3 (94% of the genes were aut omatically an notated based on prot ein
databases with an average g ene length of 1,69 1 bp), displaying a lower gene d ensity (0.32 gene/ kb)
compared to CBS 513.88 (0.42 gene/kb) (Andersen et al. , 2011) (Tabl e 2.3).
Table 2.3 Genome char acteristics of LDM3 and CBS 513.8 8
C B S 5 1 3 . 8 8
(Pel et al. , 2007)
LDM3
Size of assem bled geno me ( Mb) 34.02 35.28
GC content (%) 50.4 49.5
Protein-coding genes 14165 1 1209
Gene density (gene/kb ) 0.42 0.32
A verage gene length (bp) 1572 1691
A verage number of introns per gen e 2.57 2.21
A vera ge intron size ( bp) 97 85.6
A vera ge exo n size (bp) 370 467.61
Number of tR NA genes 269 264
N50 2,525,24 3 6,183,453
KOG analysis was empl oyed to ide ntify their bi ologica l r oles. O ut of the 11,209 predicted proteins,
9,712 ORFs ( 87%) were a ssigned t o 24 KOG funct ional categori es in total ( Figure 2.5). In LDM3,
slightly m ore genes were allocated to term G (Carbohydrate t ran sp ort and metab olis m), A (RNA
processing and modification), L (Repli cation, recombination and r e p a i r ) , O ( P o s t t r a n s l a t i o n a l
modification, protei n turnover, ch aperones) and to the un known functional g ene class (S)
compared to the control. Both LD M3 and CBS 513.88 showed a comp ar able numb er of predicted
genes distributed in e ach term, defining a high genome similari ty. Gene functio nal annotation
(Figure 2.6) confirmed that the majority of genes were signific antly allocated in catalysis, transport,
translatio n, carbohydrate metabolism, and ami no acid m etabolism , which mat ched well with t he
high-yield protein-produci ng ch aracteristic of LDM3. 264 tRNA g enes were identified in LDM3,
which wer e comparable to other enzyme-producing A. ni ger strains CBS 513.88 (269) and SH2
( 2 6 7 ) , b u t m o r e t h a n o t h e r Aspergilli strain s includi ng A. ni dulans ( 188) and A. fumigatus (179)
(Yin et al. , 2014a) .


Chapter 2 31 

Figure 2. 5 KOG analysis between A. niger CBS 513.88 an d LDM3. （ I Lipi d tran sport an d met abolism; G
Carbohydrate transport and metabolism; A RNA pr ocessing and mo d ifi catio n; O P osttra nslational
modificat ion, protein tur nover, cha perones; L R eplicatio n, reco mbinati on and repair; M Cell
wall/me mbrane/en velope biog enesi s; Q S econdary met abolites bios ynthesis, transport and catab olism; T
Signal transduction m echani sms; P Inorganic ion transport an d m etab olism; H Coen zyme transport and
metabolism; V D efense mechanisms; B Chromatin structure and dyn am ics; Z Cy toskeleton; E Amino acid
transport and metab olism; Y Nuclear s tructur e; F Nucl eotide tra nsport and metab olism; D Cell cycle contro l,
cell d ivis ion , chr omos ome partitioni ng; J Trans lati on, rib osom a l s tructure and bioge nesis; K Transcription; U
Intracellular trafficking, s ecretion, a nd vesicular trans port; C Energ y productio n and conversion; S Function
unkn own ）

Figure 2.6 KEG G annotati on of A. niger LDM3
0
20 0
40 0
60 0
80 0
IG A O L M Q T P H V B Z E Y F DJ K U C S
CBS5 13.88 LDM 3
26 00
28 00
30 00
Num b er o f genes
150
30
46
279
29
274
256
114
146
332
459
114
530
212
101
308
230
125
61
131
0 1 00 2 00 3 00 4 00 5 00 6 00
Ce ll gro wt h an d death
Cel l motil it y
Cel lu lar c omm uni t y
T r an sp or t an d c a ta b oli sm
Mem brane t ra n s port
Si gn al t ra ns du ct io n
Fo ld in g, sor t i ng a n d d e gr ad at i on
R eplicati o n and re pa ir
Tran scri ption
Tra nslati o n
Ami n o acid met ab ol ism
Biosy nthesi s of o th er seco ndary …
Ca rb o hy d ra t e me t a b o l is m
E ner g y me t ab ol i sm
G lyca n bio syn t h e si s an d m eta bo li sm
Li pid m eta bolism
Me ta bo l ism o f co f a ct ors an d v ita m ins
Met ab ol is m o f o ther ami no ac id s
Me ta bo l ism o f terp en oids an d…
N ucleot i de meta bo li s m
Cell gro wth and death Memb rane t ra n s port
Tran scri ption Ami no ac id me tab ol ism


Chapter 2 32

2.3.3 Genome structure variatio n analysis
Strains LDM3 and CSB 513.88 share widely distributed syntenic b locks, accounting for 9 6.56%
of their genomes. The dot plot depicted in Fi gure 2 .7 shows con served synteny between the two
strains, r eflecting a close phyl ogenetic relationship. However, th e synteny map ill ustrated
remarkable chromosomal rearrange ments for the 8 chromos omes (19 super contigs) of CBS 513.88
(Figure 2.8). N50 is the c riteri on used to eval uate the quality of genome assembly. N50 value for
the LDM3 geno me sequenced by hybrid approach is 6,183,453, which is much l arger than th e
control strain CBS513.88 ( 2,525,243), and th e LDM3 genome was a ssembled into 11 scaffolds,
resembling a higher quality of assemb ly t han the 19 superco ntig s of CBS 513.88. The enti re
lengths of 4 out o f 9 scaffolds in LDM3 reached a longer or sim ila r leng th co mpared to their
corresponding chromosomes in CBS 513.88. Scaffolds 1 and 2 are larger than any chromosomes
of t he reference strain (6.0 Mb i n maximum ) reach ing 7.6 Mb a nd 7 . 5 M b , r e s p e c t i v e l y . T h i s
suggests a fusion with other chromosomes, representing a notice able struct ural vari atio n in LDM 3.
In addition, scaffold 7 and 8 compose only the third superconti g in CBS 51 3.88.
Interestingly, in general , two GlaA e ncoding genes can be ident ified in most published A. ni ger
genomes, namely glaA (An03g0655 0) and gl aB ( An12g03070, shari ng 25% identit y wi th glaA ),
in which glaA is expressed stronger than glaB (A n d er s e n et al. , 2011; Schäpe et al. , 2019 ). This is
also the case in CBS 513.88 and SH2. Both have a single copy of glaA and glaB in their genomes.
However, glaB is present as the only glucoamylase encoding gene in LDM3, albe it glaA is
dominatingly expressed in t he majorit y of characterized Aspergilli . Previously r eported that glaB
displayed diverse expression p atterns i n A. oryzae under dist inct culti vation conditions, and was
strikingly expressed in s olid-state cultures but showed little or no expression i n submerged
cultivation (te Biesebeke et al. , 2005). gl aB is regulated at the transcriptional level, which could
be enhanced by starch, low water activity, high temperature and l imited h yphal extension (Kumar
and Satyan arayana, 2009). Similarly, it was also induced by iso maltose in A. nidulans (Nakamura
et al. , 2006 ). Giv en the abundant expression profiles of gl aB, its performance i n LDM3 requires
further description in submerged cultures.

Figure 2.7 Syntenic dot plot of A. ni ger CBS513.88 and LDM3

CBS513.88
LDM3


Chapter 2 33 

Figure 2.8 Synte ny m ap of the scaf folds o f A. niger LDM3 to the superconti gs of A. niger CBS 513.88 . The
coloring of the scaffolds s hows syntenic r egions in A. ni ger C BS 513.8 8. Ara bic numerals i ndicate the number
of the supercontig in A. niger CBS 513.88. Grey areas show regi ons not found in the CBS 513.8 8 genom e
sequen ce. The bl ack l ine unde rneath a section of the scaffold s indica tes i nversion seque nce. The blue
rectangular s hadow across t h e scaffolds indic a tes the transpos i tion betw een the two sequenc e fragments
sepa rated by a re d li ne in the sh adow
2.3.4 LDM3 strain-specific genes analysis
An ali gnment of the t wo genome sequences of LDM3 and CBS 513.88 sho wed some unique
regions in LDM3, incorporating 457 protei n-encoding genes. Thes e includ e 196 ORFs with
frameshift mutations, 17 ORFs in which the s tart or stop codon was lost, 81 ORFS with partial
match (coverage < 50%), 4 ORFs wi th early termination and 1 57 O RF s which did not matc h the
CBS 513.88 sequence (75 were annotated as hypothetical or of un known fun ction). To furt her
character ize these strain-speci fic genes in LDM3, we compared t heir sequences with p ublicly
available genomes of other A. niger and Aspergi lli strains, demonstrating that orthologs of 49 out
of 457 strain-specific genes were identif ied in all the compare d genomes (Table 2.4 ). GO
annotations for 2 25 out of 457 OR Fs are available, which are en riched in catalytic activity,
oxidoredu ctase activity, hydrolase activity, transfera se acti vi ty, p rotein bin ding, oxidation-
reduction process, transporter activity, and localizati on GO te rms (Figure 2.9). Among t he genes
annotated in two sign ificantly en riched GO terms o xidoreductase activity (36 genes) and
oxidation-reduct ion process (30), seven g enes could not be alig ned to the refere nce ge nome, seven
genes showed f rameshift mutatio ns, and one gene was mapped part ially to CBS 513.88 genome


Chapter 2 34

(Table B.1). The seven unaligned genes were mainly predicted to function in ami no acid m etabolic
pathways, including degradation of proline, isoleucine and leuc i ne (AN2_GLEAN_10000163 ,
mmsB predicted as 3-hydroxybuty rate hydrogenase), biosynthesis of alanine, aspartic acid, and
glutamic acid (AN2_GLEAN_10000741, gabD predict ed as succinate-semialdehyde
dehydrogenase which supplem ents succi nic acid for the TCA cy cle ( Y i n et al. , 2017), and the
biosynthesi s of arginine and proline (AN2_GLEAN_100070 14, proA predicted as glutamate-5-
semialde hyd e dehyd rogen ase).
Table 2.4 The com parison of 457 strain-specific genes (absent in CBS 513.88) in LDM3 with other publicly
availa ble Aspergillus genomes
A
A. niger ATCC101 5
A. ni ger N402
A. ni ger An76
A. niger ATCC1 3469
A. fumigatus A1163
A. f umigatus Af2 93
A. nidulans FGSC _A4
A. or yzae RIB40

Figure 2.9 Gene o ntology analysis of sp ecific genes in nonsynt enic region of A. niger LD M3
2.3.5 SNP and INDEL a nalysis
LDM3 and CBS 513.88 ex hibited a close phylogen etic relationship , sharing 97% identit y.
How ever, the unique reg ions between the two genome sequen ces ma in ly define geno me dive rsity.
Compared with the reference genome of CBS 513.88, a total of 2, 138 SNP and INDEL mutations
are present in the genome of L DM3. In t otal, non-synonymous mut ati ons w ere i dentified in 656
open readi ng frame s. KOG cl ustering analysi s unco vered that t he mutated genes are mainly
Strain No hit Homolo g
A. niger ATCC1015 279 178
A. niger N402 250 207
A. niger An76 276 181
A. nige r ATCC1346 9 252 205
A. fumigatus A1163 343 114
A. fumigatu s Af293 344 113
A. nidu lans FGSC_A4 351 10 6
A. oryzae RIB40 367 90
A. niger ATCC1015 279 178


Chapter 2 35 
clustered to A ( RNA processi ng and modification), C ( Energy pro duction and conversion), E
(Amino a cid transpo rtation and metabolism), G (Carbohydrate tra n sport and metabolism), O
(Posttranslational modi fication, protein t urnover, chaperones), J (Translatio n, ri bosomal structure
and biogenesis) and I (Lipid transport and metabolism) (Figure 2.10), all of which are very
fundamental to high-level protein expression, protein targeting, and secret ion. Sel ected genes of
intere st are depicted in Table 2.5 an d will be di scusse d as fol low s.

Figure 2.10 Th e heig ht of t he hi stogram shows t he total n umber of ge nes of A. niger LDM3 in va rious KOG
clusters. T h e grid part represents the number of muta ted gen es in each KOG category, and the white p art
means the numb er of unmu tated ge nes
Table 2.5 S ummary of functional important genes with S NP or In Del mutations
Gene ID
(CBS 513.88
nome ncla ture)
Alt
name
SNP or InDel Annotation Ref
Transcription factors
An01g07900 cpcA Insertion Transcr iption factor
impo rta nt for am ino aci d
bios ynthesi s u nder a mino
aci d starvat ion co ndi tion s
(Wanke et al. , 1997; An dersen et
al. , 2011; Yin et al. , 2014a)
An04g06910 amyR Nonsynonymous Transcr iption factor for
starch h
y

drolase
g

ene s
(Von gsangna k et a l. , 2011)
An04g06920 agdA No nsy non ymo us Secreted  -glucosidase
An15g00140 tupA Nonsyn onymo us Tr ans cri pti onal repre ss or
important for cell wall
remodelin
g

(Schachtschabel et al. , 2013)
Transporters
An15
g

04270 Nons
y

non
y

mous Su
g

ar transporte
r

An11g09600 Nonsynonymous MF S monosaccharide
transporte
r

An07g03690 Nonsynonymous Neu tral amino acid
transporte
r

An07g03970 Nonsynonymous Neu tral amino acid
transporte
r

0
20 0
40 0
60 0
80 0
S G O K I QJ U E T C A LP D M ZFB H Y
U n m u ta te d ge n e M utate d ge ne
25 00
27 00
29 00
Num b er of genes


Chapter 2 36 
Gene ID
(CBS 513.88
nome ncla ture)
Alt
name
SNP or InDel Annotation Ref
An14g07130 Nonsynonymous Neu tral amino acid
transporte
r

Protein secretion and de
g

radation
An07g02190 sec7 Nonsynonymous Gu anyl-nucleotide exchange
factor importa nt for intra-
Golgi and ER-to-Golgi
transpor
t

(Wolf et al. , 1998)
An08
g

00290 rud3 N o n s
y

non
y

mous Matrix protein o f Gol
g

i (Gillin
g

ham et al. , 2004)
An07g03880 pepC Nonsynonymous Subtilisin-lik e serine
protease

An07g08030 pepF Nonsynonymous Serin e-type
carbox
y

pep tid ase

An11
g

06350 cp
y

1p N o n s
y

non
y

mous Serine carbox
y

pept ida ses
Cell wall bios
y

nthesis
An08
g

07350
g

elB N o n s
y

non
y

mous Glucos
y

ltransferase (Mou
y

na et al. , 2005)
An02g02360 csmA Nonsynonymous Chitin syn thase (Markham and Bainbridge, 1992;
Aufauvre-Brown et al. , 19 97;
Hori uchi et al. , 19 99; T akes hita et
al. , 2002; Takeshita et al. , 2005;
Rogg et al. , 2011; Jimenez-
Orti
g

osa et al. , 2 012 )
Sporulation
An02g03160 flbA syno nymous Reg ulat or of G-prote in
signaling
(Lee and Adams, 1994; Wieser et
al. , 1994; Perez-de-Nanclares-
Arregi and Etxebeste, 2014; Yin et
al. , 2014a; van Munster et al. ,
2015)
An12g02050 wA Nons ynonym ous Polyke tide syn thase
important for pigment
b

ios
y

nthesis
(Jorgensen et al. , 2011b; Zhang et
al. , 2016)
An18
g

01170 prpA Absent Role in asexual sporulation (Yin et al. , 2014a)
2.3.5.1 Transcription factors
Among t he 872 TFs predicted i n A. niger (Par k et al. , 2008; Szklarczy k et al. , 2017), 9 TFs carry
I n D e l m u t a t i o n s i n L D M 3 ( T a b l e B . 2 ) . A m o n g t h o s e i s C p c A , w h i c h is in volved in t he d egrad ation
of misfolded p roteins and plays a r ole in RNA p rocessing and tr anslation processes b y t he
endoplasmic reticulum protein response p athway (Wanke et al. , 1997; Jorgensen et al. , 2009).
CpcA is the functional ortho log of t he Saccharomyces cerevisiae t ranscri ptional activator Gcn4p
in Aspergil li and induces t he expression of multip le genes associated with amino aci d biosynt hesis
under amino acid starvation co nditions (Vo ngsangnak et al. , 2011 ). Previous reports indeed
a s s u m e d t h a t a f r a m e s h i f t e d cp cA gene might be the cause of efficient e xpression of the Gl aA
encoding g ene glaA and othe r enzymes in A. niger (Andersen et al. , 2011; Yin et al. , 2014 a). It is
thus very tempting to speculate that this is also the case with LDM3.
Moreover, 41 out o f 60 TFs containing SNP disp lay n on-synonymou s mu tations (Table B.2). A
wide range of regul atory p athways, incl uding starch degradatio n , cell wall sy nthesis, nit rogen
assimilation, and amino acid synthesi s pathways, were a ffected. AmyR (An04g06910), for


Chapter 2 37 
example, is an essential malto se-dependent TF that regulates th e expres sion of starc h hydrolase
genes such as extracellular hydro lases includin g alpha-amylase AamA, alpha-gl ucosidases AgdA
and AgdB, and GlaA. The consensu s sequence of DNA binding of Am yR to the promoter regions
of its target genes is well known (CGGN8(C/ A)GG) (Yuan et al. , 2008). Such a sequence can
indeed be found at position -878 bp upstream of glaB, sugg esting that the glaB g e n e i n L D M 3
might be under transcriptional control of AmyR as in other Aspe rgil li .
2.3.5.2 Transporters
The majority of tr ansporters carrying SNP mutations belong to t he MFS famil y of secondary acti ve
transporters and facilitators (de Vries et al. , 2017), su ch as glucose transporters An15g04270,
An11g09600 which may be beneficia l for the uptake of substrates i n L D M 3 . I n a d d i t i o n , t h r e e
neutral amino acid transporters An07g03690, An07g03970, An1 4g07 130 had non-synonymous
mutations. Interestingly, Ala, Leu, Thr, and Ser are the top f o ur amino acids in t he composition of
GlaA, all of w hich are neutral amino acids (Table B.3). It can thus be speculated that the variation
of neutral ami no acid transporters may enhance the transport of these four major amino acids, to
support the translation of the glaB mRNA.
2.3.5.3 Protein secretion and protei n degradation
Among th e 465 genes predicted to fu nction in the secretory path ways of A. niger (Gui llemette et
al. , 2007; Jorgensen et al . , 2009; Carvalho et al. , 2012; Kwon et al. , 2012), 3 have INDEL
mutations a nd 25 carry non-synonymous SNPs in strain LDM3 (Tabl e B . 4 ) . M o s t g e n e s a r e r e l a t e d
to p rotein t ransport, l ipid metabolism, or are protease e ncodin g genes. Two genes with non-
synonymous mutations are worth m entioning: An07g02190 ( S. ce revisiae se c7 ortholog) has a
predicted r ole for vesicle traffic in int ra-Golgi and ER-to-Gol gi transpo rt (Wolf et al. , 1998).
An08g00290 ( S. cerevisiae rud3 ortholog) is a matrix protein of the Golgi and important for i ts
integrity (Gillingham et al . , 20 04).
Proteases a re capa ble of hydrolyzing prot ein-peptide cha ins, wh ich may be d etrimental for p rotein
production. I nterestingly, the three mutated protease genes ( pepC, pepF and cpy1p ) are all seri ne
proteases. Serine and threonine are two high ratio amino acids in t he GlaA pr otein (Tabl e B.3);
therefore th e reduced degradati on of seri ne enriche d proteins c aused by t he mut ations of the serine
protease ge nes mer its further exp loration in the fu ture. 
2.3.5.4 Cell wall bio synthesis
The fun gal cell wall determines hyphal morpholo gy, c ellular int egrity, an d p rotein secret ion
productivit y duri ng growth and develo pment o f A. niger ( C a i r n s et al. , 2019). Variati ons i n cell
wall compositi on and mycelial morphology of A. niger could t hus support pr otein produ ction
(Fiedler et al. 2018a, b). For example, gelB (An08g07350) encoding a GPI-anchored
glucosyltransferase important for  -1,3-glucan synthesis, is affected ( non-synonymous) (Table


Chapter 2 38 
B.5 ). Dele tion of gelB i n A. fumigat us causes the reduction of  -1,3-gl ucan c ell w all levels
accompanied by abnormal germination, decreased growth and defic ient pigment bio synthesis
during sporul ation (Mouyna et al. , 200 5). However, it has not been reported so far whet her
mutations on the gl ucan biosynthesis path way lead to the aconid ial phenotype of As per gilli .
Another interesting mutated ge ne is a chitin synthase encoding gene An02g02360 ( csmA ) with
non-synonymous SNPs in LDM 3. Orthologs are kno wn f or A. fumigat us ( chsE) a n d A. nid ulans
( csmA ) and their del etion phen otypes have been stud ied. Delet ion of the A. fumi gatus chsE gene
provokes abnormaliti es in hyphal morpholog y, sporulation, and r e duced spore surv ival rate
(Aufauvr e-Brown et al. , 1997; Jimenez-Ortigosa et al. , 2012), and knockout of the A. nidulans
csmA in duces ball oon-like swollen hyphae and i ntrahyphal hy phae form ation (Takeshita et al.
2002, 2005 ). These observations i ndicated that chitin is essent ia l for mainta ining conid iophore and
spore integrity (Jimenez-Ortigosa et al. , 2 012). However, other chit in synthase gene knock out
mutants ( chsA, chsB, chsC, ch sD, chsG, and chsF ) did not introduce abnormal spore formation in
A. fumigatus ( Mellado et al . 1996a, b, 2 003; Rogg et al. 201 1). Int eresting ly, in addition to t he
aconidial pheno type, LDM3 a lso underwent a unique morphological t ransition phase durin g the
late s tages o f submerged bioreactor cultivations, which is n ot common to other A. niger st r a i n s . I n
fed-batch cult ures, LDM3 mycelia began to swell at their tips d uri ng oxy gen limitati on, f ollowed
by mycelial fragment ation and s eparation of d ispersed mycelial structures into smaller entities
(data not shown). This finding assume d that t his morphol ogical response improved oxygen transfer.
Notably, morphology alt erations were ac companied by dramatic a c cu mulation of Gl aA in the
supernatant in t his phase, sugg esting that hy phal swelli ng and fragmentation are important for high
GlaA production or re lease into the medium (data not sho wn). It is thus tempting to speculate that
the mutation of csmA m ay be relevan t to the morphological adaptation o f LDM3 durin g fed-batch
fermentation .
2.3.5.5 Sporulation
It is well known that most species of Aspergilli repro duce asexually. A central regu latory pathway
( brlA → aba A → wetA ) is conserved in all Asperg illi a n d Peni cillium genomes and cont rols
conidial-specific gene expression and asexual sporulation (de V ries et al. , 2017). BrlA is required
to activa te ab aA a nd wetA ( Y i n et al. , 2014a). FlbA is t he central regulat or, controlling t he binding
of Fl bB a nd Brl A to the G-protein couple d recepto r, which re pre s s e s t h e g r o w t h o f v e g e t a t i v e
mycelium. T ypically, a deletio n in flbA i n filamentous f ungi induces abnormal conidiation (Lee
and Adams, 1994; W ieser et al. , 1994; Per ez-de-Nanclares-Arregi and Et xebeste, 2014; v an
Munster et a l. , 2015). Moreover, double d eletion of brlA a n d fl bA results in the fluffy p henotype
of A. ni ger (van Munst er et al. , 2015). In LDM3, only syn onymous mutations wer e distributed in
the respecti ve flbA homolog and no mut ation in brlA or flbB was iden tified. Therefore these three
genes are likely not relat ed to t he a conidial phenotype o f LDM3 . An12g0 2050 ( wA ) i s i nvolved in
the process of conidial devel opment i n A. ni ger and is required for pi gment synthesis. The del etion
of wA l e a d s t o A. niger c o l o n i e s w i t h w h i t e o r f a w n - c o l o r e d s p o r e s ( J o r g e n s e n et al. , 2011b; Zhang


Chapter 2 39

et al. , 2016). Since the wA gene of L DM3 carries a non-synonymous SNP mutation, this gene is
worth investigating in mor e detail in the futu re.
2.3.6 In vivo analy sis of two s elected genes p utatively involv ed in sporulation
Given the possible link between the aconidial and high enzyme p rod ucti on phenot ype in A. niger ,
we decided to study the function of two genes of our interest ( tupA , An15 g00140 and prpA ,
An18g01170) am ong the 656 mut ated genes in LDM3 c ompared to CBS 513.88. In doing so, we
selected the lab strain MA169.4 as the p arental strain, whi ch i s devoi d of the NHEJ p athway and
thus en sures a high er homol ogous recombination rate (Carv alho et al. , 2010). TupA (non -
synonymous) is a globally active transcriptional repressor (ort holog of the repressor Rco- 1of
Neurospora crassa and Tup1p of S. cerevisia e ). Notably , its deletion h as been show n to caus e an
aconidial phenotype and a redu ced gro wth rate in N. cra ssa ( Y a m a s h i r o y et a l. , 19 96) and A. ni ger
(Schacht schabel et al. , 2013). Thus, the present mutations in TupA prom pted us to investigate the
function of tupA in t he uniqu e phenotype of LDM3. PrpA , a g ene of unknown function, i s absent
in L DM3. Its expr ession is indu ced by brlA and ab aA - dependent reg ulatory loops in A. niger and
its ab sence is pr edicted to cause the aconidial phenotype in th e A. niger S H 2 s t r a i n ( Y i n et al. ,
2014a). Blast result indicated that the protein sequence of Tup A carries a 1 6 amino acids insertion
and one amino acid change from gl ycine (G) to aspartic acid (D) (Fi gure 2.11).










Figu re 2.11 Blast result o f Tup A protein sequences between LDM 3 (query) and CBS 513 .88 (sub ject ). R ed
blocks label mutated regions in the TupA protein sequences of L DM3 versu s to CBS513.88

T o p r e d i c t t h e f u n c t i o n o f tupA a n d prp A i n A. ni ger , we harnessed our recently published gen ome-
wide co-expression database available on FungiDB t o constr uct c o-expression networks for tupA
and prpA ( S c h ä p e et al. , 2019). The co-expression network of tup A showed an exclusively negative
correlation with the genes predicted to functi on in protein sec retion, filament ous growth, v esicle-
mediated tr ansport, cellular protein metabolic process, and fun gal-type cel l wall organization or
biogenesis (Figure 2.10A), and th is is consistent with its func tion as a transcriptional repressor .




Chapter 2 40 
Similarly, prpA showed nearly no positiv e correlations with other genes but su rprisi ngly a negati ve
association with a high nu mber o f genes (567). GO enrichment a n alysis revealed that t he prpA
network is mainly enric hed in mit ochondrion organization, prote in targeting to the mitochondrion
and protein catabolic processes, namely a ssociated with energy generation (Figure 2.10B).
Noteworthy , wherea s the tupA network contained overrepresented GO terms associated with
growth or sporulation, this was not the case for the prpA co-expression networ k (Table B.6, B. 7)
PrpA
CsnB
YTA12
Pre7
CsnA
Pre3
Mca1
Ubc1
Pre4 Sc l1
GO:protein catabolic
proces s, P< 0.05
Atp20
Ggc1 Mge1
Bsc1
Tim17
GO: Mitoch ondrion
organization, P <0.05
Tim4 4
Ssc1
Tim54 Ti m23
Hsp60
GO: Pr otein targe ting to
mit ochondri on, P<0. 01
Ate1
Pre8
Pre10
Fmp25
AB
TupA
Ypt31
KexB
GmtA
Pmt4
PkcA
Cla4
Sec7
Knh1 Aspc
GO:fungal-type ce ll wall
organization or bi ogenesis,
P<0.01 GO:filamentous growth, P<0.01
Sec23
Sec24 Se c7
Ypt31 Sec27 Se c6
GO: ve sicl e-me dia ted
transport, P<0.01
Ydj1 Snt 2 Swe1 ClxA Pkc B
GO: C ell ular prot ein
metabolism process, P<0. 01

Figure 2.12 Co-expression networ k for Tu pA- and Pr pA-en cod ing genes. The query ge ne encod ing protein is
shown in a di amond box (TupA, Prp A). Eac h circle rep resents co - expre ssed genes. T he proteins they cod e for,
are shown in the cen ter of each circ le. Only negative corre lati ons for bot h TupA (A ) and PrpA (B) are show n
as both of them only express sign ific ant negative associ ation w ith other genes. If protein names w ere not
availab le in A. nig er , th e name f or eith er the A. nidu lans or S. cerevis iae ortholog was used . Gene pa irs with in
the co-expr ession sub-networks all passed the |0. 7| Spearman co rrelat ion coe fficient cut -off. GO t erms with
functio n s in biologi cal processe s, a p- value cutoff of 0.05 , an d a Benjamini –Hochberg false discover y rate
lower tha n 0.05 were assess ed as overrepresented , and genes of interest manually fil tered
To s tudy the impact of bot h gene s on sporulation and pr otein se cretio n in A. niger , single-gene
knockout strains were constructed in M A169.4 using t he s plit ma rker approach as published
previously (Fiedler et al. , 2018a). As depicted in Figur e 2.13 and 2.14, the deletion of tupA sev erely
reduced th e myceli al growth rate and sporulation efficiency of A. nige r , which was not the case
for the prpA nul l mutant. All Δ tupA -complemented transformants obtained grew li ke the wild-t ype,
confirming that the severe growt h and sporulation defect of the m utant was caused by the tupA
deletion and the pl asmid-based tupA g e n e i s c a p a b l e t o n e a r l y r e s t o r e t h e p h e n o t y p e ( F i g u r e 2 . 1 3 ) .
However, the Δ tupA -complemented strain was still w itnessed a weaker growth rate, suggest ing
that t he cellular amount of tupA is u nder stringent cont rol. The sporulation capacity of  tupA was
reduced by ab out 35% compared to the refere nce strain FW35.1, b ut Δ prpA p r e s e n t e d n e a r l y n o
differenc e compared to the refe rence strain (Figu re 2.14). It has thus been confi rmed that knocking
out tupA i n A. niger strongly inhibits the cell growth rate, which is consi stent wit h a previo us report
(Schachtschabel et a l. , 2013).


Chapter 2 41 

Figure 2.13 P henot ypic ana lysis of Δ tupA , Δ pr pA mut ant s, a nd Δ tupA complemented stra in on solid agar
plates . S pores (5*1 04) were spot- inoculate d on the differ ent ty pes of media and i ncubate d at 30°C f or 3 days.
Strain FW3 5.1 was used as the re ferenc e strain . To fully repres s any grow th, 1 50 µ g/m l hygr omy cin w as
adopted. hyg hygromycin

Figure 2.14 Sporulation quantification of Δ tupA , Δ prpA mutan ts and Δ tupA compl emented strai n on solid ag ar
plates. (a) 1 000 spores of e ach s train were i noculat ed on the differen t ty pes o f media, respect ively, and
incubate d at 30° C f or 6 days. (b) The colo ny size of different strains af ter 6 days of culture on CM (str i pe) an d
MM (spot) me dium, r espectivel y. (c) T he number of spo res per sq uare centim eter of t ested str ains. All
experiments were conducted in bio logical triplicates . Sign ifica nce values were calculated w i th the two-ta i led t -
test wi th inde pendent varia bles ( * p <0. 05, ** p <0.01, *** p <0.0 01 )
In order to determine whether the spor ulation defect in the tupA null mut ant could im prove the
protein pr oduction capacity of A. niger , the reference strain FW35.1, Δ prpA, a n d Δ tupA w e r e
cultured at the shake flask level. Pair wise comparis on of value s for ∆ prpA and parental strain gave


Chapter 2 42 
comparable results in protein secretion. While compared to the reference strain FW35.1, the
growth rate of Δ tupA was slower in the ear ly stage o f fermentation which was consis tent wit h the
findings of a previous report (Schachtschabel et al. , 2013), but dramatically increased after 2 4 h
(Figure 2.15B). During the flask-level fermentation , the r efere nce strain showed almost no color
change, but the broth color of the Δ tu pA mutant suddenly turned yellowish after the fourth day and
b r o w n o n th e f i f t h d a y ( F i g u r e 2 . 1 5 A ) . H e n c e , d u r i n g t h e s u b m e r ged fermentation process, there
i s a v a r i a t i o n i n g r o w t h a n d p h y s iological characteristics betw een the mutant strain an d the
reference. 
To t a l s ec r et ed p r ot ei n
(mg/gDCW)
**
**
B CD
A Δ tup A
FW35 .1 ( cont rol)
Da y1 D a y2 D ay3 D a y4 Da y5
** *
*
*
** *
Dry weight (g/kg)
En zy m e a ct iv ity o f G la A
(KAG I/gDCW)
Time (h) Ti m e ( h) Ti m e ( h)
Δ tup A
Δ pr pA Δ tup A
Δ pr pA Δ tup A
Δ prpA

Figure 2. 15 Charact eristics of Δ tupA and Δ prpA m utant s during subm erged cult ivation. (a) Color change of
Δ tupA compared to FW35.1 du ring shak e fla sk culti vation in CM m edium . (b) Biomass accu mulation in
cultures of FW35.1 (square ), Δ pr pA ( diamond) , an d Δ t upA strain (tri angle). (c) Ac cumulation of total secreted
protein (normalized by per gra m biomass) in FW35.1 (black ), Δ prpA (grey) , and Δ tu pA (spot). (d ) Enzyme
activit y of Gl aA (norm alized by per gram biomass) in FW35. 1 (bl ac k), Δ prpA (gr ey) and Δ tupA (spot) . All
experiments were conducte d in biological quadruplica tes. Signif icance value s were c alcu lated with 2-taile d t -
test wi th inde pendent varia bles ( * p <0. 05, ** p <0.01, *** p <0.0 01 )
As can be seen in the Δ tupA f ermentation results, in the first t hree day s (exponent ial and e a r l y
stationary phase of fermentation), Δ tupA secreted much less protein especially GlaA which wa s
indeed not dete ctable c ompared t o the parental s train (Figure 2.15C, D) . Howeve r, a fter prolonged
cultivation (day 4-5), t he a mount of ext racellular protei n was consi derably acc umulat ed in t he
Δ tupA strain and was si gnific antl y higher than that in the cont rol ( i ncreased by about 67% at day
5). In particular, the enzyme ac ti vity of GlaA at 70 h post -ino culation showed up to 54-f old
increase compared to that at 45 h. Therefore th e dramatic accum ulation of GlaA contributed mostly
to the i ncrease of extracell ular secreted p rotein. Analysis of public ly ava ilable trans cripto me data
for Δ tupA in A. niger (Sc hachtschabe l et al. , 2013) uncovered that the maj ority of PrtT-dependent


Chapter 2 43 
proteases were significantly up-regulated during the early expo nentia l phase, for instanc e, the
expression of pepA a n d pe pB showed an up to 224- and 99-fold increase, respectively. In ad ditio n,
several GO terms associated with amino acid biosynt hetic or met abolic processes were down-
regulated, such as branched -chain family amino acid biosyntheti c processes, coenzyme
biosyntheti c process, and protein secr etion ( Schachtschabel et al. , 2013). Thi s expr ession data
indicates that the low-level protein production during the earl y fermentation phase of Δ tupA
compared to the reference strain might be due to the accum ulati on of extracellular proteases a nd
weakened a mino acid biosynthesis. It has b een reported that a l iqu id cultur e of A. nig er conidiates
abundantly at the cost of protein secretion when it enters carb on starvation (Jorgensen et al . , 201 1a).
The experimental results shown in Fi gure 2.15 mat ch this observ ation, further suggest ing that
reduced conidiation may incr ease protein production in A. niger in parallel. However, thorough
transcriptome and secretome ana lyses from bioreact or samples ar e necessary to prove o r disprove
this assumption.
2.4 Conclusi ons
We report h ere a high-quality assembled genome of an i ndustrial ly relevan t A. niger strai n LDM 3
used for GlaA p roduction. Owing to its u nique pheno type and h igh protein pr oducing potent ial, it
is o f great interest to both academia and industry to explore t he novel f unctional properties of
LDM3.  Comparative genome analysis in this study revealed several hund r eds of uni que and
mutated genes i n LDM3, some of which might be associated with i ts aconidial phenotype and thu s
its high Gla A secretion capacities. These data highlight that n ovel hypotheses regarding the link
between sporulation and protein secretion in A. niger c an be gleaned from comparative genomic s.
Other pot ential le ad genes can be leveraged in the fu ture for s ystematic optimization of protein
production capacities in di fferent A. nig er strains.

Cha pter 3 44 
Chapter 3  Glob al tran scriptio nal response of Aspergillus niger in the process
of glucoa mylase fer mentation
3.1 I ntroduc tion
Oxygen l imitation is an essential strategy for industrial enzym e production by A. niger .
Explanations of the alter ed gene expression patterns dur ing t he oxygen -limited fer mentation
process may al low us to learn valuable dynamic metabolism strat egies from how cells adapt to the
changing fermentation envi ronment and meanwhile ensure t he h igh ly efficient enzyme producti on
in A. niger .
During the growth of aerobic microorganisms, oxygen is a crucia l electron acceptor i n t he
respirat ory chain. Al ong with th e oxidation of NADH and FADH2, electr ons are transferred to
oxygen through an electr on transf er chain to release energy f or t h e f o r m a t i o n o f A T P u s e d f o r
stra in growt h and metaboli sm (Diano et al. , 2010b). Hence, oxygen avail ability is generally one
of the most key factors in t he fermen tation process of aerobic microorganisms. Ho wever, the high
density submerged cultivation of filamentous fungi generally in duced a high viscosity of broth
(Pedersen et al. , 2000a), whi ch inevitably resu lted in a natural o xygen limitat ion regardless of high
agitation and aeration, thus caus ed mass transfer res istance an d low oxygen transfer efficiency
(Diano et al. , 2010a). Pedersen et al. (2011) simulat ed the actual Gl aA industrial biomanuf acturing
process and observed the highes t yield of GlaA in the o xygen li mitation phase. Therefore, oxygen
limitation has been applied as the most e fficient strategy for industrial GlaA production presently.
Some studies have revealed the complex r elationship between the specific p roductivity ( q p ) of
GlaA and spec ific growth rate (  ). When μ is l ower than 0. 1 h -1 , higher  may confer s uperior q p .
However, q p is not coupled with  if μ is higher than 0.1 h -1 , i ndicating the fierce competition for
c e l l u l a r r e s o u r c e s b e t w e e n p r o t e i n f o r m a t i o n a n d b i o m a s s g r o w t h under high g rowth rate
(Pedersen et al. , 2000a; Pedersen et al. , 2011). T herefore, to limit cell growth through low oxygen
availa bility is a wise st rategy to force c ellul ar fl ux to wards GlaA or ev en by- products. In order to
gain a detailed picture of cel lular metabolism under o xygen-lim ited conditions, Diano et al. (201 0a)
explored the formation mechanism of different by-products i n A. niger v i a c h e m o s t a t c u l t u r e s . T h e
results showed that the pool si zes of TCA cycle intermediates a ccum ulated, but accompa nied by
weakened respi ratory activity under hypoxi c condition, resu ltin g in the accumulation of NADH
and t he consump tion of ATP. Exce ssive NADH thus l ed to t he im pr ovem ent of orga nic a cids.
Moreover, the increased formation of mannitol was the pri ncipal cellula r response to reba lanc e the
NADH/NA D rat io at low oxy gen av ail abili ty.
Temporally, available literat ure mainly focused on depicting th e physiological changes and
metabolic adap tion during the hyp oxic stage (Diano et al. , 2010b; Pedersen et al. , 2011; Lu et al. ,
2015), whi le the global response at t ranscriptional level has not been well c haracterized. In this
chapter, we analyzed the time- course transcrip tome dat a from th e fed-batch GlaA ov erproduction

Cha pter 3 45 
process sequ enced by a st rand-specific RNA-Seq app roach. Throug h gleaning the valuable
regulation informati on from the global transcriptional response under the hypoxic G laA
bioproducti on by A. niger , i t provides us w ith additi onal inspirations for the rational strain
development of this microbial platform.
3.2 Materials a nd Methods
3.2.1 Strain
A. niger DS03 043 is an indust rial hig h-yield GlaA prod ucing strain, con taining seven copies of
the GlaA coding gen e gla A, w h i c h was kindly provi ded by DSM ( Delft, the Netherlands) (Lu et
al. , 2015).
3.2.2 Flask-level cultures
In order to obtain A. nige r s p o r e s , t h e s t o r e d s p o re s o l u ti o n ( p r e s e rv e d i n 5 0 % g l y c e r ol a t -80 ℃ )
was inoc ulated onto the PDA plat es. The seed m edium consists of g lucose 22 g/L, corn syrup solid
powder 20 g/L, and the ini tial pH of t he medium was ad justed to 6 . 5 b y 3 M N a O H b e f o r e
autoclavi ng. 10 7 s pores /100 ml mediu m was i noculated into 5 0 0 ml shake flasks with b and baffle
and incubated at 34 ℃, 1 50 rpm for 24 h.
3.2.3 Bioreactor fed-batch fer mentation
This study adopted the defined m edium for all fed- batch culture s (g/kg), includ ing glucose•H 2 O
20, KH 2 PO 4 3 , N a H 2 PO 4 •H 2 O 1.5, (NH4) 2 SO 4 3 , M g S O 4 •7H 2 O 1, CaC l 2 •2H 2 O 0.1, Z nCl 2 0 . 0 2 ,
CuSO 4 •5H 2 O 0.015, CoCl 2 •6H 2 O 0.015 , MnSO 4 •H 2 O 0.04, FeSO 4 •7H 2 O 0 .3. Fed-b atch culture s
were performed on a 5 L bioreactor (Guoqiang Biological Enginee ring Equipment Technology
Co., LTD, Shanghai, China) contai ning 3 L f ermentation broth wi t h an onlin e monitoring system
for temperature , pH, agitat ion, DO, OUR, CER, and r espiration q uotient (RQ), etc. A. niger w a s
cultured at 34˚C and 375 rpm, and the aeration rate was set at 1 vvm during th e whole cultures.
The p H of fermentation broth was c ontrolled by ammonia (5% W/W) at 4.5. To avoid carbon
limitation, th e residual glucose in the broth was maintained at 5-10 g/L by adjusting the feed rate.
The experiments were performed in b i ological tripli cate.
3.2.4 Sampling for RNA-Seq
Transcriptome samples were sampled a t four time-points in the f ed-batch process, viz. 16 h (H A)
in the e xponential gro wth phase, 24 h (HB) i n the ear ly phase o f oxygen limita tion, 42 h (HC) in
the middle phase of o xygen limitat ion, and 6 6 h (HD) in the lat e p hase of oxyg en limitation.
3.2.5 Determination of biomass and enzyme activity of glucoamy lase
Please refer to 2.2.9.

Cha pter 3 46 
3.2.6 RNA Extra ction
Mycelium used for RNA extraction were frozen quickly in liquid nitrogen and stored at -80 ℃ .
The extraction and quality detecti on of transcriptional samples were performed by Sangon biotech
(Shanghai, P.R. Ch ina) Co., Ltd.
3.2.7 Strand-specific RNA-Seq
Beads containing oligo (dT) were used to isolate ploy (A) mRNA from total RNA. Pur ified mRNA
was then fragmented. Using these short fragments as templates, random hexamers were used for
first-stran d cDNA synthesi s. The sec ond-strand cDNA was synt hes i z e d u s i n g b u f f e r ,
dNTPs/dUTPs mix, RNase H and DNA pol ymerase I. Short do uble-str ande d cDNA frag ments
were puri fied wit h a QIAquick PCR extracti on kit and eluted wi t h EB buffer for end-repair, the
addition of an ‘A’ base and ligated t o Illumina sequencing adap tors. Then, the second-strand
cDNA was excised by UNG enzyme. The frag ments with expected siz e were pur ified an d then
amplified by PCR. The ampl ified l i b r a r y w a s s e q u e n c e d b y t h e I l lumina HiSeq™ 2000. The details
of the p arameters were as follow s: Expected library size: 200 b p; Read length: 90 nt; and
Sequencing strategy: pa ired-end sequencing.
3.2.8 Alignment of reads to the reference geno me and normaliza tion of gene expression
A. niger strain CBS 513.88 (Genbank IDGCA_0 00002855.2) is tak en as the refer ence genome.
Clean reads after quality control were then aligned to the refe rence genome, and no more t han 3
mismatches were al lowed in t he alignment for each read. Uni quel y m apped reads were used to
calcul ate the expre ssion level. The gene expressi on level was q uantif ied using FPKM (Dewey and
Li, 2011).
The formula is as fol lows: 3
6
10
/

10
NL
C
FPKM  , in which FPKM (A) is the exp ression level of gene
A. C is counts of fragments t hat un iquely map ped t o the gen e A. N is total counts of fragment s
that uniq uely ma pped to the reference gene s. L is th e base coun ts of the coding region of gene A.
FPKM method can eliminate the influence of gene length and diff erence of sequencing on the
precise de termina tion of gen e transcript ion level th at can be d irectly used t o compare gene
expression between different samples.
3.2.9 Differently expressed genes
EBSeq package (Leng et al. , 201 3) was adopted to identify DEGs based on the following cri te ria :
FDR  0.05 and fold chang e  2. Al l DEGs collec ted from time-cours e transcrip tom e data w ere
merged as a DEGs set for gene expression patterns an alysis. The hi erarchical cl ustering a nalysis
was performed by using the unwei ghted pair group method with ar ithmetic mean.

Cha pter 3 47 
3.2.10 Functional gene annotation
NCBI non-red undant protein database (Nr) (htt ps://www.ncbi.nlm.nih.gov), David Bio informatics
Resources 6.7 (DAVID) (htt ps://david.ncifcrf.gov) (Huang et al. , 2009b; Huang et al. , 2009a),
Aspergil lus genom e d atabase ( AspGD) (ht tp://www.aspgd.o rg) ( Cerqueira et al. , 2014), Kyoto
Encyclope dia of Genes and Ge nom es (KEGG) (http:// www.kegg.jp (O gata et al. , 2000; Kanehisa
et al. , 2016) wer e used for fu nctional ann otation of all DEGs. GOSli m in AspGD database and R
package ggplot 2 were p erformed f or Gene Ontol ogy (GO) classific ation and plotting respectively.
iPath (http://pathways.embl.de) (Yamada et al. , 2011), a user-friendly online software, is used to
visualize the distri bution of DEGs on different metabolic pathw ays.
3.3 Result and discussion
3.3.1 Growth physiology of glucoamylase o verproduction by A. niger
The typical indu strial fed-bat ch culture fo r GlaA production wa s perf ormed in thi s study. During
the whole fermentation process, t he residual glucose was mainta ined above 5 g/L to eliminate the
influence of carbon limitati on o n the metabolism of A. niger . The entire fed-batch cultures could
be roughl y div ided into t wo stages in view of the DO and OUR cu rve, namely suffi cient oxygen
supply phase (0-24 h) an d the o xygen-limited phase (24 h-72 h) (Figur e 3.1A). In the exponen tial
growth phase (8-24 h), along with the fast cell growt h, OUR inc reased dramati cally and DO
declined rapidly (Figure 3.1 A), accompanied by the continuo usly d e c r e a s e d μ i n t h e l a t e
exponential phase (Figure 3.1 B). W hen DO dropped t o the bottom (24 h), it indicates the entry o f
cultivation into the oxygen limit at ion period. Then OUR start ed to decrease because of the low
oxygen availability an d maintained at a st able level subsequent ly, but μ was conti nuously inhibit ed
during the whole oxygen-limited phase (Figure 3.1A, B). In thi s study, transcriptome samples were
taken at f our time-points based on the alteration of OUR at 1 6, 24, 42 and 66 h, co rresponding to
the exponential growth phase (HA), the early (HB), th e middle ( HC) and the late stage of oxygen
limitation (HD), respectively. In the exponen tial gro wth phase ( <18 h), t he yiel d of th e Gl aA on
biomass decreased under the h igh μ (>0.1), implying that cell a llocated more intracellu lar flux
towards biomass g r owth other than Gl aA dur ing this ph ase as pre viously reported (Pedersen et al. ,
2000a; Pedersen et al . , 2 011) (Figure E). Interestingly, wi thin 18-24 h, μ began to notably d ecline
(<0.1), accompanied by an increment of q p (Figu re 3.1D), suggesti ng t he redistr ibutio n of
metabolic flux between GlaA biosynthesis and biomass growth i n this p hase. From t he view of
GlaA prod uction, this stage may be ideal for keeping a high q p level and a moderate growth rate.
After ent ering the o xygen limitati on phase, the yield of the Gl aA on biomass di splayed a l inear
increase, re aching an i ncrease of three-fo ld than that in the e xponent ial phase (Figure 3.1E).

Cha pter 3 48 
0
10
20
30
40
0
50
10 0
15 0
20 0
0 2 04 06 08 0 1 0 0
O UR ( mmo l / kg · h )
DO
Ti m e ( h )










Figure 3.1 Growth profiles of A. niger DS03 0 43 d uring f ed-ba tch cult iv atio ns (Arrows represent f our
samp ling poin ts at 16 , 24, 42 and 66 h. (A ) O nline data of diss olved oxygen (DO) and oxygen uptake rate
(OUR). A fter the dot ted line, it mean s tha t the cul ture came in t o the oxygen-limitat ion stage; (B) specifi c
growth rate (μ); (C) Biomass; (D ) Sp ecific productivity of gluc oamylase (q p ); (E) The yi e ld of gl ucoamylase to
biomass. A ll experiments w ere conduc ted in bi o logical t riplicat es
3.3.2 Overv iew of RNA- Seq data
Table 3 .1 Seq uencing and assemb ly sta tistics for the 11 tr ansc ri ptome s amples taking fr om fo ur time-poi nts
during the hypoxic GlaA o verproduction cultures by A. ni ger DS03043
Sample
ID
Number of
clean re ads
(×10 6 )
Number of
base pairs
(×10 9 )
Number of
mapped re a ds
(to genome)
(×10 6 )
Mapped
perce ntage
to g enome
(%)
Numb er of
mapped reads
(to gene)
(×10 6 )
Mapped
percen tage
to gene (%)
HA-16h 35.43 3.54 29. 95 84.54 21.71 61.2 8
HB-24h 35.1 2 3.51 29.23 83.22 21. 81 62.08
HC-42h 33.6 3 3.36 28.60 85.04 20. 96 62.31
HD-66h 33.83 3.38 28.53 84.32 20. 92 61.8 2
Biologi cal tripl icate transcriptom e samples taking at four time -points were co llected in t his study,
and 11 qualified RNA samp les were th en sequenced. Each sample g enerated a pproximately 0.35
billion base clean reads (90 bp paired-end reads) a fter quality control . Then, the high-quality reads
were mapped to the p ublished CBS 513.88 genome. The aligned tra nscriptome sequences to t he
reference g enome or genes accounted for 83.22-85.04% and 61.28- 62.31% respectively. In wh ich,
79.52-83.37% or 57.6 7-61.01% of the aligned reads were uniq uely mapp ed to t he reference
genome or reference g enes, respectively (Ta ble 3.1).
C DE
A B
HA HD
HC
HB
0
0.04
0.08
0.12
0.16
0.2
0 2 04 06 08 0 1 0 0
µ (h-1)
Time (h )
0
5
10
15
20
25
0 2 04 06 08 0 1 0 0
Biomass (g/kg)
Time ( h)
0
0.5
1
1.5
2
2.5
0 2 04 0 6 08 0 1 0 0
q p (KAGI/gDCW·h)
Time (h )
0
10
20
30
40
50
60
70
80
0 2 04 06 08 0 1 0 0
Yi e l d o f G l a A
(KAGI/gDCW)
Time ( h)

Cha pter 3 49 
3.3.3 Functional and g ene expre ssion pattern analyses of DEGs
Correlation analysis revealed th at the Pearson correlation coe f ficient (PCC) among r eplicates was
high up to 96.1% to 99.6%, demonstrating the high reproducibili t y of replicates. Hierarchi cal
clustering b a sed o n gen e FPKM (Figure 3.2) displayed that all 1 1 sample s were clust ered into tw o
branches. The most significant transcriptional alteration was w itnessed between 24 h and 66 h,
suggesting a n oticeable effect of the hypoxic envir onment on th e global transcriptional network.

Figure 3.2 Hier archi cal cluste ring plo t f or all transcr iptome sa mples
FDR≤0.05 was used to def ine DEGs bet ween two-time point s. A tot al of 515 DEGs were collected
after the combination of all DEGs from the two-points compari so n (Figure 3.3). Th e most number
of DEGs were identified between t he l ate oxygen- limited phase ( 66 h) and the early limite d phase
(24 h), which is c onsistent with the hierarchy clust ering (Figu re 3.2). While it was not witnessed
an ap paren t transc ription al div e rsity between the middle and la te st ages of oxygen- limitation.

Figur e 3.3 DEGs gener ated by two-time points comp ariso n

Chapter 3 50

To determine th e function of these DEGs, 515 DEGs were then clu stered by gene ontology (GO)
annotation and KEGG enrichment analysis (Figure 3.4) to identif y th e strongest resp onded
metabo lic activ itie s. Results show ed that th e maj ority of DEG s were enriched in the followi ng GO
terms with functions in biol ogica l pr ocesses: transport, RNA me tabolic process, response to stress,
lipid metabolic process and cell ular ami no acid metabolic proce ss. As for KEG G path way s, most
DEGs were o verrepresented in translation (40 genes), followed b y th e car bohydrate metaboli sm
(35 genes), amin o aci d metabolism ( 29 genes) and l ipid m etaboli sm ( 23 g enes). Above evidence
reflect the flexible transcrip tional machi nery against the low energy input in t he hypoxic condition.

Figure 3.4 G O (A) a nd KEGG (B) function enrichme nt analys is of all D EGs

For the purpose of well characteriz ing the gene expression patt ern related to the c hanged oxygen
supply, the expression profiles of all 515 DEGs were illustr ate d via heat map (Figure 3.5 A) and
the hierarchical clusteri n g analysis was pe rformed using unwe ig hted pair group m ethod with
arithmeti c m ean (Figure 3.5B). All DEGs were clu stered into 12 expression patterns which
represented a class of statistical trend of gene expression in continu ous four sampling point s. We
put the most concern in clusters c1, c2 (continuously do wn-regu lated) and c9, c10 (consistently
up-regulated) which may repr esent the direct t ranscriptional re sponse to the continuous decrease
of DO (Figure 3.5B).
Given the GO and KE GG analysis of these two expression patterns (Table 3.2), 171 d own-
regulated genes i n Prof ile c 1and c 2 a re mainly enrich ed in tran slation such as ribosome, tRNA
synthase, ribonucleoprotein compl ex, aminoacyl-tRNA biosyn thesi s, and tRNA am inoacylation .
This well matched the suppressed biomass growth under oxygen limitation. Profiles c9 and 10
(Table 3.2) cont ain 221 up- regulated genes but with lower stati stic significance co m pared to
profiles c1, c2. I n sum, albeit the general inhibited cell tran slation efficien cy, the activation of f atty
a c i d d e g r a d a t i o n , s y n t h e s i s o f a s p a r t a t e a n d p r o l i n e , t r a n s p o r t er activity, a nd transcripti on regulator
B
A

Cha pter 3 51 
activity, in particular the high transcript ion level of glaA (Figure 3.6), are pr imary transcri ptional
alterations to under pin the effic ient accumulation of extracell ular glucoam ylase.

Figure 3.5 Hier archic al clu stering analysi s of 51 5 DEGs dur ing GlaA fermenta tion. (A) Each hor izontal line
displays the expr ession data for on e gene a fter normaliz ation ( the mean of FPKM v alue at one tim e point
divided thr ough the maxim um of mean F PKM value for each gene) a t time p oints as indic ated. The c olor scale
at the top left represents t he normalized e xpression l evel (0-1 ). The highest expression of each gene is
therefor e defined as 1 (dark red color). Co lor bars in the midd le indic ate define d sub- branches acco rding to the
height of th e tree. The genes assigne d to each cluster ar e list ed in Tabl e c1-c12; (B) Gene express ion profiles
analysis. C 1 to c1 2 represe nt the twe lve clusters i ndicated ord erly b y the color b ars. Mean and s tandar d error
of the normal ized expression data of genes in each cl uster at t he four time points wer e used to plot the curves.
The numbe r of genes foun d in each cluster is ind icated b y the v alue of n
Table 3.2 Gene ontolog y (GO ) enrichment analysis of four overr epresented clusters c1, c2 an d c9, c10
Profile c1, c2 ( down- regula ted)
GO Term Description GO term Count Ontology p Value
GO:0005840 ribosome 11 CC 1.13E-07
GO:0030529 ribonucleoprotein comp lex 11 CC 9.56E-07
GO:0003735 stru ctural constituen t of ribosome 11 MF 1.57E-06
GO:0006412 trans lation 14 BP 2.29E-06
GO:0005198 stru ctural molecul e activity 11 MF 8 .26E-06
GO:0043228 non -membrane-bounded o rganelle 12 CC 1.03E-05
GO:0043232 intracellular non-me mb rane-bounded 12 CC 1.03E-05
GO:0008654 pho spholipid biosynt hetic process 3 BP 0.059
B
A

Chapter 3 52

GO Te rm Descri ption G O term Count Onto logy p Value
GO:0006399 tRNA metabolic process 4 BP 0.055
GO:0043038 amino acid activation 3 BP 0.088
Profile c 9, c10 (up- regulated)
GO Term Description GO term Count Ontology p Value
GO:0008080 N-acetyltransferase acti vity 3 MF 0.19
GO:0016410 N-acyltransferase activity 3 MF 0.20
GO:001 6407 acetyltransfer ase activity 3 MF 0.27
GO:0044271 nitrogen compound biosynthetic p rocess 8 BP 0.099
GO:0008652 cellular amino acid bi osynth etic process 3 BP 0.41
GO:0009309 amine biosynth etic process 3 BP 0.43
GO:0016053 organic acid biosynthetic process 3 BP 0.51
GO:0046394 carboxylic acid bios ynthetic pro cess 3 BP 0.51
GO:0000166 nucleo tide binding 23 MF 0.21
GO:0032555 purine ribonucleotide binding 16 MF 0.29
GO:0032553 ribonucleo tide binding 16 MF 0.29
GO:0017076 p urine nucleotide binding 18 MF 0.36
GO:0005524 ATP binding 13 MF 0.43
GO:0032559 adenyl ribonucleotide binding 13 MF 0.43
GO:0 0018 83 purin e nucleo side bi ndin g 15 MF 0.50
GO:0030554 adenyl nucleotide binding 15 MF 0.50
GO:0001882 nu cleoside binding 15 MF 0.51
GO:0015399 primary active transmem brane transporte r activity 3 M F 0.44
GO:0005694 chromosome 3 CC 0 .19
GO:0003677 DNA bindin g 15 MF 0.39
GO:0030528 transcription re gulator activity 11 MF 0.42
CC,

cellul ar component; BP, biologi cal process; MF, molecular funct ion

Figur e 3.6 T ranscriptiona l le vel of GlaA encoding gene gla A dur ing the f ermentat ion
0
1000
2000
3000
4000
5000
6000
0 2 04 06 08 0
FPK M of g l a A
Time
FPKM of glaA

Chapter 3 53

3.3.4 Key transcriptional res ponse during glucoamyla se overpro duction

Figure 3.7 KEG G and iPa th analysis of DEGs in 66 h (HC) vs 24 h ( HB). (A) K E GG enrichment ana l ysis; (B)
iPath enric hment anal ysis of DEG s. Blocks r e present over represe nted pathways. 1. Fatty acid biosynthesis; 2.
Fatty acid meta bolis m; 3. Pyruv ate metabol ism; 4. Valine, leuci ne, and isoleucine biosynthesis; 5. T CA cycle;
6. Oxidative phosphoryl ation. B lue means down- regulate d pathway s, and r ed rep resen ts up -regul ated pathways
1 
2 
3 
5
4 
6

Cha pter 3 54 
As depicted in the physi ology prof iles , DO remai ned at the lowe st level acc ompanied by a sharp
decrease of µ, OUR, and q P from t he early (24 h) to the late peri od of o xygen limitatio n (42 h)
(Figure 3.1), suggesting that c ellular metabolism gradually ada pted to the hypoxic stress. Based
on the comparison between 24 h and 42 h, t he iPath m ap visualiz ed that fatty acid catabolism
pathways wer e signific antl y activated , while t he fatty a cid ana bolism and oxidative
phosphorylati on were en tirely inhi bited (Figure 3.7). Since the c ompetition between fatty acid and
amino acid biosynt hesis for the same cytosol ic cofa ctor NADPH a nd t he liberation of important
metabolite precursors and energy such as AcCoA from fatty acid catabolism (Rat ledge and W ynn,
2002), we c an c onclude th at the degrad ation of fatty acid may c ont ribute to t he pr ovision of
cytoplasmic reductant and metabol ite precursors required for t h e biosynthesis of amino acids as
building blocks for proteins.
Ser, Th r, Ala, Leu, Gly, Asp, and Val take up more than 50% of the amino acid composition of
GlaA (Figure B.3). Remarkably, pyruvate kinase (An07 g08990) and asparagine synthase
(An04g01340) encod ing genes were contin uously up- regulated and t ranscribed at a h igh level,
which m ay suppo rt for the p rovision o f PYR an d OAA as t he car bo n sk eleton of Thr, Ala, L eu and
Val. Si milarly, as one o f t he genes on the Ser biosynthesis pat hway, the high transcription level of
An11g01390 (gl ycolate dehydrogenase) may facilitate the high bi osynthesi s efficiency of Ser and
Gly. Nevertheless, expect f or above six a mino acid s, the expres sion level on othe r amino acid
biosynthesis pat hways were not wi tnessed visible alteration.
Moreover, it can also be noted that a broader rang e of h ydrolas e and oxid oreductase were induced
under o xygen-limited phase, but t heir e xpression tendenci es are obscure. In addition, 29 out of 865
identif ied transport ers (Pel et al. , 2007) were differently expressed, and most of them (21 genes)
were up-regulated, indicating th e activation of transporter act ivity.
3.3.5 Transcriptional changes re levant to t he secretion pat hwa y
As an ex cellent expre ssion ve ct or for a variety of homogeno us a nd heterogenous protei ns, the
secretory mechani sms in A. nig er h as obt ained extensi ve attent ion by researchers (Guillemette et
al. , 2007; Jorgensen et al. , 2009; Carvalho et al. , 2012; Kwon et al. , 2012).
BipA , encoding the main chaperon e prot ein in the en doplasmic reticu lum (ER), was up-regulated
before 2 4 h and d own-regulated su bsequently, which correlated w ell wit h t he chan ge of specifi c
productivit y o f GlaA. It is also a m arker gene on the unfolded p rotein response (UPR) pathway
(Määttänen et al. , 2010). The induction of bipA relies on th e high heterolo gous protein loads and
the deletion of ERAD gene (Carvalho et al. , 201 1). Thus, i ts down-regul ated expression pattern in
this study might indicate that UPR was not induced in the proce ss of GlaA overproduction. Three
DEGs (An0 4g06180, An02g01690, An0 8g10650) as components of COPI I coated vesicles that
mediate the transportation of protein from ER to Golgi were als o u p-regulated. The improved
biosynthesis of COPII coated vesicles m ight be advan tageous for cells to co pe wit h high protein

Cha pter 3 55 
traffic. Among genes encoding extracellular protease, An02g13410 (up-regul ated), encodi ng a
putative AcCoA transporter, is involved in the transport of mem brane permeability CoA from the
cytoplasm to t he inner membran e of ER to transiently acetylate resident proteins on ER, which is
associated with the optimization of the folding efficien cy of s ecretory protein (Kwon et al. , 2012).
Compared with data sets from ot her hi gh protein secret ion lit er ature, secret ory pathways did not
display a strong t ranscriptional response dur ing Gl aA o verprodu ction in this thesis, as the majority
of g enes on secreto ry pathways s uch as UPR, ERAD di d not differ ent ially express. Overall , protein
secretion does not play a limitin g role in extracellular GlaA a ccumul ation in A. niger DS03043.
3.3.6 Transcriptional changes rel evant to transcri ption factor
Table 3.3 Summary of important u p-regu lated transcription fact ors in t he process of GlaA f ermenta tion
Gene I D Alte r
name
FPKM value of each gene in the 4
samplin
g

tim e p oin ts in thi s stud
y

Annotation in A. niger Reference
16 h 24 h 42 h 66 h
An04g06940 prtT 267.5 105.1 335. 1 362.5
Member of the fungal-specific
Zn2Cys6-binuclear cluster
protein family; transcrip tional
activator of major secreted
proteases and also enhances
the expression of genes
involved in iron uptake
(Guillemette et
al. , 2007; Punt
et al. , 2008;
Sharon et al. ,
200 9)

An14g02540 srbB 382. 6 292. 3 792.7 1095. 7
HLH DNA binding domain
protein, sterol regulatory
element-binding
prot ein respons e to hyp oxia;
SrbB co-r egul ates ge nes
involved in heme biosynthesis
and demethylation of C4-
sterols with SrbA
(Park et al. ,
200 8; Ce rqu ei ra
et al. , 2014;
Chung et al. ,
201 4)
An11g07350 885.3 1635.8 407 .6 267.4
Ort holog (s) ha ve role in
positi ve regul ation of
secondary metabolite
biosynthetic process;
Function- GABA utilization is
amdR-dependent in A. oryzae
(777 aa)
(Park et al. ,
200 8; Ce rqu ei ra
et a l. , 2014)

An17g01370 Ada 2 6.3 95.8 326.9 3 06.6 DNA repair and transcriptio n
factor Ada
(Punt et al. ,
200 8)

Among the 869 identi fied tran scripti on f actors (TFs), 4 TFs, viz. srbB (An14g 02540), prtT
(An04g06940) , An1 1g07350 and An17g013 70, were strikingly improv ed durin g fed-ba tch
cultures ( Table 3.3). Particularly, the transcri ption level o f srbB and prtT i n t h e m i d d l e p h a s e o f
oxygen li mitation reached 23 f olds higher than that in the ex po nential growth phase. More notably,
the most r emarkably altered TF is An17g01370, whose expression level at 42 h is more than 10-
fold of that at 16 h . According to t he NCBI anno tation, An17g0 1 370 pl ays a role in DNA rep air,
carrying Zn-bi nding and two AraC-type DNA-bindi ng d omains, but the function of this TF has
not been well c haract erised yet in A. niger . PrtT, a member of the Zn (II) 2Cys6-binuclear cluster

Cha pter 3 56 
family, is the o nly identified specifi c prot ease transcription factor in A. niger , which controls the
expression of m any extracell ular proteases and r egulates iron u ptake as well (Ha gag et al . , 2012).
Through r andom mutagenesi s, Pu nt et al. (2008) obtained a prt T mutant , leading to deactivated
protease activit y. The ortholog of prtT regulat es the ma jor alkalin e protease AlpA and th e neutral
protease Np1 in A. oryzae . To achieve a more det ailed regulatory rol e of prtT i n A. fumigatus ,
Sharon et al. ( 2 0 0 9 ) c o n s t r u c t e d a prtT null mutant, resulting in a dramatic reduction of six secreted
proteases (ALP, MEP, Dpp4, CpdS, AFUA_2G17330, a nd AFUA_7G062 20 ). In our
transcriptome data, several extracellular proteases such as pepA , pepB, and pepE d i s p l a y e d a s l i g h t
up-regulation tendency under the control of the activated prtT . To avoid t he degradation of GlaA
resulted from secreted p roteases, knockout or kno ckdown of prot ease genes or PrtT coul d be a
wise str ategy to prof it the accumulation of GlaA or o ther extr a cellular secreted proteins.
SrbA (Sterol-regulatory element b inding protein, SREBP), the t r anscription factor of Helix-Loop-
Helix ( bHLH) famil y, points in t he resistance to antifung al dru gs and the virulen ce of Aspe rgil lus
fumigatus . Chung et al. (2014) identified the gene sets subj ected to the di rect contro l of sr bA i n A.
fumigatus t hrough ChIP-seq an alysis. They c onfirmed the versat ile biolog i c a l r o l e s o f S r b A a s a
global transcription factor under hypoxia conditi ons, invo lves in t he regulation of sterols synthesis,
iron uptake, hyphae growth, mycel ium polarization, assimilat ing nitrate, components o f the cell
wall, vi rulence of pathogens, and t he biosynthesis o f amino aci d s , f a t t y a c i d s a n d l i p i d . S r b B ,
sharing high homology with SrbA, co-reg ulates genes involved in heme bio synth esis a nd
demethylation of C4-sterols wi th SrbA in hypoxia conditi on in A. f umigatu s . Besides, SrbB can
also independently regulate carb ohydrate metabolism. We found t hat both srbA and srbB show ed
an up-regulated expression pattern during the whole cu ltivation s, but the expression level of srbA
was consistent ly l o w. Noteworthy, srbB expressed at a high level a nd dramaticall y up-regulated
from th e middle to the late o xygen-limitation phase, i ndicating that SrbB probably functions as a
more important transcriptional regulator for hypoxia fermentati on of A. niger . Orthology of all 30
genes directly boun d to SrbA in A. fumigat us were a lso identified i n A. nige r genome and shared
a high identity. However, when searching for genes in A. nig er genome containing the same SrbA
binding motif (5 ’-(A/G) TCA(T/C/G)(C/G)CCAC(T/C) -3’) (Chung et al. , 2014) as reported in A.
fumigatus , the mat ching rate was l ow, sug gesting that the bindi ng motif of SrbA in A. niger m a y
be altered. Due to th e invisi ble alteration of mo st enzymes inv olved in sterol biosynthesis pathways,
it demonstrated that the low oxygen availability i n this study did not cause a strong tran scriptional
response of this importan t metabolic act ivity in A. niger . The logical explanation is tha t the
environmental oxygen content du ring the GlaA production by A. niger was entirely different from
the extre me hypoxic co ndition (1%) as A. fumigatus faced in Chung’s study.
3.4 Conclusi on
In this study, we implemented a time-course comparative transcr iptome a nalysis at f our time-
points to study the glo bal transcriptional response in the pro c ess of oxygen-limited GlaA

Cha pter 3 57 
fermentation by an indust rial GlaA producing strain A. niger DS03043. Oxygen availability plays
an essential effect on the physiolo gical characteristics and ph enotype of A. niger . Low oxygen
availabili ty caused by the hig h-density A. ni ger f ermentation r educed t he O 2 input for the
respiratory chain, leading t o the lower oxi dative p hosphorylati on level and weakened release of
ATP. Along with t he l imited cell growt h, it was o bserved a wide r a n g e o f c e l l u l a r m e t a b o l i c
redistrib ution, su ch a s the sev e rely inhibit ed fatt y acid biosynt hesis, continuo us up-regulated
tr anscr ipt ion of gl aA , and the activated biosynthesis of dominating amino acids, whi ch are
conducive to diverge more metabolite p recursors and energy subs t r a t e s t o m a n d a t e t h e d e s i r e d
GlaA biosynthesi s. Compar ative transcriptome analy sis draws a d etailed map of how A. niger
relies on highly fl exible metab olic networks t o cope wi th adver se e nvironments or cellular burdens
and to ensure a strong pull towards GlaA biosynthesis.


Ch ap te r 4 58 

Chapter 4  Exploring the effect of N ADPH supply on enzym e
bioproduction in low-y ield GlaA producing Aspergillus niger
4.1 Introduction
Nicoti namide adenine dinucl eoti de phosph ate (NADPH) is a n i mpor tant cofactor ensuring
intracellular redox balance, anabolism and cell growth in all l iving system s. PPP, cyt osolic NADP-
ICDH, and NAD P-ME a re well-defined NADPH source s. To m eet the h i gh demand of NADPH
for heterologous protein expression in fun gi, a wealth of metab olomic and fluxomic data
demonstrated that cell triggers a s ignificant metabolic flux re distributi on to b oost the fl ux towards
the PPP (Driouch et al. , 201 2; Ni e et al. , 2014). For the cell factory A. niger , several genetic
approaches have been shown the potential to maximize its enzyme pr oduction capability i ncluding
protein carrier approaches, t unable Tet-on driven gene expressi on and morphology e ngineering, to
name but a few ( Cairns et al ., 2018; Cairns et al., 2019; Fiedl er et al., 201 8a,b). Notably, cofactor
engineering, i.e. t he rebalancin g of the intracellular redox st atus, has been reported to improve
productivities in the bacterial cel l factories Esche richia coli (Li et al. , 2018; Yao et al. , 2019) and
Corynebacterium glut amicum , as well as in the yeast cell factory Yarrowia lipolytica (Wasy lenko
et al. , 2015). However, the impact of cofactor engineering on enzyme bioproduction has not been
systematically studied in A. niger .
Table 4.1 Su mmary of reactions p roducing NADPH wh ich could be used for strain growth and enzyme
biosynthesis
Rn
name Rn descriptio n Formula Gene
R25 Glucos e 6- phosphate -
dehydroge nase (
g

sdA, G6PDH)
G6P + NADP => D6PG L + NADP H
+ H An02g12140
R27 Phosphogluconate
dehyd rogenase (
g

ndA, 6PGDH)
6PGC + NA DP => Ru5P + C O2 +
NADP An11g02040
R36 Isocitrate dehydrogen ase
(NADP+) ( icdA, NADP-ICDH)
ICIT[m] + N ADP[m] => A KG[m] +
CO2[m] + NADPH[m] An02g12430
R38 Isocitrate dehydrogen ase
(NADP+ , NA DP-I CDH)
ICIT + NADP => AKG + CO 2 +
NADPH An02g12430
R55 Malic enzyme (NAD P-specif ic)
（ maeA, NADP-ME ）
MAL + NADP => PYR+ CO2 +
NADPH An05g00930
R57 Malic e nzyme ( NADP-specific)
（ maeA, NADP-ME ）
MAL[m] + NAD P [m] => PY R An05g00930
R110 (S )-3-Hydr oxybuta noyl-
CoA:NADP+ oxidoreductase
[m] + CO2[m] + NADPH [m] An1 4g00430
R125 dihydrofolate:NADP+
oxido reduc tas e
3hbcoa[m] + NADP [m] <=>
aaccoa[m] + N ADPH [m] + h[m] An12g04590
R190 Alcohol dehydrogenase NADP [m] + DHF[m] <=> NADPH
[m] + FO LATE[m] An16g02510
R775 NAD(P) transhydrogenas e NADP + NADH => NA DPH +NAD An0 2g09810
- NADPH oxidase reg ulator
NoxR ( riaA ) - An16g05550
[m] represent reactions take pl ace in the mitochond rion


Ch ap te r 4 59 

In light of our previous multi-omics analysis for GlaA overprod uctio n process, fermentative
production of enzymes could be lim ited by the low availability of NAD PH in A. niger . We thus
mined our recently published gen ome-scale m etabolic network mod el developed for the A. niger
protein production reference strain CBS 513.88 (Lu et al. , 2 0 1 6 ) t o p r e d i c t s e v e n p o t e n t i a l N A D P H
generating enzymes a nd examine their effect o n GlaA production in A. niger (Tabl e 4.1 and Fig ure
4.1). The follow ing e nzymes were s elected: two enzymes of the c ytosolic PPP (glucose-6-
phosphate dehydrogena se, G6PDH; 6-p hosphogluconat e dehydrogenas e 6PGDH), t wo c ytosolic
NADP-dependent enzymes (NAD P-ICDH and NADP-ME) and three uncha r acterized op en
reading frames (ORFs). An12g04590, An14g 00430 show high homo log y t o NADP+
oxidoredu ctases and An16g02 510 displays homology t o alcohol deh ydrog enase. Addi tionally,
An02g09810 (NAD(P)-dependent transhydrogenase) a nd riaA (NADPH oxid ase regulator)
predic ted fr om th e av ail able l it erature were also incorporated.
G6P 6PG L 6PG
RU5P
PEP
Gluc ose
PYR
AC -Co A
IC I T
OA A
MA L
AK G
S UCC-Co A
FU M
gsdA
1.1.1.49
gndA
1.1.1.43
ic dA
1.1.1.42
maeA
1.1.1.40
NADP+ NADPH
NADP+
NADPH
NADP+
NADPH
PYR
MAL
CI T
Mi t o ch o n d r i o n
HIS
OAA
CIT
ICI T
AKG
CIT
OAA
NADP+ NADPH
F6P
AC AL
ETH
An16g02510
1.1.1.2
NADP+
NADPH
DH F Fol a te
A n 1 2g04 590
1.5. 1.3
NADP + NADP H
AC
AA C- Co A 3HB - C o A
N ADP +
NADP H
A n 1 4g00 430
1 . 1.1. 157
pgi
pyr
CO
2
CO
2
NAD+
NADH

Figure 4.1 P athway ma p hi ghlig hti ng a ll se ven genes modifi ed d uring t his study i n red
In order to evaluate whether cof actor engineering could open a new avenue to break the bottleneck
of protein biosynthesis in A. niger , we integrated an additi onal copy of th ese g enes under the
control o f the strong and tunabl e Tet-on gene switch into the pyrG locus of a lab est ablished ur idine
auxotrophic model A. niger s train AB4.1 (only with one c opy of glaA ). Flask-leve l cultures were
impleme nted to r eveal the direct i nfluence o f diverse cofactor r egulation strategies on intracellular
NADPH abundance and prot ein capacity in A. niger .


Ch ap te r 4 60 

4.2 Materials a nd methods
4.2.1 Strain and medium
A. niger AB4.1 is the lab established uri dine auxotrophic strain with o nly one glaA c opy (van
Hartingsveldt et al. , 1987), which was used to con struct all transformants i n this chapter (Table
4.2). FW35.1 works as the reference strain for flask-l evel cult ures in this chapter (Wanka et al. ,
2016).
Table 4.2 Strains use d in this Chapter
Strain
name NO. Background
strain Relevant genoty pe/description Source
A B 4 . 1 cspA1 -, kusA ::DR- amdS -DR, pyrG −
(van
Hartings veldt
et al. , 1987)
FW35.1 AB4.1 cspA1 -, pyrG + (Wanka et
al. , 2016)
OE gsdA
(G6PDH) YS7.4 AB4.1 Overex pression of An02 g12140 ( gs dA ) via
tet-on, pyrG + This ch apter
OE gndA
(6PGDH) YS9.9 AB4.1 Overexpressio n of An11g02040 ( gndA )
via tet- on, pyrG + Th is chapte r
OE icdA
(NADP-I CDH) YS10.6 AB4.1 Overexpression of An02g12430 ( icdA ) via
tet-on, pyrG + This ch apter
OE An14g00430 YS11.8 AB4 .1 Overexpression of An14g00430 via tet-on,
pyrG + This ch apter
ΔAn14g00430 YS16.1 YS11 .8 An14g00430:: hygB This chapter
OE maeA
(NADP-ME) YS12.16 AB4.1 Overexpression of An05g0093 0 ( maeA )
via tet- on, pyrG + Th is chapte r
OE An12g04590 YS13.4, AB4.1 Overexpression of An12g04590 via tet-on,
pyrG + This ch apter
OE An16g02510 YS14.4 AB4 .1 Overexpression of An16g02510 via tet-on,
pyrG + This ch apter
ΔAn16g02510 YS15.7 YS14 .4 An16g02510:: hygB This chapter
OE riaA Y S 1 8 . 2 0 A B 4 . 1
Overexpression of An16g05550 ( riaA ) via
tet-on, pyrG + This ch apter
Δ riaA MA75.2 MA70.15 Δ kusA , Δ riaA ::Ao pyrG (Kwon et
al. , 2011)
OE An02g09810 YS19 .27 AB4.1 Overexpression of An02g09810 via tet-on,
pyrG + This ch apter
Media for strain cu ltivat ion: 121°C autocl ave
1) Comp lete medium (CM) (g/L): 20 ml 50% glu cose, 20 ml 50  ASP+N, 2 ml 1 M MgSO4, 1
ml 1000  Tra ce elem ents, 1 g casa mino aci ds, 5 g yeast extract, 15 g Ag a r, pH set to 5.8
2) Minimal mediu m (MM) (g/ L): 20 m l 50% g lucose, 20 ml 50  ASP+N, 2 ml 1 M MgSO4, 1
ml 1000  Trace elements, 15 g Agar
3) MM medi um with urid ine: add 10 mM fil ter-sterilize d uri dine t o the autoclaved MM medium
4) Shake flask medium: 3% Maltose·H 2 O, 10 g try ptone, 5 g Yeast extract, 1 g KH 2 PO 4 , 0.5 g
MgSO 4 ·7H 2 O, 0.03 g ZnCl 2 , 0.02 g CaCl 2 , 0.0076 g MnSO 4 ·H 2 O, 0.3 g FeSO 4 ·7H 2 O, 3 ml
Tween 80, pH is adjusted to 5.5 b y 1 M HCl.


Ch ap te r 4 61 

Media for A. niger transformation: 121°C autoclave
1) Transfo rmation plates: 0.95 M sucrose in MM medi um (without gl u cose) with 1.2% agar
2) Top ag ar: t he same as tra nsfo rmatio n plates but w ith 0. 6% agar
3) Transformation medium with hyg romycin: add filter-sterilized 20 0  g/ml hygromycin and
500  g/ml caffei ne in t ransformation plates a s well as top agar to a voi d the growth of
background strain
4.2.2 Solutions
Solutions used to prepar e the ab ove media: (1)- (9) are subjecte d to 121°C autoclave
1) 0.9% PS: 9 g/L NaCl
2) 200 mM MES buffer (g/L): 19.52 g MES, pH se t to 5.8 by NaOH, f ilter st erili ze
3) SMC sol ution (g/L): 242.32 g sorbitol , 10 ml 5 M CaCl 2 , 1 00 ml 200 mM MES buffer
(pH=5.8), s et pH t o 5.6 by 1 M Na OH
4) TC solution (g/L): 10 ml 5 M CaCl 2 , 10 ml 1M Tri s/HCl ( pH 7.5)
5) STC solution (g /L): 242.32 g sorbitol, in 1 L TC solut ion
6) 50  ASP+N (g/ L): 297.5 g NaNO 3 , 2 6.1 g KCl, 74.8 g KH 2 PO 4 , set pH to 5.5 by 5 M KOH
7) 1000  Tr ace el ements (g/L): 10 g EDTA, 4.4 g ZnSO 4  7H 2 O, 1.01 g M nCl 2  4H 2 O, 0.32 g
CoCl 2  6H 2 O , 0 . 3 1 5 g C u S O
4  5H 2 O, 0.22 g (NH 4 ) 6 Mo 7 O 24  4H 2 O, 1.11 g CaCl 2 , 1.0 g
FeSO 4  7H 2 O, 50  ASP +N ( g/L): 297 .5 g NaN O 3 , 26.1 g KCl, 74.8 g KH 2 PO 4 , set pH to 4.0
by 1 M NaOH and 1 M HCl
8) 1 M MgSO 4 (g/L): 246 .5 g MgSO 4  7H 2 O
9) 50% glucose (g /L): 500 g glucose
10) PEG- 6000 (g/L): 7.5 g PEG-6000 in 30ml TC soluti on, prepare fr eshly before use
11) Protoplastationsoluti on : 200mglysing enzymein10ml S MCpH5.6,filtersterilize 
12) hygromycin (100 mg/ml): 1 g hygromycin in 10 ml deionized water , filtersterilize ,storeat
‐20°C
13) Caffeine (50mg/ml): 5 g caffeine in 100 ml deionized water, filter sterilize
14) Uridine (1M): 22 .4 g uridine in 100 ml deionized water , fi lters ter ilize
15) Proline (1M): 115 g pr oline in 100 ml deionized water, filter steri li ze
16) 8 M pota ssium ac etate (g/L): 3 9 .26 g potassium acetate in 50 m l deioni zed water
17) DNA e xtrac tion buffer: 2.5 m l 10% SDS solut ion, 10 m l 1 M Tris H C l ( p H 8 . 0 ) , 6 . 2 5 m l 2
M NaCl in 50 ml deionized water
18) RNase A: 100 mg RNase A i n 50 ml deionize d water, s tore at -20° C
4.2.3 Plasmids const ruction
Integrative plasmids used fo r overexpressi ng NADPH genera ting g e nes in th e A. niger strains were
cloned by Gibson assembly or AQUA cloni ng (Beyer et al. , 2015). The backbone vect or was
obt ai ned b y d ig esti ng pF W22 .1 wi th Pme I, and then the 8.1 kb fragment was p urified by gel


Ch ap te r 4 62 

extraction. Plasmi d pFW22.1 carri es the Tet-on inducible gene e xpression system, and an
unfunctional pyrG , which was used as a selective marker for transformant screeni ng (Wanka et al. ,
2016). Open re ading frames ( ORF) of NADPH generating enzy mes (6 PGDH, G6PD G, NADP-
ICDH, NADP-ME, An14g00 430, An15g04 590, An16g0 2510, An 02g09810, and riaA ) were
amplifie d by PCR, using genomic DNA of A. niger B36 strain as the templa te. PCR p roducts were
integrated into pFW22.1 at Pme I si te under the control of the Doxycycl ine-inducible tet-on
expression system. Primers carryin g overlapping regions used fo r PCR and plasmids constructed
in t his stu dy are li sted in Tabl e A.4 and Table 4.3, respective ly.
Table 4.3 Plas mids constru cted in this chapter
Plasmi d Containing gene
pYS1.4 An12g12140 （ gndA ）
pYS4.1 An11 g02040 （ gsdA ）
pYS5.1 An 02g12430 （ icdA ）
pYS6.1 A n05g0093 0 （ maeA ）
pYS7.1 A n12g045 90
pYS8.1 A n14g004 30
pYS9.1 A n16g025 10
pYS10.1 An16g 0555 0 （ riaA ）
pYS11.1 An0 2g098 10
4.2.4 Gibson cloning and AQUA cloning
Solut ions u sed in Gib son assemb ly
1) 5 х isothermal (ISO) buffer: 25% PEG-8000, 5 00 mM Tris-HCl pH 7 .5, 50 mM MgCl 2 , 50
mM DTT, 1 mM dNTPs and 5 mM NAD, store at -20  。
2) assembly master mix: 320 µl 5× IS O buffer, 0.64 µl of 10 U/µl T 5 exonuclease (Epicentre,
USA), 20 µl of 2 U/µl Phusio n DNA polymerase (New England Biola bs, UK), 160 µl o f 40
U/µl Taq DNA li gase (New Engl and Biola bs, UK) in 2ml, stor e at -20  。
Primers used for Gibson cloning and AQUA cl oning carry 1 5-20 overlappi ng base pair regions.
For Gibson assembly, 15 µl assembly master mix was i ncubated wi th 5 µl DNA for 1 hour a t 50 °C.
In general, 12 ng per kb of the vector was used. The amo unt of insert DNA fragmen ts used for the
cloning depending on the s ize. Small fragments ( <1/4 of the lar gest fragment) were added in 8 f old
excess and fragments between 1/2- 1/4 of t he largest fragment we re added in 4 fold excess. In the
case of AQUA cl oning, dei onized wat er i nstead of the Gi bson mi x wa s u sed. 1 0 µl DNA mix ture
was prepared, includin g backbone, insert fragment (molar r ation : 1:3 -1: 10) an d de ioni zed wat er.
Then inc ubated the 10 µl DNA mixtur e for 1 h at room tem peratur e.


Ch ap te r 4 63 

4.2.5 E.coli colony PCR
E. coli si ngl e c ol onies w ere rand omly pick ed wi th au tocla ved toot hpic k s . T h e n t h e y w e r e
inoculated on a new re scue plate and put into 20 μl of dei onize d water to obtain strain suspension.
2 μl of the strain suspension wa s taken as the templat e for col ony PCR. System for E. coli colony
PCR is listed as follows.
Table 4.4 System f or E. co li colony PCR




4.2.6 Construction of ΔAn14g0 043 and ΔAn1 6g02510 disruptants
upstrea m
An14 g00430/ An16g 02510
Wild type downstream
upstre a m hyg F1
downstream
hyg F2
upstrea m hyg down strea m Transgenic Strai n
hyg
Fus ion PCR
F1
upstream hyg
F2
downs t ream
+ +

Figure 4.2 An14g00430 an d An16g02510 gen e knockout cassettes a nd hom ologous r ecombina tio n str ategy.
Primer pairs 004305’_fwD/004 30_5 ’_rev and 025105’_fwD/0251 0_5’_ rev were used to amplify the upstream
flank of An14g00430 and An16g02510, respectively. In addition, primer pairs 00430_3’_fw/004303'_revD
and 02510_3’_fw/025103'_rev D were used to amplify the downstrea m flank of An14g00430 and
An16g02510, respectively. Primer pairs 442 hygP6f /443 hygP7r w ere used to am plify the hygrom ycin
fragment. Split markers were generated by Fusion PCR using the primer pairs 004 30_5’_ fw/445 hy gP9r and
02510_5’_fw/445 hygP9 r for the f ir st spilt marker and primer pa irs 44 4 hygP8f /00430 _3’_re v and 44 4
hygP8f/02510_3’_rev for the sec ond split marker. The two split m arkers (1 µg for ea ch) were t ransfo rmed into
YS11.8 (OE An14g00430 strain) or YS14.4 (OE An16g0251 0 strain). hyg re presents the hygromycin
resistance gene.
Gene deleti ons were obtained using the split marker method (Ni e ls en et al. , 200 6). PCR product s
containing either ~1.5 kb 5’ or 3’ flanking regions of the corresponding gene a nd a part of the
select ive mark er hygrom ycin were us ed for tra nsforma tion (Figur e 4.2). To avoid l ethal phenot ype,
all deletion st rains were const ructed on the background of thei r corr esponding overexpression
Template 2μl
wat e
r

17 µl
Prime r1 ( 10 μM ) 0.4 µl
Prime r2 ( 10 μM ) 0.4 µl
dNTP s 0.5 µl
10*Ta
q

Buffer 2
µ

l
Taq polymerase 0.1 µl
Total volume 2 0 µl
94 °C 3min
94 °C 20 s
30× 56 °C 20 s
72 °C 90 s
72 °C 2 min
4 °C ∞


Ch ap te r 4 64 

st ra in s. T he 5′ an d 3 ′ f la nks we re a m pl if ie d by P CR wi th th e p r imers described in Supplementary
Table A.5. Primer pairs used t o ampli fy DGI-l abelled probes use d for Southern blot analysis are
listed in Table A.3.
4.2.7 Preparation of A. ni ger spore suspension
A. niger strains were inoculated on CM plates and incu bated at 30°C for three days. Then spores
were collected by a cotton stick with 10 ml 0.9% NaCl and quan t ified by b l ood coun ting chamber.
One ml of spore suspension was mixe d with 0 .5 ml of 50% glyce ro l for storage at -80°C.
4.2.8 A. niger transfo rmation
To achieve A. ni ger m ycelium, 2.5  10 8 spores were inoculated into CM li quid medi um, 
supplemented with 1 mM uridine when necessary, and incubated at 30  C 50-80 rp m for 16-18 h.
A. niger was transformed using PEG mediat ed protoplast transformation a c cording to Arentshorst
et al. (2012). 8-10 µg plasmid DNA were used whe n using pl asmid as t h e donor DNA. When
adopting the spl it marker method for g ene deletion, 1 µg of eac h spli t mar ker (gel extra cted PCR
products) was co-transformed. For uridine prototrophy screening , pAB4.1 wit h functional uridine
was used a s a positive co ntrol. In t he case of hygro mycin selec tion, pAN7.1 was adop ted.
Transformants were subcultivated twice on selective plates and then conducted diagnostic PCR
and Southern analysis. Integration of a con struct at the py r G locus was verified by diag n ostic PCR
using primer pairs of 393 and 392 (listed in Table A.4) before Southern analysis. Integrated
transformants g ive a signal at 3 kb, while negative trans forma n ts show no si gnal.
4.2.9 Southern analysi s
Solutions used f or Southern blotti ng
1) Denaturation buf fer: 0.5 M NaOH, 1.5 M NaCl
2) Neutrali zation buffer: 1.5 M NaCl ， 0.5 M Tris , pH set t o 7.0 by HCl
3) 20 х SSC: 3 M NaCl, 300 mM sodium c itrate, pH set to 7.0 by HCl
4) Hybridizati on buffer: 1 M NaCl, 1% (w/v ) SDS, 10% dextrane sul p hat e
5) High St ringe ncy buffe r: 25 ml 20 х SSC, 0.1% SDS
6) Low Stringency buffer: 100 ml 20 х SSC, 0.1% SDS
7) Washing buffer: 0.3% Tween 20, in 1 L maleic acid buffer
8) Maleic acid buffer: 1 00 mM maleic acid, 150mM NaCl , pH set to 7 .5 by NaOH
9) 10x blocking buffer: 10% Blocking reagent (Roche), heat dissol ved in 100 ml maleic acid
buffer, then autoclave, stor e at 4 ℃ . 1x blocking buffer was diluted by malei c acid
10) Anti body solutio n: add 1 µl Anti-Dig-AP (P32 ) antibody in to 10 ml 1x blocking buffer,
prepare fr eshly
11) Detecti on buffer: 100 ml 1 M T ris-HCl (pH=9.5) , 100 mM NaCl
12) Strippingsolution : 200mMNaOH,0.1%SDS


Ch ap te r 4 65 

Southern anal ysis was performed as previou sly described in Aren tshorst et al. (20 12). System for
genomic DNA digestion i s as descri bed in Table 4.5. After diges tion for overnigh t, the restricted
DNA m ixed wi th l oading dye was app lied onto an 0.7 5% ( w/v) agar ose gel and t ransferred to a
nylon membrane. PCR system to obtain DIG-labeled probes is li st ed in Table 4.6 and primer pairs
t o a m p l i f y p r o b e s a r e l i s t e d i n T a b l e A . 3 . G e n e r a l l y , t r a n s f o r m an ts were an alyzed with at l east two
different restricti on enzymes.
Table 4.5 S ystem for g enomic DNA dig estion



Table 4.6 PCR system to obtain DIG-labeled probes

4.2.10 Analysis of broth
As described in 2.2.9.
4.2.11 NBT staining procedures
p -Nitro Blue Tetrazolium Chloride (NBT) st aining method was perf ormed to deter mine reacti ve
oxygen sp ecies (ROS) according to Kwon et al. (2011). In total 1 0 6 /ml*20 spores was i noculated
into 20 mM MM li quid medium (supplied with 0.003% yeast extract t o i nduce germination). Put
cover slides into steri le plates, then added the mixed MM mediu m i n t o i t , i n c u b a t e d a t 2 5 ° C f o r
overnight. For NBT s taining, germlings were incubated for 1 h i n 50 mM sodi um p hosphate buffer
containing 0.5 mg ml NBT (Sigma-Aldrich, USA), washed once wit h 100% methanol and once
with water and immediately subjected to microscopy.
4.2.12 qRT-PCR
Mycelium harvested for RNA extr action was ground in li quid nitr ogen and then extract ed by the
Fungal Total RNA Isolation Kit (Sangon, Sh anghai, China). About 1 μ g o f t o t a l R N A w a s u s e d
for cDNA synthesi s using the PrimeScript TM RT reag ent Kit with gDNA Eraser (Takara, Shiga,
Japan) according t o the manufac turer’s instructi ons. The real-t ime PCR reacti on system was
prepared with TB Green TM Premix Ex Taq TM II (Takara, Shiga, Japan) in a volume of 2 5 μl with
diluted c DNA as a te mplat e. Dilut ed cDNA was used to keep mea n Ct (threshol d cycles) values
Genomi c DN A 6-7 µl
wat e
r

10-11 µl
Buffe
r

2 µl
Enzyme 1 µl
Total volume 20 µ l
Genomic DNA 0.2 µl
wat e
r

18 µl
Primer 1 1 µl
Primer2 1 µl
DIG dNTPs 2.5 µl
10*Taq Buffer 2.5 µl
Taq polymera se 0.25 µl
Total volume 25 µ l


Ch ap te r 4 66 

between 20 and 30. Each reaction was c arried out in triplicates . Oli gonucleotide primers used for
qPCR are listed in Table A.6. The 28S rRNA and 18S r RNA were us ed as t he internal standard.
PCR cond itions were as follows: 95 °C for 3 min, followed by su bsequent 40 cycl es of the t hree-
steps: 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s.
4.3 Result and discussion
4.3.1 Construction of integ rati ve p lasmids used for gene overe xpression
gs dA
Pme I Pme I
2.4 kb
pYS 4.1
gn dA
Pme I Pme I
2.1 kb
pYS1 .4
ic d A
Pme I Pme I
1.9 kb
pYS5.1
mae A
Pm e I Pme I
2.2 kb
pYS6 .1
An12 g04 590
Pm e I Pme I
0.9 kb
pYS 7.1
An14 g00 430
Pm e I Pme I
1.1 kb
pYS8 .1
An16 g02 510
Pm e I Pme I
1.2 kb
pYS 9.1
ri aA
Pm e I Pme I
1.8 kb
pYS10. 1
An02 g09 810
Pm e I Pme I
4.2 kb
pYS11.1
Pme I
pFW22 .1
10029 bp
AmyR
An16 g 04 690
rt TA2 S-M 2
T cg r A
Pt etO7
Pmin_ gpdA
Lu c
T tr pC
py r G *
Pm e I
Pm e I
pFW22.1- gsdA
10511 bp
Am yR
An 16g 04 690
rt TA2 S-M2
T cg r A
Ptet O 7
Pmin _ gp dA
gs dA
T trpC
pyr G*
Pm e I
Pm e I

Figure 4.3 Plasmids con struction strat egy used for gene ov erex pression. Vector pFW 22.1 was us ed as an entr y
vecto r, digested wi th Pme I. Genes of inter est were amplif ied with primers contain ing ove rla pping r egion s to
the plasmid backbone
The ORF regions of above i nterested genes were indi vidually ins erted i nto the plasmid pFW22.1
(containing the Tet-on i nducible gene switch) at Pme I site. Tet -on gene swi tch is indu cible b y the
additi on of doxyc ycline (DOX) t o t he cult ure m edium, i s t ight i n the absence of DOX and
metabolism-independent in A. niger ( M e y e r et al. , 2 011). It has furthermore been shown to
strongly induce gene expression up to levels above the glucoamy lase ge ne, whi ch is o ne of the
highest expressed genes in A. niger (Mey er et al. , 2011; Wanka et al. , 2016; Sc häpe et al . , 2019).


Ch ap te r 4 67 

Plasmid constructi on strategies are shown in Figure 4.3. Aft er ve rified by E.coli colony PCR,
plasmids were extr acted for enzym e restriction, then su bjected to sequencing. Single enzyme
restriction results for the v alidation of c loned plasmids ar e shown in Fi gure D.1, a nd all expected
plasmids have b een successfully c onstructed.
4.3.2 Characte rizing the effect of NADPH engi neering on enzyme production in the low-
yield Gl aA producer
4.3.2.1 Construction of NADPH generating enzymes engineered A. niger strai ns
The above cloned plasmids were individually transformed into th e A. nige r AB4.1 based o n the
PEG medi ated protoplast transformation as depicted in 4.2.8. St r a i n A B 4 . 1 i s u r i d i n e - a u x o t r o p h
due to a def ective pyrG * gene carrying a fra me shift muta tion ( py rG 378 ) (Arentshorst et al. , 2015).
Introduction o f an unfunctional pyrG ( pyrG Ba m HI ) on the plasmid pFW22.1 at pyrG * locus occu rs
efficiently and confers uridine prototrophy in A. ni ger (van Harti ngsveldt et al. , 1987) (Fi gure 4.4).
Southern analysis manifested that all engineered st rains on the AB4.1 background in case o f single
integrat ion have been achieved (Figure C.3 and Table 4.6).
R eci pi ent str ain
( pyrG -) pyrG *
Transgenic
strai n
An16g0 4690 Tcg rA Pm in_ gpdA
OE gen e py rG * py rG
rtT A2 S- M2
T trpC
Singl e-cros sover

Figure 4.4 Genotype of pyrG in the transg enic strain after integ rated with th e cloned plas mid
Unlike the typical four NADPH generating enzymes located on the central m etabolic network, the
effect of An12g04590, An14g004 30 (homologous to A. nidulans ha dA ) , and An 16g02510
(homologo us to S. cerevisiae adh7 ) on t he NADPH ava ilabili ty has not bee n char acterized i n A.
niger . We finally also decided to d elete the nati ve ORFs of An14g004 30 and An16g02510 in their
respective Tet-on driven overexpression strains in order t o ana lyze their deletion phenotypes in the
absence of DOX (Figure 4.2). However, on ly An12g0459 0 nu ll st ra ins failed to construct even
after anal yzed 40 transformants. Considering its role as a mito chondrial NADPH provider, we
excluded the focus on it. An14g0 0430 functions as the  β-hydro xybutyryl-coenzyme A (CoA)
dehydrogenase (BHBD) involv ed i n but yryl-CoA synthesis, cat alyz ing (s)-3-hydroxybutanoyl-


Ch ap te r 4 68 

CoA + NADP (+) = 3-acetoacetyl-CoA + NADPH. An16g02510 ,  an alcohol dehydrogenase,
catalyses the reaction alcohol + NADP + = a ldehyde + NADPH. Based on gene annot ation,
An16g02510 is more likely to act a s an ald ehyde reductase than as al cohol dehydrogenase.
Southern analysis of Δ An14g00430 and ΔAn16g02510 d isruptan ts are shown in Figure C.4.
4.3.2.2 Weak impac t of NADPH perturba tion on enzyme for mation in the low-yield GlaA
producer
All engineered strains were subjec ted to batch cultivat ions in shake-flask format, where by a
medium containing maltose as GlaA-inducing carbon source was us ed. FW35.1 (a pyrG +
derivative of AB4.1) was taken along as reference strain. Induc t i o n w a s s t a r t e d a t t h e e a r l y
exponential phase about 18 h after inoculation by add ing 20 µg/ ml DOX. Due t o th e light
sensitivity of DOX, it was refilled every 12 hours. Samples were taken at 24, 4 8, and 72 h after
inoculati on. Physi ological parameters (dry w eight, tota l se cret ed pr otein, r esidu al gl ucose , a nd
e n z y m e a c t i v i t y o f G l a A i n t h e b r o t h ) w e r e m e a s u r e d . S a m p l e s f o r NADPH measurem ent a nd
qRT-PCR were taken in the exponentia l phase. Experi ments were p erf ormed in biological
quadrupli cates.
As depicted i n Figure 4.5A, modif ied strains h ad near ly n o chan ged bio mass g rowth com pared to
the control strain FW35.1 on the flask-level. DOX-induced gene expression in all eight AB4.1
derivatives was about 1.5-2.7 times higher compared to the refe rence strain FW35.1 as examined
by qRT-PCR (Figure 4.5B), in whi ch maeA showed the highest overexpression level. However,
the im plement ation of dive rse NADPH regulation strate gies did n ot induce the transcription of
glaA, and ove rexpre ssion of NADP- ME even repr essed the e xpressio n of gl aA . Overexpression or
deletion of An16g02510 both notably i nhibited the gla A, b u t i t k e e p s un k n ow n a b o u t t h e r e as o n .
Although this le d to an elevat ed NADPH pool of about 30 % when gndA , icdA or An16g02510
were overexpressed, no significan t increase in GlaA enzyme acti vity was observed for all of the
seven strains compared t o the F W 35.1 reference (Figure 4.5 C, D , E). The best performance of
protein bio synthesis was observed in the eng ineering of icdA conditi on, where the yield of t otal
secreted protein a nd GlaA were increased by 5.9 % and 7.52%, res pectively. H owever, in line with
the transcri ptional alteration of glaA , overprod ucti on of mae A r educed th e yield of GlaA by 5.57%.
NADPH engineering displayed an unex pected mild influence on pro tein ov erproduction in this
weak GlaA pr oducer. The logical explanations could be as follow s. For the nativ e GlaA producing
strain AB4.1, which carries only one glaA copy, we a ssume th at the e xcess of NADPH might not
be allocated to increased ami no acid biosynt hesis but was chann el ed to other NADPH c onsumpt ion
pathways, e.g. for the produ ction of steroids, lipids an d nu cle otides and thus biomass formatio n.
Future 13 C metabolic flux analyses targeting sugars, amin o acids, steroids, li pids and nucleotides
in th ese strains will prove or d isprove this hypothesis. These analyses will also clarify whether


C hap t er 4 69


D
0
0.5
1
1.5
2
2.5
3
3.5 glaA engi neered gen e
Relative gene
express ion lev el
***
***
***
***
***
***
***
***
**
**
**
*
N A D P H s u p p l y i s a b o t t l e n e c k f o r p r o t e i n b i o s y n t h e s i s a s p r e v i o usly proposed for the yeast Y.
lipolytica (Zhang et al. , 2014).

Figur e 4.5 Flask-l evel ferme nta tion results of engineered stra ins in the background of AB4.1. A. Dry cell
weight, the addition of doxycyclin e and samples taken for RNA i solatio n and NADPH determination are
indicate d with a purple arrow. The right plot presents m ore det ails of the strain gr owt h from 16 h to 42 h; B.
Relative gene express ion level of glaA and the engineered genes in com p arison to the control strain F W35.1;
C. Total secreted protein per gram biom ass at 72 h after inoc ul ation; D. Enzyme activit y of Gl aA per gram
biomass at 72 h aft e r inoculatio n; E. Intracellular NA DPH level in the exponential phase. All exp eriments were
conducted in biological quadrupl icates . Total sec reted protei n , enz yme acti vity of GlaA, a nd NADP H were
normal ized by dr y cel l wei ght ( DCW). Sign ifica nce v alue s were c alculated with the two-tailed t -test with
independent v ariables (* p <0 .05, ** p <0.01, * ** p <0.001). FW35.1 is the control . OE represents overexp ression
B C
E
0
5
10
15
20
0
5
10
15
20
0 1 02 03 04 05 0
Resi dua l gl uco se (g/l )
Dry weigh t (g/kg)
Time (h)
Dr y w e ig h t (g /k g) Res idual glu cose (g/L)
0
5
10
15
20
25
0 2 04 06 08 0
D r y w e i g h t ( g/ kg)
Tim e (h )
FW35.1 OE gs d A OE g ndA
OE ic d A OE m ae A O E A n 14g0043 0
ΔAn14g00430 OE A n 16g02510 ΔAn16g02510
Dox
RNA/NADPH
Dox
RNA/NADPH
0
0.0 5
0.1
0.1 5
0.2
0.2 5
0.3
NADPH (µm ol/g·DCW)
*
A
OE
g

sdA OE
g

ndA
OE maeA
OE icdA
OE An16g02510
OE An14g004 30
Dry cell weight
（ gDCW/kg ）


C hap t er 4 70


4.3.3 Explora tion of the functi on of NADPH oxidase re gulator R ia A on protein format ion
in
A. nige r

Due t o the weak eff ect of abov e NADPH genera tion s ystems, we a t tempt to focus on al ternative
reported NADPH regulators to uncove r their role in protein accu mulation in A. ngier . NADPH
oxidase r egulator riaA ( noxR , An16g05550) is the Rho GTPases RacA-speci fic downstream
effector, which oxid izes NADPH to generate ROS (React ive ox ygen species) at hyphal apices and
is also involved i n hyphal tip extension as well as ( a)sexual r eprodu ction (Kwon et al. , 2011 ).
Additionall y, knockout riaA is also assumed to prof it the cellulase production, as riaA remark ably
down-regulated d uring cellulase overproduction in A. nige r (Patyshakuliyeva et al. , 2 016).
However, the speci fic molecular mechanism of RiaA in cellulase prod uction has not been well
defined. It is thus speculated that the loss of riaA ma y s upport t o maintain int racellula r NADP H
level, so as t o m eet the h i gh requirement of NADPH for cel lulos e biosynthesis. To investiga te this
hypothesis, we took th e p reviously established riaA null strai n MA75.2 (Kwo n et al. , 2011 ) and
the riaA overexpression st rain constru cted in this study as t argets, in order to observe t heir
behaviour on the NADPH ox idizat ion and protein production.
4.3.3.1 Phenotype of riaA o verexpression and knockout s trains and NBT staining
RiaA overexpressio n strain was identified by Sout hern analysis (Figure C.5) . T o perform the
phenotypic anal ysis of engineered strains, a series of dil uted spore suspension (10
7
-10
3
/ml) of the
control and en gineered strains were inoculated on MM and MM wi t h DOX plat es (Figur e 4.6) .
There is no phenotypic difference between OE riaA and the control, but Δ riaA is acco mpanied by
a reduced sporulation, suggesting th at riaA is i nvolved in the asex ual r eproduction as previou sly
depicted (Kwon et al. , 201 1) .





Figure 4.6 Dot-in oculation of FW35.1 (control), Δ riaA and OE riaA on MM and MM+DOX plates. 5 μl of a
series of a ten-fold dilution started with 10
7
conidia per ml were p oint-in oculated on MM and MM+DOX
plates and inc ubate d at 30° C for 3 da ys
R O S i s a k e y p l a y e r i n t h e r e g u l a t i o n o f c e l l d i f f e r e n t i a t i o n a s well as fungal growth (Cano-
Dominguez et al. , 2 008). p-Nitr o Blue Tetrazolium Chloride (NBT) was u sed t o ch aracteriz e ROS
abundance at tips of hyphae under Leica d igital microscope. As shown in Figure 4.7,  the blank d id
 
5*10
4
5*1 0
3
5*10
2
5*10 5

Control

MA75.2 Δ
riaA

YS18.2 0 OE
riaA

MM MM+DOX

5*10
4
5*10
3
5*10
2
5*10 5


C hap t er 4
71


0
0.5
1
1.5
FW 35.1 Δri a A OEr i aA
gl a A riaA
12
14
16
18
Relative ge ne
expression level
FW35 .1 Δ ri aA OE riaA
***
***
glaA riaA
not disp lay any blue

fluorescence as expected. The blu e fl uorescence intensity f or t he Δ
riaA
s t r a i n
was signifi cantly lower than that of the control str ain, demons trating that the elimination of
riaA
could reduce the accumu lation of ROS by su ppressing the oxi diza tion a cti vity for NADP H.
Nevertheless, we could not o bserve distinct fluor escence intens ity bet ween OE
riaA
a nd th e w il d
type, which might be relevant to the cellular regulation for RO S to maintain the intracellular redox
balance (O’Donnell
et al.
, 2011).

Figure 4.7 p-Nitr o Bl ue Tetrazolium Chlori de (NBT) staining fo r the con trol strain, Δ riaA and OE ri aA
4.3.3.2 Complex regulatory mechanism of
riaA
on protein biosynthesis

Figure 4.8 F lask-level fermentati o n of Δ riaA and OE riaA strains. (A ) Dry cell weight (DCW) ; (B) Rel ative
expression level of gl aA and ri aA ; (C) Total secret ed protein at 72 h after ino culation; (D) Enz yme activity of
glucoamylase at 72 h after inocu lation; (E) Intracellula r NADPH level in the middle of the exponential p hase.
All expe riments were c onducted in biolo g ical quadru plicates. To tal sec reted pr otein, enzyme a ctivity of GlaA,
Blank Control Δ
riaA
OE
riaA

0
5
10
15
20
25
30
24 h 48 h 72 h
D r y w e i g h t ( g/ k g )
FW35.1 OE r i aA Δ ri a A
A B
C D E
0
0.05
0.1
0.15
0.2
0.25
0.3
F W 35 .1 M A 7 5 .2 YS18 .20
NADPH (µm o l/gDCW)
FW35.1 Δ riaA OE riaA
OE riaA Δ riaA
Dry cell weig ht
（

g

DCW/ k
g

）


Ch ap te r 4 72 

and N ADPH were norma lized by dry cell weight (D CW). Signif icanc e val u es were calc ulated wit h the two-
tailed t -test w ith independent var iables (* p <0.05, ** p <0.01, *** p <0.001). O E represents overexpress ion
This st udy employed the same fl ask-level culture st rategy as de scribed in 4 .3.2.2 to uncover the
impact of riaA on the intr acellular NADP H pool . Ce ll gro wth was n ot influenc e d by the g enetic
perturba tion (Figure 4.8 A). Knockout of NADP H oxi dase regula to r o nly mildly up-regulated the
transc ription level of glaA for 1.2 folds. However, DOX-induced g ene expression o f riaA w a s
strikingly improved for 14 folds, accompanied by severe t ranscr iptional inhibition of glaA f o r 3
folds (Figure 4.8 B). This indica tes the inhibit ion role of riaA gen e prod uct i n glaA tra nsc ript io n.
The alterat ion of glaA o n the transc ription lev el co rre lated well with the c hanged pr otein capacity
in e ngineered strains and t he parental s train. As shown in Figu re 4.8 E, Δ ri aA null strain enhanced
the NADPH po ol by 34% compa red to t he refer ence stra in, while O E riaA onl y displaye d a slightly
reduced NADPH a bundance for abo ut 12%. Ho wever, consi stent wi th the unchan ged protein
performance re gulated by othe r NADPH gener ation sy stems in AB4. 1, the enzyme acti vity of
GlaA was only elevated by 8.22% in Δ riaA . Here may und erpin t he two speculations in th e section
4.3.2.2 as well. I n ad dition, the delicat e metabolic regulati on in filamentous f ungi may also limit
the efforts of gene knockout . Nevertheless, the prot ein b iosynt hesis was noticeably suppressed in
OE riaA , wh ere the yield of total s ecreted protein and the enzyme acti vity of GlaA were dropp ed
by 5 4.15% and 71.06%, respectively (Figur e 4.8 C, D). The a bove evi denc e impli es that riaA may
negati vely r egulate the secr eted protein accum ulation from mu lt iple dimen sions, in part icular at
the transcriptional level.
Table 4 .7 GO enrichment analysis for co- expression ne twork of RiaA encoding gene
GO ID Name Count in
th is G O term FDR
GO:0006412 translation 142 2.41E-63
GO:0034641 cellular nitrogen com pound metabolic process 504 1.23 E-51
GO:0042254 ribo some biogenesis 120 2.27E-48
GO:0006399 tRNA metabolic process 67 4.66E-19
GO:0007005 mitochondrion org anization 68 7.34E-17
GO:0006520 cellular amino acid metabolic process 85 6.67E-13
GO:0065003 protein-containing complex assembly 84 1.99E-09
GO:0006605 protein targeting 41 1.02E-07
GO:0 006 457 prote in fol din g 24 9.03E- 06
GO:0009056 catabolic process 143 5.30E-05
GO:0006810 transport 261 0.0005 41068
GO:0006629 lipid metabolic process 98 0.000541068
GO:0006091 generation of precursor metabolites and energy 2 6 0.0 08789279
GO:0051186 cofactor metabolic process 52 0.011151314
Co-expression networ k analysi s (Table 4.7) revealed th at RiaA i s i nvolved in compl ex m etaboli c
regulations. RiaA showed a positive correlati on with 78 genes b ut negatively correlates with a
surprisingly high number of g enes ( 2446), indicating t he role o f RiaA as a versatile transcrip tion


Ch ap te r 4 73 

repressor. RiaA not on ly participates in energy metabolism path w a y s s u c h a s c o f a c t o r m e t a b o l i c
process but also i nhibits protein biosynthesis and transport, f or i nstance, translati on, cellular amino
acid metabolic process, protein folding and transport ation. Ov e rall, co-expression network
analysis of RiaA suppor ted our hypothesis that the severel y inh i b i t e d p r o t e i n f o r m a t i o n i n O E riaA
was a ttri buted to the NADPH o xidat ion in parall el wit h the in hi bitory role of Ria A in protein
transcripti on and secret ion proc ess.
4.3.4 Explorati on of the func tion of the NADP+ de pendent trans hydrogenase oxidase in A.
niger
I r r e s p e c t i v e o f t h e k e y r o l e o f t h e P P P o n N A D P H r e g e n e r a t i o n , an efficient carbon economy i s
only guaranteed w hen the carbon flux ent ers the glycolytic path way (Embden-Meye rhoff-Parnass
pathway, E MP) instead of the PPP, because the PPP releases one carbon a s CO 2 when oxidizing 1
mole of hexose. NAD ( P)+-dependen t tr anshydrogen ase driv es the reducti on of NADP+ from
NADH thro ugh th e electrochemical pr oton gradien t across the cyt oplasmic membrane ( Kabus et
al. , 2007), without causing the loss of carbo n fl ow. This pat hway behaves as a major s ource of
NADPH in E. coli, con tributing to 35–45% of the NADPH required for anabolism, whi ch is
comparable to that produced by the PPP (Sauer et al. , 2004) . E. coli contains two transhydro genase
isoforms, showing differen t physiological functions: PntA (Pnt) i s a membranebound and proton-
translocating transh ydrogenase, an d UdhA is a soluble and energ y-i ndependent transhydrogenase
found in some h eterotrophi c bacteri a ( Jan et al . , 2013). UdhA was reported to promote th e yield
of pr oducts, whil e PntAB functioned reverse ly (Sanchez et a l . , 2010; Jan et al. , 2013). In contrast,
Yin and Xu heterologously expresse d pntAB in C. glutamicum , which effectiv ely impr oved the
intracellular NADPH pool and the y ield of proline and lysine, i n parallel (Yin et al. , 2014b; Xu et
al. , 2015). Based on the curre nt annotati on database of A. niger , onl y one homolog of E. coli
NADP+ dependen t transhydrogenase (An02g09810 ) was identified in A. niger , cat alyzi ng t he
cytoplasmic reactio n nadp + nadh => nadph + n ad. An02g098 10 i s the homo log of t he proton-
translocating transh ydrogenase PntAB, con taining a conser ved PN TB superfamily domain. Since
this transhydrogenase has not been wel l characterized in Aspergilli , its r ole as an NADPH supplier
in A. niger remains unclear. To test the above hyp othesis, Pnt overexpressi on st rai n was
const r uc ted in AB.1 . P ositiv e tr ansformants were identified by Southern analysis (Figure C.6).
Due to the inoculati on error, the biomass of OE An02g09 810 at 2 4 h was lower than the control
(Figure 4.9 A). DOX-initi ation drove an increase of 1 3 fol ds fo r the transcript level of An02g0 9810
in OE An 02g09810 abo ve that in B36. Inter estingly, it can be no ted that the transcriptional strength
of either riaA or An02g09810 gene that not locates o n the central metabol ic n e t w o r k c o u l d b e
strikingly i nduced by the tet-on switch. However, even induced under the same geneti c control,
expression strength of those engineered genes on the central ne twork was far less notable than riaA
or An02g09810, suggesti ng the r obust regulati on of core carbon metabolism.


C hap t er 4
74


FW35.1 OE
An02g098 10
FW35. 1 O E
An02g09810
The tran scription of
glaA
was sl ightly inhibited, which is inconsi stent with the protein performance
(Figure 4.9 B). NADPH pool wa s extended by 33% after the ov erex pression of An02g09810
(Figure 4.9 E), accompanied by a rise of the yield of total sec reted protein and GlaA b y 26 .45%
and 23.45%, respectively (Figure 4.9 C, D).
pntAB
functions in a reversible reaction, whose
direction is controlled by culture conditions, environmental di sturbances a nd g enotypes (Jan
et al.
,
2013). Hence, An02g09810 may behave as an NADP+- dependent trans hydrogenase to con vert the
reaction from NADH as t he hydro ge n donor to produce NADPH in AB 4.1. This thesis provides a
possibility that An02g09810 may point i n a similar regulatory r ole for NADPH regeneration as
PntA in
E. coli
. However, it s clear regulation picture i n
A. niger
re quires further cha racterizati on
in o ther ap prop riate hos ts.

Figure 4.9 F lask-level cultur es of NADP- transhydro genase over expression is olate. (A) Dry cell weight; (B)
Relative expression level of glaA and An 02g09810; (C) Total secret ed p rotein after 72 h of inocu lation; (D)
Enzy me activ ity of gl ucoamyl ase after 72 h of ino culation; (E) Intracellul ar NADPH level. All e xperiments
were conducted in biological quadr uplicates. Total secreted pro tein, e nzym e activ ity of G laA, and NADPH
were nor maliz ed b y dry cell weig h t (DCW). S ignificanc e values w ere calculat ed with t he t wo-tailed t -t est with
independent variables (* p < 0.05, ** p <0.01, *** p <0. 001) . OE re presents overexpr essi on

A B
C D E
0
2
4
6
8
10
12
14
24 h 48 h 72 h
Dry w e ight (g/k g )
FW35.1 OE An02g09 810
0
0.5
1
12
14
16
glaA An02g098 10
Relative gene
expression level
***
0
0.1
0.2
0.3
0.4
NADPH (µ mol/g·DCW)
FW35.1 O E
An 02g09810
FW35.1 OE
An02g09 810
Dry cell weight
（

g

DCW/k
g

）


Ch ap te r 4 75 

4.4 Conclusi on
In or der t o break t he bottleneck of en zyme overproduction cause d by the lo w NADPH ava ilabi lity
in A. nig er , this study performed the NADPH eng ineering in a low-yield Gla A producer AB4 .1, t o
evaluate the pe rformance of ni ne NADP H genera tion systems predi ct ed by GSMM . Diverse
NADPH regul ation strat egies sligh tly induced the accumulati on o f NADP H poo l but showed
nearly no support for the protein producti on. This could be exp lained by the following reasons.
Since AB4.1 is not an opti mal cell factory for pro tein overprod uction, the accumulated NADPH
may be all ocated to other NADPH competitive pathways, such as b iomass gr owth, ster oids, and
lipids format ion. Besides, NADPH supply coul d not be the bottle n eck for GlaA biosynthesis due
to the l ow NADPH requirem ent in AB4.1 (Zhang et al. , 2014). Furthermore, this study u ncovered
the comp lex inhibition role of NAD PH oxidase regul ator RiaA in protein biosynthesis in A. niger .
We also obser ved that NADP+-depen dent tra nshydrogenase could be a putative alternative target
for t he cofactor engineering in A. niger, beside s the well-defi ned t argets 6PGDH, G6PDH, NADP-
ICDH a nd NADP-ME. Ove rall, the low-yie ld GlaA h ost strain AB4.1 m i g h t n o t b e t h e a p p r o p r i a t e
predictor to highlight the impact of c ofactor engineering in A. niger , since the no altered protein
capacity after genetic perturbation. In t he subsequent studies, a hi gh-yield GlaA producing
platform B3 6 wi ll be targeted as a new host to apply t he identi cal NADPH re gulat ion st rategies as
in t his chapter, t o obser ve wh ether the NADPH p erturbation migh t play a more effective role under
a higher intracell ular pull towards GlaA produc tion.


Ch ap te r 5 76 

Chapter 5  Construction of a u ridine auxotrophic mutant of a high-yield Gl aA
producing Aspergillus niger strain B36 via CRISPR/Cas9 gene e diting
5.1 Introduction
In order t o comp are the effect of the seven selected genes on G laA production in an A. ni ge r s t r a i n
carrying one gl aA (AB4.1) or seven glaA (B3 6) gene copies, we first had to ensure that the
introduced genetic modifications woul d allow us to directly com pare the observed phenotypes.
This required that the i ntroduced genes would be under t he same genetic control and furthermore
introduced at t he same genomic l ocus in both recipi ent strains. As depicted a bove, all candi date
genes were overexpres sed under the control of the synthetic Tet -on gene swi tch a nd integrated at
the defective pyrG locu s in the native uri dine-auxotrophic AB4.1. However, strain B3 6 d o e s c a r r y
an intact pyrG gene (Verdoe s et al. , 1993). We thus fi rst mutated the pyrG locus in this strain in
order to apply the same g ene targeting strategy.
PyrG is a wi ldly used counterselected marker in Aspergilli, a nd it s auxotrophic strain ( pyrG — ) is
the foundation for the uridine prototrophy based transformants s c r e e n i n g , w h i c h h a s b e e n
successfully a pplied in a wide range of fung i (Du et al. , 2014). The last two steps in the
biosynthesis of uridine a re cataly zed by orotate phosphoribosyl t r a n s f e r a s e ( E C 2 . 4 . 2 . 1 0 ; O P R T a s e )
and orotidine-5’-phosphate decarboxylase enco ded by pyrG gene (EC 4.1.1.23; OMPd ecase).
Mutations i n either OPRTase or OMPdecase result in uridine auxo troph y and the resistance to 5-
FOA.  Antimetabolite 5-fluoroorotic ac id (5-FOA), an analogue of uri d ine, is not toxic. Whil e it
can be converted to the toxic 5-fluorouracil nucleoti de (5-FUMP ) under the catalysis of OPRTase
and OMPdecase to block the biosy nthesis of thymidylate dTMP, l e ading to a severe inhibiti on on
biomass growth through disruptin g the RNA structure. However, o nly those 5-FOA-resistant
mutants keeping the unmutate d OPRTase could be used as pyrG — chassis cells, w hich i nduces the
difficulty for pyrG — mutant selection (Du et al. , 20 14). 5-FOA was employed to i solate pyr G
disruptants through a seaml ess ge ne deletio n approach without i n troducing additional selective
markers. Furthermore, count erselection o f pyrG under the pressure of 5 -FOA allows t he reuse of
t h i s s e l e c t i v e m a r k e r f o r s e q u e n tial gene knockout in fil amento us fungi (Tadashi et al . , 20 08;
Kaneko et al. , 2009).
It has b een r eporte d tha t in vivo c ons titutive ly exp ressed Cas9 and sgRNA allows high efficiency
of genome editing, but a higher probability o f off-target effec ts as well (Shi et al. , 2017).
Nevert heless, transient ex pressi on of Cas9 an d sgRNA is suffici ent to mediate e ffi cient gene
manipulation with lo w o ff-target p ossibility ( Cao et al. , 2016), which coul d be ob tained by i n vitro
purified Cas9 protein and sg RNA (Pohl et al. , 2 016), by t ransient expressio n plasmid AMA1
vector (Zhang et al. , 2016), or by the inducible tet-on syst em (Weber et al. , 2017). Production
strains carrying resistance marker are inhibited in i ndustrial applications. Thus, the self-replicating


Ch ap te r 5 77 

plasmid containing the AMA1 seque nce (autonomous maintenance in Aspergil lus ) could be an
extraordinary tool for the mar ker-free gene- editing in industri al pro ducing strains (Verho et al. ,
2003; Nodvig et al. , 2015; Zhang et al. , 2016). Additionally, in vitr o transcribed gRNA avoids
utilizing endogenous U6 p romoter, without cloning efforts, thus e nables to accelerat e the
application of CRI SPR/Cas9 medi ated gene manipulatio n in fi lame ntous fungi (Liu et al. , 2015;
Pohl et al. , 2016; Zhang et al . , 2016; Zheng et al. , 2017). Based on the a bove merits of in vitro
CRISPR/C as9 system, we decided to edit the pyrG gene in B36 by foll owing a CRISPR strategy
that employed ribonucleoprotein parti cles. This method was fi rs t published for t he penicillin
producer Penicillium chrysogenum ( P oh l et al. , 20 16) and has l ater been successfully establi shed
in other fungal cell factories (Kwon et al. , 2019).
5.2 Materials a nd methods
5.2.1 Strains and media
Table 5.1 A. n iger strain s used in t his c hapter
Strain name Backgr ound strain Re leva nt ge notype/de scrip ti on Ref eren ces
N402 W ild typ e
MA169.4 AB4.1 cspA 1-, kusA :: DR- amdS -DR, pyrG −( C a r v a l h o et al. , 2010a)
B36 N402 W ith multi copies of glaA
，
amdS+ (V erdoes et al. , 1993)
MM and CM medium (as depicted in 4.2.1) were used to culture B3 6 in t his chapter.
Media for A. niger transformation: 121°C autoclave
1) Transformation medium with hygr omycin: please refer t o 4.2.1
2) Transformation medium with 5-FOA: add f ilter-sterilized 0.75 g/ L 5-FOA, 10 mM uridine,
and 10 mM prolin e in both autoclave d trans formation plates an d top agar
Subculture media: 121°C auto clave
1) MM medium with hy gromycin : please refer to 4.2.1
2) MM medium with uri dine: add 10 mM fi lter-sterilized uridine in autoclaved MM medium
3) M M m e d i u m w i t h 5 - F O A : f i l t e r - s t e r i l i z e d 0 . 7 5 g / L 5 - F O A , 1 0 m M u ridine, and 10 mM proline
in autoclaved MM medium
Prepare 5-FOA sol ution: 0.75 g 5- FOA was d issolved in 100 m l de ioniz ed wa ter, he ated at 50-
60°C a nd stirred until dissolved. Then immediately added the di ssolved 5-FOA solution i nto 1 L
transformation m edium or subculture medium a fter filt er s terili ze, otherwise 5-FOA may
precipitate when cool down.
5.2.2 Homologous recombination strategy to construct t he uridi ne auxotroph of B36
Primers used to construct disrup tion cassettes are listed in Ta ble A.7.
1) The first homolog ous recombination strategy: MA169.4 carrying u nfunctional pyrG * was used
as a template. Primer p airs 393/514 were used to amply the 2.3 kb fragment containing the
promoter and ORF region of pyrG* , and 542 /514 were used to ob tain the 1 kb ORF region of


Ch ap te r 5 78 

pyrG* . 1-2 µg of each fragment was i ndividually t ransformed int o B36 through PEP-mediated
protoplast transformation.
2) The second homologous recombination strateg y: Also taking MA169 .4 as the template, primer
pair 1 339/1340 were used to amp lify the 2.2 kb fragment F1 cont aining t he promoter, ORF
and terminator region of unfunctional pyrG* , whic h was inserted int o pAN7.1 at Xba I site by
Gibson assembly t o obtain pl asmid pYS2 .1. Primer pair 1337/1 338 were used to ampl ify t he
1.5 kb fragment F2 containing the ORF and termin ator region of functional pyrG , which was
inserted into pAN7.1 at Nhe I locu s by Gi bson assembl y to obtain plasmid pYS3.1. 442/443
were used t o amplif y hygromy cin fragment from pAN7.1 . Gene deletions were obtained using
the split marker method. Then spli t marker 1 (including F1 an d a part of the select ive marke r)
and 2 (including F2 and an other part of the select ive marker) w er e obtain ed by f usion PCR
through primer pairs 444/1340 and 1334/4 45, respecti vely, and 1 -2 µ g of t hem were
cotransformed into B36. Diagnostic PCR and So uthern analysis we re a dopted to identify
positive transfor mants.
5.2.3 A. niger PEG-me diated protopl ast transformation
As described in 4.2.7
5.2.4 Biosynthesis and in vitro purification of Cas9 protein
5.2.4.1 Plasmid and media
Plasmid pET28aCas9cys (Addg ene: 53261) was used to express Cas9 protein in E. coli.
2× TY medium (g/L ): 16 g T rypton, 1 0 g Y east extrac t, 5 g N aCl, 121°C autoclav e.
Single colony or glycerol stock was inoculated i nto 2×TY m edium (containing 50 µ g/ml
Kanamycin) and incu bated at 37°C, 200 rpm overnight
5.2.4.2 Solutions used for protein purif ication
1) SDS-PAGE gel:
Table 5.2 SDS-PA GE g el
8% separation gel V olume (5ml) V olume (1ml)
30% Acr -Bis ( 29:1) 1.3 0.17
1M T ris-HCl (pH 8.8) 1.9 0.13
10% SDS 0.05 0.01
10% Ammonium
persul fate 0. 05 0.01
ddH 2 O 1.7 0.68
TEMED 0 .003 0.001
2) Binding buff er: 5 mM imidazole, 50 mM NaPO 4 buffer, 0.3 M NaCl, p H=7
3) Ni-NTA protein pu r ific ation :
Washing buffer: 20 mM imidazole, 50 mM NaPO 4 buffer, 0.3 M NaCl, pH=7


Ch ap te r 5 79 

Elution buffer: 250 mM imidazole, 50 mM NaPO 4 buffer, 0.3 M NaCl, p H=7
4) FPLC protei n purification:
Buffer 1: 50 mM Tris-HCl, 0.3 M NaCl, pH=8
Buffer 2: 50 mM Tris-HCl, 0.3 M NaCl, 50 mM imidazole, p H= 8
Elution buffer : 6 2.5 mM imidazole, 125 mM im idazole, 312.5 mM i mi dazole, 500 mM
imidazole
5) Protein st orage bu ffer: 50 mM sodium phosphate buffer pH 7, 200 mM KC l, 0. 1 mM E DT A,
1 mM DTT, 0.5 mM PMSF, 20% glycerol
6) activity buffer: 4.77 g HE PES, 1 .12 g KCl, 0.077 g DTT, 0 .029 g E DTA, 2.03 g M gCl 2 ∙7 H 2 O,
mix everything except DTT in 800 ml H2O, adjust pH to 7.5 using KOH, a dd DTT, adj ust to
1 L and filter sterilize
5.2.4.3 E. coli main cu lture
Measured t he OD600 of the overnight ( O/N) culture from 5.2.4.1 and inoculated it into 20 ml
2×TY wi th 50 µ g/ml Kanamycin in a 100 ml f lask to a final OD600 of 0.05, at 3 7°C, 250 rpm.
When OD600 reached 0.4-0.6, added 4 mM IPTG to in duce the expre s s i o n o f C a s 9 , a n d i n c u b a t e d
at 18°C, 200-250 rpm for O/N.
5.2.4.4 Protein sample preparation
Measured the OD600 of the O/N culture from 5.2.4.3. Took 2.5 ml broth and spin down at 5,000
rpm for 3 min to harvest the biomass. Resuspended the biomass i n 60 µl buffer (1×laemmli sample
buffer, diluted in 50 mM sodium phosphate buffer pH =7), boiled for 5 min at 95°C, and then spin
down at 12,000 rpm for 10 min. Appli ed 10-1 5 µl supernatant onto the SDS-PAGE gel and run
gel at 200 V for 1 h.
5.2.4.5 E. coli ultrasonication
Resuspended sediment fro m t he 200 ml culture from 5.2.4.3 in 10 ml ice-c old binding buffer.
Homogenized twice for 1 mi n on ice (Braunschweig 300S, Germany) , an d then spin down at
22,000 g, 4°C for 10 min. App lied supernatant as well a s sedime nt on the SDS-PAGE gel.
5.2.4.6 FPLC protei n purification
Applied the supern atant of the hom ogenate on to a histrap co lumn ( G E H e a l t h c a r e ， USA). Set
four el ution gradients: 62.5 mM imidazole, 125 mM imid azole, 31 2.5 mM i midazole and 50 0 mM
imidazole
5.2.4.7 Cas9 protein storage
Slide-A-lyzer 10 kDa (dialysis cassette) was used for dialysis and removal o f imidazole, dial ysed
twice, at a dilution factor of 1:200. Then the Cas9 protein was s t o r e d i n t h e s t o r a g e b u f f e r , a t -
20°C.


Ch ap te r 5 80 

5.2.5 In vitro sy nthesized sgRNA
The sgRNA were selected online us ing t he Cas-Designer website ( htt p://www.rgeno me.net/cas-
designer/), based on the f ollowing standards: The out-of-frame score val ue of primers should be
higher than 66 and mismatch is not allowed. Two sgRNAs were des igned to test the gene t argeting
efficien cy, which located at 205 bp and 393 bp after the start codon of py rG . DNA te mplates for
in vitro sgRNA synt hesis (MegaScript T7 Transcription Kit (Thermo Fishe r Scientif ic, USA))
were construct ed as DNA oligos incorporatin g a T7-promoter sequ ence, 20 bp prot ospacer and a
77 bp sgRNA tail (Table A.8 ).
Table 5.3 System f or in vitro sgRNA tr anscriptio n
Component Volume (  l)
Nuclease- free w ater 3.25
10  RNA synthesi s bu ffer 1
ATP 1
CTP 1
GTP 1
UTP 1
T7 RNA p olymerase 1
DNA template 100 ng
In total 10
Incubate d in the P CR machine f or 8 h at 37 °C. The synthesized s gRNA was stored at -20 °C.
5.2.6 Gene editing strategy for pyrG via CRISPR/Cas9
For in vitro CRISPR/Cas9 gene- editing system, 5 µl in vit ro pur i f i e d C a s 9 p r o t e i n , 1 µ l o r 2 µ l
sgRNA, and 2 µg pMA171.1 plasmid carrying hygromycin as a selec tive marker (C arvalho et al.,
2010) wer e cotransformed in to A. niger B36 as e stablished i n (Pohl et a l. , 2016) wit h sli ght
modifications. Transformants were subcu ltured t wice on MM plate s containing uridine, pr oline,
and 5-FOA. Consequently, t he ORF region of py rG fr om sporulating single col onies on 5- FOA
medium was amplified to confirm mutants by sequencing.
5.3 Result and discussion
5.3.1 Homologous recombination strategies to construct the B36 uridine auxotroph
Before adopting the CRISPR/Cas9 t echnology, two convention al ho mologous recombinat ion
methods were firstly attempt to obtain a B36 uridine auxo troph wi t h t he i d e n ti ca l py rG* genotype
resembling in AB4.1.


Ch ap te r 5 81 

upstrea m pyrG
Wild type
B36 dow nst ream
upstrea m pyr G*
upstrea m pyr G* d ow nst ream Trans genic Strain
MA169.4 pyrG * OR
5-FOA
39 3 51 4 514 54 2

Figure 5.1 Strateg y on e to build B 36 uridi ne aux otrophi c mutan t via homologous recombination
As depicted in Figur e 5.1, t wo kinds of do nor DNA contai ning un functional pyrG* amplif ied from
MA169.4 were individual ly tran sformed int o B36 und er t he select ion of 5- FOA. Even though
several transformant s were harvested fro m the 5-FOA tra nsformat ion plat es, n o transforman t
carrie s any muta tions on pyrG after verified by sequenci ng. Besi des un functional pyrG - , mut ations
on other genes involved in uridine biosynthesis pathways may also result in uri dine auxotrophic
phenotype, like pyrE (Du et al. , 20 14). This could provide a reasonable expl anation for our ca se.
Since the expect ed transformant was no t yielded by thi s method, an alternative method was then
tried.

upstream pyrG
Wild type
B36 downstrea m
dow nstream
downstrea m pyrG*
upstream pyrG* down stream Transgenic Str ain
5-FOA
pyrG hyg
upstream hyg
downstrea m downstrea m
pyrG*
pyrG hyg upstr ea m Transgeni c Strai n
444 1340
445
1334

Figure 5.2 Strateg y two to co n struct B3 6 uridine auxotrophic m utant via hom ologous rec ombi nat ion. hyg
represents hygromycin


Ch ap te r 5 82 

8224 bp
9056 b p
py rG* PpyrG Tp yr G
Xba I
Xba I
py rG Tp yrG
Nhe I
Nhe I
Xba I Nh e I
hyg
hyg
hyg
F1 F2
13 39
13 38
13 40 13 37

Figu re 5.3 Const ruction of pla smids p YS2.1 and p YS3.1
In strategy two , pAN7 .1 was chosen as the vector t o construct t he f ollowing t wo plasmids as
depicted i n section 5.2.2 (Figure 5.3). Then we cotransformed t he bipartite substrates obtained
from the split ma rker method to achie ve the entire pyrG-hyg-p yrG * knockout cassette i n vivo that
was integ rated at the inta ct pyrG l ocus of B36 (Figure 5.2). As long as obtai ned the expected
transformants carryi ng the entire pyrG-hyg-pyrG * kn ockout cassett e, we exp ected to loop out the
functiona l pyrG a n d hyg f r a g m e n t s u n d e r t h e 5 - F O A s e l e c t i v e p r e s s u r e t o r e m a i n t h e u n f unctional
pyrG * ( Figure 5.2). After verified b y diagnostic PCR (Figure C.7) a nd Southern analysis ( Figure
C.8), YS5.1, YS5.4, YS5.9, YS5.13 are four expect ed transf orman ts carrying t he entire pyrG-hyg-
pyrG * kno ckout cassette. Then, 2*1 0 4 ~2*10 6 spores/ml spore susp ension of YS5.1 and YS5.4
were inoculated on 5-FOA medium in order to l oop o ut the funct i ona l pyrG and hyg regions. After
incubated for three days, sporulati ng singl e colonies wer e p icked a nd inocul ated simultaneously
on MM medium a nd uridin e medium w ith hygromycin. Expected colon ies shoul d not survive on
both these two media. However, no expected transforman ts were o btained even after two tries,
suggesting that the loop out failed.
I n s u m , b o t h t h e a b o v e t w o a p p r o a c h e s f a i l e d t o o b t a i n e x p e c t e d B36 uridine auxotrophic mutants.
The possible reason could be the minor difference between the pyrG* genot ype (only carryin g 1
bp deletion) in AB4.1 or MA169.4 and the wil d-type py rG, which is not easily distinguished by
the cell when attempting t o substitute the wild-type pyrG w i t h pyrG * through homologous
mediated repair. H ence, we subsequently transferred our f ocus o n in vitro CRISPR/Cas9 approach
employing ribonu cleoprotein parti cles to edit ORF of pyrG in B36.

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C hap t er 5
83


5.3.2 In vitro purific ation of Cas9 protei n
The pET2 8aCas9cys vector was use d t o ex pr es s t he C as 9 pr ot e i n ( 1 60 kDa) in
E. col i
. As sh own
in Figure 5 .4, Cas9 protein succ essfully expressed after IPTG i nduction (Figure 5 .4 lane 3, 4) and
the blank strain displ ayed nearl y no signal at 160 kDa (Figure 5.4 lane 2). However, the sediment
samples after homogenization also showed a clear signal at 160 kDa (Fi gure 5.4 lane 6), indi cating
an incomplete homogenization whic h could b e improved by prolong ing the homogenization time.
In a ddition, it should be n ot ed that pro t ein s uffered degradati on after f reezing and thawing (Figure
5.4 lane 7, 8, 10). Therefore, to k eep the protein activity, re peat ed freezing an d thawin g should be
avoided.

Figure 5.4 SDS-PAGE for samples a fter ho mogenized. 1. Nega tive contr ol; 2. Bl ank; 3. Su pernatant after
homogeni zation (7.5 µl); 4. Su per nata nt after homogenization (7 .5 µl) ; 5. P age ruler #266 14; 6. sediment a fter
homoge nization; 7. sedim ent a fter freez e-th aw; 8. superna tant a fter f reeze-thaw (7. 5 µl); 9. page ruler
unstained (#26614); 10 . supernatant after f reeze-th aw (7.5 µ l)
FPLC (fast protein liquid chroma tography) allows the high t hro ughput puri fication of Cas9
protein. 20 ml supernatant after homogenization was applied t o the Histrip purification column
(GE17-5248-01, GE Healthcare, USA). After elut ed with a gradien t concentration of imidazole
(62.5 mM imidazole, 125 mM imidazole, 3 12.5 mM imidazole and 50 0 mM imidazole) (Figure
5.5), t he elution samples were subj ected t o SDS-PAGE g el to eva luate th e p urific ation effect o f
Cas9 p rote in
.

Figure 5.5 The elutio n process of C as9 prote in by F PLC. A7 an d A8 eluant contains Cas9 protein
15 0 k D a
200 k Da
1 2 3 4 5 6 7 8 9 10


C hap t er 5
84



Figure 5.6 SDS-PA GE for FPLC elution samples. (A) 1. page rule r un stained (#26614); 2. after
homogenization; 3. flo w through (A 1 to A3 FPLC ex ample); 4. 6 2. 5 mM imida zole; 5. 62. 5 mM i midazol e; 6.
125 mM imidazole; 7. 312.5 mM imidazole; 8. 312.5 mM imidazole; (B) 1. supernatant after homogenization;
2. flow through; 3. page ruler unstained (#26614); 4. 125 mM im id azole; 5. 3 12.5 mM imid azole; 6. pag e rule r
SM0431; 7. 500 mM imidazo l e; 8. 500 mM i midazole
As s hown in Figure 5.6, ther e was nearly no Cas9 protein signal before elution (Figure 5.6A lane
3). When eluted with 62.5 mM imidazole (Figure 5.6A column 4, 5 ), it could be witnessed a str ong
absorbance peak (Figure 5.5), while the elution samples were mi xed wit h abundan t unexpected
proteins. Cas9 protein start ed t o be eluted when the imidazole concentratio n reached 125 mM
(Figure 5.6A lane 6, Figure 5.6B l ane 4). Notably, when eluted by 312.5 mM imidazole, it can be
observed an abundance peak accompanied b y the most prominent Ca s9 protein signa l, indicat ing
the optimal retention time of Cas9 (Figure 5.5, Figu re 5.6A lan e 7, 8 and Figure 5.6B lane 5).
When turned to 500 mM imid azole, there was no longer any abun da nce peak, only causing a weak
protein si gnal (Figure 5.5, Figur e 5.6B lane 7, 8). Tak en toget her, 312.5 mM imidazole is the
optimal concentrat ion for Cas9 p rotein elution. After dialyzed twice by the dia lysis ca ssette slide-
A - l y z e r , t h e c o n c e n t r a t e d C a s 9 p r o t e i n w a s k e p t i n t h e s t o r a g e buffer, at -20 °C, used for the
subsequent in vitro CRISPR/Cas9 gene editing.
5.3.3 In vitro CRISPR/Cas9-mediated
pyrG
gene editing
To ensur e th e gene e diting effici ency, two sgRNAs were selected o n l i n e a t t h e C a s - D e s i g n e r
website, which located at 205 bp and 393 bp after t he start cod on of
pyrG
, respectively. 0.5

l in
vitro synthesized sgRNAs were applied to the gel (Fi gure 5.7), i n w h i c h s g R N A 1 d i s p l a y e d a
stronger si gnal intensity compared to sgRNA2. The weak s ignal o f sgRNA2 c ould be the wrong
sample loading. Due t o the dif ferent concentration of sgRNA1 an d sg RNA2, 1 -2

l of sgRNA1
and 2-3

l of sgRNA2 were used for
A. niger
transform ation.

A
1 2 3 4 5 6 7 8
B
1 2 3 4 5 6 7 8 9 10


C hap t er 5
85



Figure 5.7 s gRNA1 an d sgRN A2 after in vitr o syn thesi s. Loading volume is 0.5  l
Given t he previ ous optimi zed CRI SPR/Cas9 gene edi ting strategy in
A. niger
in our lab, this study
adopted the following setup for
pyrG
gene editing: 5 μl of in
vitro
purified Cas9 protein, sgRNA1
( 1 o r 2 μ l ) o r s g R N A 2 ( 2 o r 3 μ l ) , a n d 2 μ g o f s e l f - r e p l i c a t i n g p l a s m i d A M A 1 ( a u t o n o m o u s
main tenan ce in
Aspergillus
) containing hygromycin resistan ce m arker. In vi ew of the resul ts in
section 5.3.1, 5-FOA selectiv e pressure is l ikely t o int roduce mutati ons to other genes in uridin e
biosynthesi s pathways, instead of
pyrG
. Therefore, hygromycin instead of 5-FOA was used as the
selective marker here.
MM pl ate
sgR NA 1
YS20.1
sg R NA 1
YS20.2
sgR NA 2
YS20.3
sgR NA 2
YS20.4
5-F O A plate
A B
C

Figur e 5.8 Generation of a pyrG
-
mutant of B3 6. (A) Primary tran sformation p lates supplemented with
hygr omyci n. ( B) Growt h of pr imary tr ansf ormants on MM p lates. ( C) Subcultiv atio n o f p rimary transformants
on 5-FOA plates. Posit ive tran sforman ts w ere sporul ati ng.
After incubated for 5 days at 37

C, more coloni es were obta ined when 2 μl sgRNA was used
compared to 1 μl, whil e 1 μl sgRNA is suffici ent to ob tain enou gh coloni es for subsequent
screening (Figur e 5.8 A). Several primary transformants were un able to grow on MM medium as
M
100
cc

bp


C hap t er 5
86


expected (Figure 5.8B). Then they were subcultivated on 5-FOA p lates, a nd positive transformants
could thrive with sporul ation (Figure 5.8C). C onsequentl y, PCR on
pyrG
open reading frame from
all po ssible t ransformants de fined t hat B36 prefers to repair g enomic double-strand breaks by
visible long fragment insertion or deletion (Fi gure 5.9). After al igning with t he wild type
pyrG

from B36, results show that YS20.1 carries a 462 bp insert (289 bp fr om AMA1 sequence) at 4 bp
upstream of the PAM site, and YS2 0.2 carries a deletion of 195 bp at 101-295 bp after the start
codon of
pyrG
. Both disruptions inactivated the functi on of
pyrG
i n B36. However, YS20.3
isolated from the sgRNA2 transform ation plate pr esented no
pyrG
s ignal. W hen incubated on CM
medium at 30°C, all above three mut ants exhibited similar growt h r ate as AB4.1 (4 days) but
slower than wild type B36 (3 days).

Figu re 5.9 PCR on pyrG open readin g fra me. 1 . YS20 .1 ； 2. YS20 .2; 3. Y S20. 3; 4. U nmuta ted pryG
To ensure the screening effici ency, an alternative method was e mployed simultaneously to screen
pyrG
-
mutants. Al l spores on sgRNA1 trans formation plat es were elute d to obtain a spo re
suspension by 10 ml 0.9 % NaCl. Then 100 µl spore suspension wit h a gradient concentration of
10
8
~10
6
/ml was inoculated on 5-FOA pl at es. Sporul ating single colonies coul d b e harvested after
3 days (Fi gure 5.10). Similarly,
pyrG
OR F from 7 ou t o f 11 screened transforma nts (unab l e to
grow o n MM plates) sh owed visibl y discrepant signal versus to t he control (Figure 5 .11). All B36
uridine auxotroph ic mutants ide ntified by CRISPR/Cas9 are liste d in Table 5 .4.

Figure 5.10 Inoculated 100 µl 10
8
, 10
7
, or 10
6
spore/ml spore solut ion on 5-FOA pl ates (left) and the growth
of potential B36 pyrG - mutants on MM plates (righ t)
10
7
10
5

10
6

1 2 3 4 L a d d e r
1 kb
YS20.1: 462 bp i nsertion

YS20.2: 195 bp deleti on


C hap t er 5
87



Figure 5.11 ORF of pyrG ampli fie d from transfor mants Y S20.5~YS2 0.15 .
Table 5.4 Summary of B36 uridin e auxotrophic mutants generated in this ch apter
Strain

Insertion siz e posit ion

YS 20.1 460 b
p

4b
p

u
p

stream of PAM
YS 20.3 456 bp 4bp upstream of PAM
YS 20.5 118 b
p

2b
p

u
p

stream of PAM
YS 20.7 236 b
p

2b
p

u
p

stream of PAM
YS 20.11 6 20 b
p

2 b
p

u
p

stream of PAM
YS 20.13 6 37 b
p

2b
p

u
p

stream of PAM
Stra in De leti on size po sit ion
YS 20.2 195 b
p

101 b
p

~295 b
p

afte
r

py

r
G

start codon
YS 20.4 195 bp 1 01 bp~295 bp after pyrG start c odon
YS 20.12 1 95 bp 101 bp~295 bp after pyrG start codon
After multiple rounds of cultiva tion under non-selective condit ions, we observed t hat the
hygromycin resistance in all the above-mentioned B36 uridine au xotrophic mut ants was lost,
leading to the reuse o f hyg romycin s elective marker for subs equ ent stra in construction.
5.4 Conclusion

In this study, we successfully obt ained the uridi ne auxotrophic mutants of B36 via CRI SPR/Cas9
modification us ing the ribonucle oprotein (RNP) appr oach, prov id ing an appropriate microbial
chassis used for rational strain design with a strong pull t owards GlaA biosynt hesis.
1 kb

YS20.3
YS20.4
YS20.5
YS20.6
YS20.7
YS20.8
YS20.9
YS20.10
YS20.11
YS20.12
YS20.13
PC
1.5 k b

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C hap t er 6
88


Chapter 6 Engineering cofac tor metabolism for improved prote in and
glucoamylase produc tion in Aspergillus niger
6.1 Introduction
The design-build–test –learn ( DBTL) cy cle is a systematic metabo lic engineering strategy t o
achieve t he desired outcome t hr ough reconstructing heterologo us m e t a b o l i c p a t h w a y s o r t o
rewiring native metabolic activiti es (Niel sen and Keasling, 20 1 6; Ando and Garcia Martin, 2018).
Rational strain devel opment of ce ll factor ies can be improved b y the i terative application of the
DBTL cycles, which not on ly co ntributes to the opti mization of biomanufacturing processes, it is
also advantageous t o build a c omplete metabolic model of engine ered cells to deep en our
understanding of cellular metabolism. The advance o f genetic en gineering has speeded up th e
turnaround time of DBTL cycle in metabo lic engineering ( Liu and Niels en, 2019).

Figure 6.1 Schematic illustrating the design–build–test–learn cycle for NADP H eng i neering. As NADPH
generation was hypothesized as t he bot tlene ck for Gl aA p rodu cti on in A. niger , this cycle starts with using
GSMM to des ign o ptim al co nstruc t s. CRISPR/Cas9 is adopted to ac hieve the chas sis cel l and t he Tet- on
indu cibl e gene exp ress ion sy ste m is incorp orate d to ov erex press genes o f inte rest in the bu ild phase . The
engineered str ains are tested in f lask- an d bioreactor -level to p rovide insight into the metabolic adaption
mechanisms under g enetic p erturb ation. Key d esign factor s are i denti fi ed in the l earn step to a dvan ce ratio nal
engineering in the next round
In light of the results fro m Chapter four, NADPH eng ineering pe rformed i n the low-yi eld Gl aA
production strain AB4.1 on ly ar oused neglectable improvement fo r protein capacity. It is
speculated that the competition of other NADP H requiring pathwa ys and the sufficient NADPH
provision i n AB4.1 could b e responsible for th is scientific iss ue. As r eported that the effect of
genetic perturbation coul d be strain-dependent (Li
et al.
, 2013; Rodriguez-Fromet a
et al.
, 2013).


Ch ap te r 6 89 

In or der to investigate whet her the model predict ion is strain- dep en dent, this chapter applied
identic al NADPH engin eering stra tegies i n a high-yi eld GlaA pro d ucing strai n B36 carrying sev en
glaA gene co pies, to under stand if a strong pull towards glaA biosynthe sis (seven gene copies)
mandates a hi gher NADPH supply com pared to the native conditio n (one gene copy). We present
the implementation of the classic DBTL cycle integrating t he re cently developed new techni ques
regarding GSMM prediction, h ighly efficient genetic modificatio n and omics analysis to accele rate
this framework of metabolic engineering (Fi gure 6.1), thus to m ake A. niger into an efficient
enzyme production cell fact ory.
6.2 Materials a nd methods
6.2.1 Strain and media
A. niger B36 was used as the reference st rain for all fermentation exper iments i n this chapter
(Wallis et al. , 1999). YS20.2 is the uridi ne auxotrophic mutant of B36 engine ered by
CRISPR/Cas9 genetic modification, which was used as the recipie n t s t r a i n t o c o n s t r u c t a l l
modified strains in this chap ter (Table 6.1).
T able 6.1 Stra ins used in this cha pter
Strain
name NO. Background
strain Relevan t genotype/d escription Source
B36 Multi copi es of glaA , amdS + (Verdoes et
al. , 1993)
Y S 2 0 . 2 B 3 6
pyrG -, with 195 b p deleti on at 101 bp~295
bp after pyrG start codon Chap ter 5
OE gsdA
(G6PDH) YS23.20 YS20.2 Over expr ession of An 02g1 2140 ( gsdA ) via
Tet-on, pyrG + This ch apter
OE gndA
(6PGDH) YS22.17 YS20.2 Overexpression of A n11g02040 ( gndA )
via Tet- on, py rG + Thi s chapter
OE icdA
(NADP-I CDH) YS37 .10 YS20.2 Overexpression of An02g12430 ( icdA ) via
Tet-on, pyrG + This ch apter
OE An14g00430 YS24.9 YS20.2 Overexp ression of An14g00430 via Tet-
on, pyrG + This ch apter
ΔAn14g00430 YS26.3 YS24 .9 An14g00430:: hygB This chapte
r

OE maeA
(NADP-ME) YS21.14 YS20.2 Overex pression of An05g0 0930 ( maeA )
via Tet- on, py r
G

+ This ch apter
OE An16g02510 YS38.2 YS20.2 Overexp ression of An16g02510 via Tet-
on, pyrG + This chapter
ΔAn16g02510 YS35.9 YS38 .2 An16g02510:: hygB This chapter
Media used in thi s chapter:
1) fla sk-l evel cult ure medi um: as depicted in section 4.2.1
2) Batch cult ure medium (g/L): glucose·H 2 O 27, KH 2 PO 4 1.5, NH 4 Cl 4.5, KCl 0.5, MgSO 4 ·7H 2 O
0.5, 1000×Trace element 1ml, 0. 01% PPG 2000, set pH to 3.0 by 1 M HCl
3) Maltose-limited chemostat medium : the same as the batch culture m edium only the carbon
source was changed to maltose·H 2 O . T h e f i n a l c o n c e n t r a t i o n o f m a l t o s e w a s 1 % ( w / v ) i n b a t c h


Ch ap te r 6 90 

culture and 0.8% (w/v) in chemos tat cultivat ions. G ermina tion w as induced by the addition of
0.003% (w/w) yeast extract.
6.2.2 A. niger colony PCR
Step 1 ： Small amount of spores were picked up by a sterile toothpick t o pick up a and then put
into 25 μl EpI sol ution
PCR system ：
T e mperatu re T ime Cycle
65°C 6min
98°C 2min * 2
12°C ∞
Step 2 ： touchdown PCR
system T emperature T ime Cycle
template 5 µl fro m step1 95 °C 5 min
water 9 µl 95°C 3 0s
20* (-0.4°C/cycle) dNTP s 2.4 µ l 62°C 30s
Primer1 (10m M) 1.5 µl 72°C 3min
Primer2 (10m M) 1.5 µl 98°C 30s
10* 10*T aq buf fer 3 µl 56°C 20s
T aq 0.3 µ l 72°C 3min
4 M mono hy dra te 7. 5 µ l 4° C ∞
T otal 30 µl
6.2.3 Batch cultures
Batch cultivations were adapted from Kwon et al. (20 12) and performed in 6.6 L BioFlo 3000
bioreacto r with an elect ronic bal ance (New Brunswi ck Scient ific , NJ, USA). 4 kg ammonium-
based minimal medi um and 10 9 conidia/L was ino culated into 6.6 L fermenter. To avoi d loss o f
the hy drophobic conidia, t he culture was aerated only th rough t he headspace of the re actor a nd the
agitation rate was set at 250 rpm during t he first two hours of c ultivation. Aft er the majority of
conidia germinated, the aeration and agitation were increased t o 2 L/min and 750 rpm, respectively .
The temperature was 30°C, and the broth pH was maintai ned at 3 by comp uter-controlled addition
of 2 M NaOH or 1 M HCl.
6.2.4 Chemostat cult ures
Submerged cultivations were carr ied out in 5 L bioreactors ( NCB IO, Shanghai, C hina). All
parameters were set the same as in 6.2.3. Chemostat cult ivation s were initiated in the late
exponential g rowth phase when OUR (Oxygen Uptake Rate) or CER ( Carbon-dioxide Escape Rate)
started to decr ease and DO (Dissolved Oxygen) st arted to increa s e . T h e d i l u t i o n r a t e ( D ) w a s s e t
a t 0 . 1 h -1 . The stead y-state was reached afte r approx imately three residence times (≈ 30 h) a nd


Ch ap te r 6 91 

indicated b y constant CO 2 , O 2 and biom ass concent rations. Sampl es were ta ken re gularly ( 6-8 h)
to monitor growth and to determine if a steady-state had been r eached. All samples were quickly
frozen in liquid nitrogen. 10 µg/m l DOX was added when the biom ass reached 1-2 g/kg. To ensure
inducti on, 10 µg/ mL DOX was a lso applie d t o the feed mediu m. Sa mples used for extracting
intracellular metabolites were tak en during steady-state and in tr acellul ar metab olites w ere
quantified by GC/ LC-MS. Sa mples for NADPH m easurement and qRT-P CR were taken both in
the mid-expone ntial phase (4 h after DOX induc tion) and at stea dy-state.
6.2.5 Analysis of culture brot h
Please refer to 2.2.9.
6.2.6 Analysis o f GlaA by dot blot
Quantification of ex tracellular GlaA by dot b lot was adapted fr om Fiedler et al. (2018a).
6.2.7 Quantification of e xtrac ellular o rganic aci d
By-product s such as ex tracellular oxalic acid were dete rmined b y HPLC (Sh imadzu, Kyoto, Japan).
5 mM H 2 SO 4 w a s u s e d t o r i n s e t h e V A R I A N M e t a c a r b H p l u s c o l u m n a t a f l o w rate of 0.4 ml/min
at 50 °C. The wavelength of the spectrophotometer was set at 21 0 nm. A 1 ml sample was fil tered
over a 0.2 2-µm f ilter daily t o d e termine ext racellular or ganic aci d and glucose concentr ations in
the extra cellular m edium.
6.2.8 Fast extraction of intracellular metabolites
Quantification of intracellular me ta bo li te s wa s bas e d o n Lu et al . (2015), and slight modifications
were made. This study adopted the Isotope Dilution Mass Spect ro metry (IDMS) met hod to
accurately quantify intracellular metabolites (Wu et al. , 2005). 1–2 ml broth was rapidly tak en
from bioreactors by a fast-sampling equi pment to tubes with 10 ml preco oled quench solution
(−27.6 °C 60 % v/v m ethanol solu tion) at steady-state. T o preci s e l y d e t e r m i n e t h e a m o u n t o f b r o t h
taken, the tubes were wei ghed be fore and after sampling. In o rd er to remove the e xtracellular
metabolites promptly, the mixture was filtered wit h a v acuum pu mp. Then, 20 ml precooled
quench solution was used to rinse the filter cake. The washed f ilter cake a nd 1 00 µl 13 C i nternal
standard solution was added to p rew armed 25 ml 75 % (v/v) ethan ol solution and extracted 3 min
at 95 °C. The tubes were cooled down on ice to room temperature . The filtrate was collecte d after
vacuum filtratio n a nd concentrated to 600 µl by rotary e vaporat ion. Subsequently, the metabolite
pools wer e quantifi ed with the L C-MS/MS (Thermo Fisher Scientif ic Corpo ration, USA) and GC-
MS (Agilent, Santa C lara, CA, USA).
6.2.9 GC-MS
150 μl concen trated filtrate from 6.2.8 was add into a 2 m l via l with insert, then freeze -dried for
overnight. 7 5 μl of acetonitrile was used to d issolve the fr eez e-dried intracellular metabolites when


Ch ap te r 6 92 

measuring amino acids, or 75 μl 1% pyri dine methoxyamine was us ed for organic acid, sugar-
phosphate and polyols measurement. Incubated for 1 h at 70 ℃ . The n 75 μ l deri vating age nt was
added .  N-tert -Butyldimethyl silyl- N -met hyltrifl uoroacetamide (Si gma-Aldr ich, USA) with 1%
tert-Butyl dimethylchlorosilane and N -Me thy l- N -(trim et hylsily l)trifluo roacetam ide (Sig ma -
Aldrich, USA) was used for amino acid and sugar-phosphate quant ificat ion, respe ctively. Then,
after incub ated for 1 h at 70 ℃ , i n s e r t s w e r e p l a c e d i n t o a n e w 1 . 5 m l E P t u b e s , c e n t r i f u g e d a t
12000 rpm for 2 min. 100 μl of th e supernatant was carefully pi petted i nto a new vial wi th insert
and used f or measurement. 78 90 A GC coup led with 5975 C MSD si n gle quadrupole mass
spectromete r (Agilent, Santa Cla ra, CA, USA) was used to quant i fy intrace llular amino acid s and
part of phosphocarbohydrate metabolites. HP-5MS 30 m × 0.25 mm × 0.25 μm (5% Phenyl M ethyl
Siloxane) ultra in sert GC column (Agilent, Santa Clara, CA, USA ) was used to separate
metabolites. The temperature-risi ng procedure for the column wa s as follows: the original
temperature was set at 100 ºC a nd kept for 1 min, then heated u p to 300 ºC at 10 ºC /min and kept
for 10 min . High purity helium wa s the mobile phase at a flow r ate of 1 ml /m in. 1 µl sample was
used fo r inject ion. The temperat ures of i njector, qua drupole an d ion source were set at 250 ° C,
150 °C, and 230 °C, respect ively . The ions were generated by a 70 eV electron beam .
6.2.10 LC-MS
The co ncentrate d 1 50 μl of the fi ltrate from 6.2.7 was fi ltered over a 0.22-µm filter , then applied
to the UPLC syst em (Thermal Ultimate 3000, Thermo Fisher, USA) coupled with m ass
spectrometry system (Thermal TSQ Quantum Ultra, Thermo Fisher, USA) for quantification.
Xcalibur (Thermo Fish er, USA ) was used to process data.
6.2.11 Quantification of intracellul ar NADPH
1 ml brot h was quic kly taken from the shake flask or bioreactor and frozen immediately in liquid
nitrogen. Samples wer e t hawed before measurement and dil uted 2 o r 5 t i m e s w i t h 1 × P B S , a n d
centrifuged at 12,000 rpm f or 5 minutes to remove th e supernata nt . Intracel lular NADPH an d
NADP+ were quantified b y E nzyFluo TM A s s a y K i t ( B i o A s s a y S y s t e m s C o r p o r a t i o n , U S A )
according to the manufacturer’s manual.
6.2.12 Quantitative real-tim e PCR (qPCR)
Please refe r to 4.2.12.
6.2.13 Multivari ate statistical analysis of intracellular meta boli tes
Hierarchy clustering analysis o f metabolomics data a t steady st ate was plotted using the R package
pheatmap. Principal component a na lysis and partial least square s d iscriminant analysis (PLS-DA)
were then performed by R p ackage ggbiplot. Pathway enrichment
analysis was pe rformed by the online metabolo mics analysis webs ite Me taboAnal yst 4.0
((https:/ /www.metabo analys t.ca/MetaboAn alyst/face s/home.xhtm l).


Ch ap te r 6 93 

6.3 Result and discussion
6.3.1 Transformati on efficiency of B36 uridine auxotroph
Through in vitro CRISPR/ Cas9 gene editi ng, we achieved several B36 uridine auxotrophic mutan ts
with diverse unfunctional pyrG genotype validated by gene sequencing. To t est whet her B36 pyrG -
mutants displayed a shared trans formation efficiency as the nat ural urid ine auxotrophic mutant
AB4. 1, mae A overexpression strains were constructed on the YS20.2 carrying truncated pyrG
genotype. Cloned plasmid pYS6. 1 used for overex pressing maeA under the co ntrol of Tet-on
syst em, was tran sforme d in to Y S20.2. Sin ce the proto plast t ran s formation process of B36 h as not
been optimized, only l imited tr ansformants were harvested on th e original t ransfor mation plates
(Figure 6.2), showing a low tran sformati on rate of 1 CFU/ µgDNA .

Figure 6.2 I nitial transformatio n plates after tr ansforma tion whe n YS20. 2 as th e backgrou nd str ain
All 14 transformants were further tested by diagnostic PCR and Southern analys is (Figure C.9).
Southern blotting confirmed that 4 out of 5 PC R-verified transf ormant s carry single plasmid
integration as e xpected, while one with multi-copies integratio n, indicating a positive transformant
rate of 28% resembling that in AB 4.1 . Taken together, YS20 .2 co uld be the transformation host
for fu rther gene fun ction studies in this high protein secretio n strain, but t he tr ansformation
efficien cy remains to be optimized.
6.3.2 Optimization of the proto plast transformation effic iency of B36
I n t h e p r e v i o u s A. niger PEG-mediated protoplast transfor mation process (as describ ed i n 4.2.8),
young mycelium was harvested after the submerged cultivation at 50 r pm f or overnight. However,
when incubated YS20.2 under the same conditions for 16-18 hours , a large number of
ungerminated spores remained in the broth . This could be explai ned by the lower growth rate of
YS20.2 th an AB4.1. A l arge am ount of ungerminated s pores caused difficult ies in disti nguish ing
spores and proto plasts during th e subsequent protoplasat ion. Th ere fore, we at tempted to in tensify
the incubation speed from 5 0 rpm to 80 rpm to promote the germi nation of B36 spores.
Polyethylene glycol (PEG) (Boni and Hui , 1987),  as an ideal cell fusoge n, could affect the
permeability of the cell membrane . Here we introduced a higher concen t ration of PEG (6 0% PEG
4000 was used instead of 25% PEG 6000) as described in Pohl et al. (2016), to obser ve its impact
on the transformation efficiency of YS20.2. Then plasmids pYS4. 1 and pYS8.1 were transformed


Ch ap te r 6 94 

individual ly in to YS20.2 by adopti ng t he newly optimized PEG-me diated protoplast
transformation method to construct overexpression strains of gsdA and An1400430, r espectively.
It was f ound that nearly no r esidual spor es remained i n the bro th after increasin g incubat ion speed,
making it easier to define protopl asts, so as to better control the lysing time during the
protoplasation. Besides, it might lead to higher absorp tivity f or donor DNA whe n cells were
exposed to 60% PEG 4000 instead of 25% PEG 6 000, whi ch was c onf irmed in other fungal cell
factorie s as well (Kwon et al. , 20 19). As shown in Figure 6.3, the transformati on efficiency wa s
improved for 4~5 folds co mpared to the orig inal strategy, reach i ng 4~ 5 cf u/µgDNA. Thi s
optimized protoplast transformation method was used for all A. niger transformation experiments
in thi s chap ter.

Figure 6. 3 Transform ation pla tes after optimizing the A. niger PEG -med iate d pro topla st transf ormation
proc esse s
6.3.3 The impact of NADPH engin eering on GlaA production is st rain-dependent
Besides the above three constructe d modified strains, other integrative plasmids (pYS1.4, pYS4.1,
pYS5.1, pYS8.1, pYS9.1) were used to obtain gsdA , gndA , icdA , An14g0043 0, An16g02 510
overexpression strains, and ΔAn 14g00430 and ΔAn16g02 510 null st rains were engineered via
split marker approach as described in section 4.3.2.1. Southern analysis of all modifi ed strain s is
listed in Table 6.1 and Figu re C.9-C10.
Like that in AB4.1, genetic perturba tions did not induce growt h bur den in B36 engi neered strains
(Figure 6.4A). When all seven candidate genes were o verexpresse d in the YS20.2 b ackground
strain containing seven glaA gene copies, increased transcript levels were similar as in AB 4.1, but
for An16 g02510 higher transcription levels (a ri se of 4-fold) w as o bserved (Figure 6.4B, Tab le
6.2), an d two d eleted g enes showed nearly no signal. Noteworthy, overproducti on of 6PGDH
displayed t he most notable regulatory effect on t he transcripti on of glaA , leading to a rise of 2.4
folds than in B36.

OE An14g0043 0 OE gsdA


C hap t er 6
95


A B
C D
E F
* * *
**
** ***
Dr y w e i g h t ( g/k g )
Relat ive g ene
expres s ion le vel
Enzy me activ ity of GlaA
(KAG I/gDCW)
Tota l secret ed protein
(mg/gDCW )
NAD PH
( μ mol/g DCW)
Time (h)
Dox
RNA/N A DPH
**
OE gsdA
Cont rol
OE gndA
OE icdA
OE maeA
OE An14 g00430
OE An16g 02510
Δ An16g0 2510
Δ An14g 00430
AB4.1
YS20. 2
ho st
** *
** **
** *
** *
*
*
** *
** *
glaA eng ine er ed ge n es
1 2 3 4 5 1 2 3 4 5
1- gl aA t ransc ript le ve l 2- engi nee re d ge ne t rans cript leve l
3- NADPH level 4- Total se creted p r o tein
5- E nz y me ac ti vi ty o f G l aA
B36
OE icdA
OE maeA OE gndA
OE gs dA
Δ An14g00 430 Δ An1 6g02510
OE An14 g00430
OE An16 g02510
Figure 6.4 F lask-lev el fer menta tio n results of engineered stra ins in th e background of B36. A. Dry cell w eight;
B. Relative e xpressio n level of glaA an d engi neered ge nes; C. T otal se creted pr otein per gram bioma ss at 72 h
after i noculati on; D. Enzym e activit y of GlaA per gr am bioma ss at 72 h after inoculation; E. Intracellular
NADPH concentration in the expon enti al p hase; F . Comparison be tween engineered strains on the AB4.1 and
B36 backg round, resp ectively . T otal sec reted prote in, enzyme ac tivi ty o f Gla A, and NA DPH w ere no rmalize d
by dr y cell we ight ( DCW) . All experim ents wer e con ducte d in bio lo gical quadruplicates. S ignifica nce va lues
were cal culat ed with t he two -taile d t -te st with ind epe ndent varia bles (* p <0 .05, ** p <0.01 , *** p <0.001 )
An NADP H pool i ncrease of abo ut 30% was measur ed i n t hree str ains overexpressing
maeA
,
gndA
,
gsdA
, respectively (Figure 6 .4E). Even tho ugh identical NADPH gener ation strategi es performed
differ ently on regul ating the NADPH conce ntrat ion i n two rec ipi ent strains, onl y the engineering
of 6PGDH c ould sta bly improve the NADPH pool by 30% in both rec ipient strains. Wit h r egard
to Tet-on driven gene expression of
maeA
,
gndA
,
gsdA
, about 10% increase in sec reted tot al protein
0
5
10
15
20
25
0 2 04 06 08 0
0
0. 5
1
1. 5
2
2. 5
3
3. 5
4
4. 5
0

20
40
60
80
10 0
12 0
14 0
16 0
0

20
40
60
80
100
(g )

0
0. 1
0. 2
0. 3
0. 4
0. 5
0. 6
E
Dry cell weight
（ gDCW/kg ）


Ch ap te r 6 96 

and about 10 -18% hi gher GlaA a ct ivit y was observed for these st rains ( Figure 6 .4C, D). Thes e
observations, therefo re, encourag ed our hyp othesis that NADPH e ngineering might be a strategy
to improve GlaA production bu t suggested that such a n approach is glaA gene copy n umber
dependent. This d ata furthermore impl ies t hat the impact is not linearly corre lated wit h increased
transcripti on of NADP H producing en zymes.
Table 6.2 Summar y of flas k -level r esul ts of all enginee red str ain s on the AB4.1 and YS2 0.2 backgro und
Gene Strains Relative
expression
level of
g

laA
Relative
expression
level of target
genes
Relative
NADP H
Relati ve
total
secret ed
protein
Relative
enzyme
activity
Gla A
OE gsdA
(G6PDH )
YS7.4
YS23.20
1.3
1.6
2.3
2.3
12.4%
* , 29.5%
4.0%
*,7.4%
-1.0%
**,9.7%
OE g ndA
(6PGDH)
YS9.9
YS22.17
1.2
***, 2.4
*, 2.0
*2.2
28.8%
* , 28. 0%
2.9%
**,12.8%
2.9%
***, 17.7%
OE icdA
(NADP-IC DH)
YS10.6
YS37.6
1.0
1.1
**, 1.5
***, 1.6
29.5%
12.1%
6.0%
12.8%
7.5%
4.1%
OE mae A
(NADP-M E)
YS12.16
YS21.14
**, 0 .7
**, 1.6
***, 2.7
**, 1.4
13.3%
*, 30.4 %
4.0%
*, 11. 6%
-5.6%
11.8%
OE An 14g 0043 0 YS11.8
YS24.9
***, 1.3
*, 1.4
**, 1.9
2.6
-1.0%
37.5%
6.6%
3.6%
-0.4%
-0.9%
ΔAn14g00430 YS16.1
YS36.3
***, 0.8
0.9
***, 0.1
***, 0.1
9.9%
11.9%
-6.9%
13.5%
-0.3%
9.5%
OE An 16g 0251 0 YS14.4
YS38.2
***, 0.5
0.9
2.0
***, 4.0
*,32.0 %
11.1%
2.1%
2.9%
-10.3%
-5.6%
ΔAn16g02510 YS15.7
YS35.4
***, 0.4
0.7
***, 0.1
***, 0.02
5.4%
-9.6%
-3.1%
-1.2%
-9.5%
-11.6%
Signif icance values were calculate d with the two-tailed t -test with independent variables (* p <0. 05, * * p <0. 01,
*** p <0 .0 0 1 ). T h e u p p e r r o w w i t h i n o n e block represents relative val u es from the engineere d strain comp ared to
the r eference strai n FW 35.1, and the lower row shows re lative values compared t o B36. OE represent s
overexpressio n
6.3.4 Effect of diverse NADPH ge ner ation systems in batch c ult ures
Based on the above fl ask-level fe rment ation results, several mo dified strains showing superior
protein cap acity wer e subjec ted to b ioreact or-level bat ch cultu res, to observe the influence of
NADPH engineering in the ex ponential phase under the well-contr olled condition. 2 .7% glucose
was taken as the carbon sou rce. When 18 ml of NaOH had been add ed to the batch cul ture, 10
µ g/ml DOX was ad ded to initia te the induct ion. Then samples were t a k e n e v e r y 2 h t o m e a s u r e t h e
specific gro wth rate of strains. To en sure induction efficiency , DOX was refilled every 12 h.
It can b e see n fro m Figu re 6.5A th at spe cific gro wth rates o f t ested strains were not a lter ed un der
genetic perturbatio n. Dot b lot wa s used to quantify the GlaA co ncentration in the broth at the end
of the batch cul tures. However, t he r eproducibility of duplicat es is not perfect as the large deviation
between replicates in Figure 6.5C, which may be caused by inhom ogeneous sampling because of
the high viscosity of broth. According to the box plot (Figure 6.5C), t he yield of GlaA reduced


C hap t er 6
97


when overex pressed
gsdA
and
maeA
, but t here was a ri se of 38% and 27% in An14g00430 and
gndA
overproduction strai ns v ersus to B36. However, the alteration o f total secreted protein was
inconsistent with t he yield of GlaA (Figure 6.5D), where th e to tal secreted prot ein was
accumulated by about 30% in OE
gsdA,
but inhi bited i n OE
gndA
.
A B
C D
Total s e creted protein
(m g/ gD CW)
Dr y w e i g ht ( g/ kg )
R esi d u a l gl uco se
(g/L)
Yi e ld of Gl aA (m g/ gD CW )
B36
(h ) 2 5 2 7 4 2 2 5 27 42 2 5 2 7 4 2 25 27 42
OE maeA
OE An14g00 430
OE
gn dA
OE
gs dA
0 0.01 0.02 0.05 0. 1 0.25 0. 5 0.75 1 (mg/m l)

Figu re 6.5 Batch cultures for N ADPH generating gene overexpres si on st rains. (A ) The gr owth rate and
residual glucose during batch cu ltur es; (B) Dot blot ana lysis t o quantify t he GlaA durin g ba tch cu ltures; (C)
The yiel d of GlaA at the end o f batch c ultures; (D) The yield o f total s ecreted protein at the end of batch
cultures. Total secret ed protein and enzyme activ ity of GlaA we re normaliz ed by dry cell weight (D CW). All
measureme nts w ere base d on biologi cal duplicates
Since th e fie rce com petitio n for cellular resources bet ween c el l growth a nd protein biosy nthesis
during the exponential phase, 8 0% of t he i nput carbon is mainly consumed by biomass growth and
maintenance. However, GlaA is largely accumulated when cell gro wth enters th e stationary phase
(Sui
et al.
, 2017). Therefore, due to the o bvious int erference of biomass growth on protein
accumulation during t his p hase, batch cult ures cou ld not provid e us with a reliable e xplanation
about the correlation among genetic pert urbation, NADPH level, and en zyme production.
Subsequent studi es will con duct hi ghly reproducible chemostat c ultivati on t o ch aracterize diffe rent
engineered strains.
0
5
10
15
20
25
30
0
2
4
6
8
10
12
14
16
0 1 02 03 04 05 0
Ti m e ( h)
B3 6
OE maeA
OE gnd A
OE An14 g00430
OE gsd A
B36 gluc os e
OE maeA glu cose
OE g ndA g luco se
OE An14 g00430 gl u co s e
OE gsd A gl ucose
Dry cell weight
（ gDCW/kg ）


Ch ap te r 6 98 

6.3.5 Impact of NADPH generati on on physiol ogical changes and metabolic adaption
6.3.5.1 Physiol ogy a nd gene expressi on during maltose-l imited c hemosta t cultu res
To shed fu rther lig ht on t hese phenomena, we c ultivat ed the t hr ee most promising strains
overexpressing maeA , gndA , gsd A , respectively, in the YS20.2 b ackground under chemost at
conditions and compared their performance with chemostat data o btained for B36 as reference.
Strain B36 and the three strains overexpressing maeA , gndA a nd gsdA , respectively, were run in
duplicate malto se-limited chemostat cultures. To induce the exp ression of t hese three ca ndidate
genes, 1 0 µg/ml DOX was added during t he early expon ential grow th phase, when the biomass
reached 1-2 g DCW /kg. After about 22 h, the cultivation process was swit ched t o the chemost at mode
with a dilution rat e D = 0.1 h -1 , as described in Kwon et al. (2012) and the Materials and Methods
section. DOX was continuou sly added throug h the feed medium. Du ring exp onential and steady
state conditions, samples were taken for biomass determination, g ene expression analyses (qRT-
PCR), intracellular metabolite qua ntification (GC/LC-MS) and se creted protein determination
using an in-house developed fast-quen ching sampling device (u np u b l i s h e d ) . C a r b o n w a s
accounted fo r in carbo n balances of the feed medium, effluent c ulture broth and exhaust gas. The
carbon dio xide evolution r ate (CER), the ox ygen uptake rate (OU R), and bi omass concentrat ions
reached constant levels after a bout t hree-vo lume chang es (Figu r e 6.6A, C).
The genetic modification did not lead to visibl e growt h burden during the exponent ial p hase, in
line with that reported in sectio n 6.3.4 (Table 6.3 ). I nteresti ngly, chemostat cu ltures hig hlighted
the impact of genetic pertur bation on strain growth. Overexpres si on of gsdA o r gndA i n c r e a s e d t h e
amount o f biomass d uring the chemostat as previously d escribed (Park et al. , 201 4), wher eas
overexpression of maeA r educed it (Figure 6.6 C). This is also reflected by the final car bon-
recoveries (Table 6.3). They were hi gher in both gsdA o r gndA overexpressi ng str ains (110%,
104%), but lower in mae A overexpressing strain (91%) compared to 9 9% of t he B36 strain. Similar
to shake flask-level cultivations, transcript levels of all thr ee overexpressed genes were a bout 1.3
( gsdA , gndA ) or 2.7 ( maeA ) times above their respective transcript levels in B36 during the
exponential phase. They c onsiderably increased to 3.3 ( gsdA , g ndA ) or 8.2-fold ( maeA ) during
steady s tate condi tions (Figure 6.6 E, F), likely because of co nti nuous DOX feed ing. This data
might suggest that although all t hree genes are under the same Tet-on driven tr anscriptional control
a t t h e s a m e l o c u s ( pyrG ), other regulatory mechanisms, e.g. mRNA turnover or metabolic f eedback
regulation, might additionally control the acti vity of these th ree can didate genes. The yi eld curve
of tot al secreted pr otein (Figur e 6.6D) well described the impa ct of d iverse NADPH regulation
strategies on protein biosynt hesis. Notab ly, overexpression o f gsd A increased biomass
accumulation (Table 6.3) but inhibited the yi eld of total secre ted protein and GlaA by 40% (Figure
6.6D), suggest ing a competit ion between growth and protein prod uction as previously proposed
(Park et al. , 2014). In contrast, ov erexpression of gndA a n d maeA positiv ely impa cted pro tei n
secretion by 60% and 30%, respectively (Table 6.3). Thi s data w as consi stent with NADPH pool


Ch ap te r 6 99 

measurements duri ng steady state co nditions: Over expressi on of gndA a nd maeA i n c r e a s e d
NADPH levels (46% and 66%, respectively), whereas o nly wild-typ e NADP H leve ls were
observed for the strain overexpressing gsdA (Figure 6.6G).
Taken together, t his t est step well d efines the direct associat ion of cofactor engineer ing with
protein biosynthesi s. 6PGDH behav ed a s the optimal t arget for r egulating enzyme production in
cell factory A. niger , which highlights the scalabilit y of this cofactor engineering to guide
molecular br eedi ng of other hom ologous or heterogenous protein cell factories.
Table 6.3 Kinetic parameters for A. nig er B36 and recombination strains a t steady state . All pa rameters
were measure d based on biological duplicates

Data for strain B36 and three Te t-o n dr iven over expre ssion str a ins derived thereof are shown for ch emostat
cultures with m altose as growth-l imiting substrate. Standard de v i a t i o n s ( ± ) a r e g i v e n f o r m e a n v a l u e s o f d u p l i c a t e
independent steady state results which were measured in technic al tr iplicat es. C biomass , biomass concentr ation
(dry cel l weigh t ( DCW)); q CO2 , speci fic carb on di oxide evolution rate ; q O2 , specific oxygen uptake rate; RQ,
respiratory quotient calculated as th e ratio of CO 2 produ ction and O 2 consumption rates; q protein , sp ecific
prod uctio n rate of extrac ellu lar protein; q s , specific substrate consumption rate; Y Gl aA /X , yield of total
glucoa myla se act ivity; C -recov ery, ca rbon re cove ry.

B36 OE
g

sdA OE
g

ndA OE ma eA
µ (exponential phase)
(

h -1
)

0.21±0.01 0.2 1±0.02 0.21±0.01 0.19±0.01
C bio ma ss
(g DCW /kg) 5.66±0.1 6 7.5±0 .36 6.46±0.1 7 4.74±0.29
q CO2
(mmol/g DCW ·h) 1.33±0.0 4 1.00±0.0 3 1.1± 0.03 1.45±0.09
q O2
(mmol/g DCW ·h) 1.6±0.18 1.1 9±0.15 1.51±0.11 1 .79±0.20
RQ 0.83±0.09 0 .81±0.1 0.73±0.06 0.812±0.09
q Protein
(mg/g DCW ·h ) 3.71±0.7 2.27±0.37 5 .62±1 4.94±0.49
q s
(mmol maltose /g DCW ·h) 0.39 ±0.011 0.31 ±0.009 0.35±0.031 0.47±0.011
Y Gla A/X
(U/g DCW ) 23.84± 1.08 14. 38±0.82 38.88± 3.6 30.83 ±0.1
C-recovery 99% 110% 104% 91%


Ch a p t e r 6 100 

A B
C D
EF
G
B36 OE gs dA OE gndA OE mae A
NAD PH
(μmol/g· DCW )
Re l at iv e ex pre ss io n le ve l of gl aA
B3 6 O E gs dA OE gn dA OE mae A
Rel a t i ve ex press ion le ve l of e ngine ered genes
Rela tiv e g ene
expr es sion le v el
Relativ e gene
expression level
ba t c h ch em o s t a t
Exponential Chem ostat
Ch em os tat cul tiva tion
El ution ti m e (h )
El u t i o n t i m e ( h )
CER (mm ol/L·h)
D

ry ce
ll

we
i

g
h

t
(gDCW /kg)
Oxa lic ac id
( μ mol/g DCW)
T otal s ecret ed
p rotei n ( m g/gDCW)
NA DPH l evel
DO X
RN A/ NA DPH
RN A/NA DPH/ Met abol it es
Chemost at cu l tivat ion, t=0
B36 OE gs dA OE gn dA OE maeA
Exponential Chemos tat
B36 OE gndA OE gs dA OE maeA B36 OE gndA OE gs dA OE maeA
B36 OE gndA OE gs dA OE maeA B36 OE gndA OE gsdA OE mae A
Elut io n time (h)
Bat ch
cult ure Ch em ostat cultivat ion, t=0
Batc h cult ur e Chem ostat cultivat ion, t=0
Elut io n time (h)
Elut io n time (h)

Figure 6.6 Phy siol ogic al pa rameters f or A. nige r B36 a nd three engineered str ains at di lution ra te 0.1 h -1 . (A)
CO 2 product ion rate (CER) ; (B) By-produ ct oxalic acid; (C ) Dry cel l weight; (D) The yield of t otal secrete d
protein; (E) glaA relative gen e expression lev el at the exponential gr owth phase and steady-state phase; (F)
Target genes relative g ene expr ession leve l at the exp onential g rowt h phas e and stea dy-state p hase ; (G )
Intracellular NADPH level at steady state. Data represent th e m ean± S D from two ind ependen t cultur es. Tot al
secreted protein, oxa lic acid, NA DPH and enz yme activity of Gla A were normalized by dry cell weight
0
0.1
0.2
0.3
*
**
3.6
4.6
0
1
2
***
**
**
*
**
0

1
2
3
4
7.5
9.5
*
**
*** ***
***
***


C h a p t e r 6
101


(DCW). Significance va lues were calculated with the two-tailed t -test with i ndepend ent variables ( * p <0.0 5,
** p <0.01, *** p <0.001). S traight l ines in A -D a re trend lines for two independ ent cu ltu res

6.3.5.2 Multivariate statistica l analy sis of intrace llular met abolites

Figure 6. 7 Bi oplot (A), VI P score of 42 intracel lular metaboli tes calc ulated usin g PLS model (B), a nd
respec tive pathway impact an alysis (C–E) for metab olic profilin g at steady state. Three r eplicates from each
strain were denoted with the same color
To systematically characterize a nd evaluate t he effect of mutat ions o n th e metabolism, we also
examined the m etabolomics for samples of eight chemostat runs. In total, 42 intracellular
metabolites were identified and quantified for all eight chemo s tat runs. Th ese included nine sugar
phosphat es, eight organic acids, 19 amino a cids, and six curren cy metabol ites ( NAD, NADH ,
NADP, NADPH, ADP, ATP). Princip al Component An alysis (PCA) unco vered that samples from
all four strain s separat ed i nto four disti nct groups as shown in the score pl ot of Partial Least
Squares Discrimination Analy sis (PL S-DA) (Figure 6.7A).

Especially the strain overexpressi ng
maeA
d i s p l a y e d t h e s t r o n g e s t m e t a b o l i c c h a n g e s , w h e r e a s t h e s t r a i n s overexpressing
gsdA
a n d
gndA
, respecti vely, showed on ly subtl e differences when compared to t h e m e t a b o l i c p r o f i l e o f
strain B36. The loading map unco vered representative metabolite s which mainly contributed to
the sep aration of thes e four strain s. Vari ations of re lative ab undances of pyruvate (PYR), succinate
(SUC), histidine (HIS), maltose ( MAL), 6-phosphogluconat e (6PG) m ainly contributed to
distin guish bot h
gsdA
a n d
gndA
overexpr essing strains, whereas the majori ty of variables
contributed to t he separation of OE
mae A
s train (Fi gure 6.7A). A v ariable imp ortance plot (VIP)
displaying the relative contributi ons of these representative m etabolites, demonstrated that VIP
Pathway impact
OE gsdA
Gly ci ne, serin e and
threonine metab olism
Pentose phosphate pathway
Glyoxyl ate and
dicarboxylate meta bolis m
Glutathio n e metabol ism
Alan ine, asp artate and
glu tamate metab olism
0.0 0.1 0.2 0.3 0 .4 0.5 0.6 0.7
0 2 4 6 8 1 0
-log(P)
OE gndA
Pen t os e ph osp h ate p ath way
Glyo xyl at e a nd
di carbo xyl ate m etabol ism
Glu tat hion e meta bolism
0 1 2 3 4 5 6 7
-log(P)
OE maeA
Glycine, serine and
th reo n ine m eta boli s m
Arg i nin e and pro line
met abolis m
Glu tat hion e meta boli s m
Al an ine , as p ar tat e a nd
glutamat e met abolism
-log(P)
VI P sco re of 42 intr ac el lu l a r
metabolites
B36 OE gsdA OE maeA
PC1 (48% expla ine d v ar.)
PC2 (18.6% expla ine d var.)
OE gndA
AB
C D
E
Path w ay imp act Pathway impact
0.0 0.1 0.2 0.3 0.4 0 .5 0.6 0.7 0.0 0 .1 0.2 0.3 0.4 0. 5 0.6 0.7
0 2 4 6 8 1 0
E
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6


Ch a p t e r 6 102 

values of 18 metabol ites out of th e 42 metabolite s were abov e 1 (Fig ure 6.7B), suggest ing that
these 18 me tabolites co uld be con sidered a s potentia l markers t o discri minate all four strains.
Pathway en richment analyses highlighted that th e pento se phosph at e path way, the glyoxylate
bypass and dicarbo xylate metabo lism had a signific ant im pact i n t he strains overexpressing gsdA
and gndA , respectively. However, glycine , serine, and threonine metabol ism was enriched in the
strains overexpressing gsdA and mae A . The l atter in general show ed overrepresentation of amino
acid meta bolic pat hways inclu ding alanine, a spartate and glutam ate metaboli sm, arginine and
proline metabolism b esides glyci ne, s eri ne and threonine metabo lism (Figure 6.7 C-E) .
6.3.5.3 Metabolic pro filing of amino acid pools and central ca rbon metabolism
Metabolome bri dges a link betwee n genotypes and p henotypes, whi ch allows to i ntuitively reflect
the me tabolic leve l altera tions a rou sed by genetic perturbation s , p r o v i d i n g t h e o r e t i c a l b a s e s f o r
uncovering the d ifferences in phy siological characteristics and pro duction capacity of diverse
engineere d strains. In gener al, Ala, Glu, Gly, L eu, and Lys are t he top five amino acids i n the
biomass of A. niger , whereas Ser, Thr, Ala, Leu, and Gly account f or about 50% of the total amino
acids in GlaA (Sui et al. , 2017). Our metabolomics analys es uncov ered that in all four s train s,
amino acids from the glutamate family are most and aromatic ami no acids are least abundant,
which is in general agreement to p reviously reported amino acid pools in A. ni ger (Lu et al. , 2015).
Overall, the amino acid pool i n the gndA over expression strain was slightly reduced compared to
B36 (4%) but was increased in strains overexpressing gsdA (22%) or maeA (30%), respect ively
(Table 6 .4).
A general observation was also t ha t the histidin e pool dramatic al ly increased when gsdA (4 fo lds)
or gndA ( 1.6 folds) were overexpressed. T wo out of the fi ve amino acids dominating A. niger ’s
biomass (Glu, Lys) accumul ated in the strain overexpressing gsdA , while the pool sizes of three
top GlaA composing amino acids (Ser, Th r, Gly) were l ess abu nda nt compared to B36. This data
might explain reduced GlaA production in this strain. In the ca se of ma eA o verexpression, nearly
all amino acids where higher abu ndant when compared to strain B36 (Tabl e 6.4), especially t he
pools for the GlaA domin ating amino acids Thr, Ala, Leu and Val , suggesting that i ncreased a mino
acid pools might cause the extra driving fo rce for Gl aA fo rmati on i n a maeA overex pressing strain.
In contrast , the overall amino acid pools of the strain overexp ressing gndA displayed only moderate
reduction compared to B36 (Table 6.4).


C h a p t e r 6
103



Figure 6.8 The pool siz e of amino a cids for t he A. niger reference strain B36 and th ree o verexpression str ains
at steady state . Blocks i n the heat ma p represent B36, OE gsdA , OE gndA an d OE maeA from le ft to righ t.
Amino acids label ed in yel low are the to p fiv e amino acids in t he compositio n of GlaA. Red arrows indicate
the enzymes overexpressed


Ch a p t e r 6 104 

Table 6.4 Pool sizes of in tr acellular amino acids of A. niger B36 (control) and three opt imized s trains at
steady st ate. The unit is µmol/g DCW . A ll a mino acids we re measured in te chni cal tri plicate
Name B36 OE gsdA O E gndA O E maeA
Gln 155.52±2.50 204.01±2 7.37 153 .52±18.31 2 02.97±17.40
Glu 91.5 4±1. 23 101.2 1±5. 12 87.90± 6.18 110. 50±12.41
Leu 4 5.21±4.96 54.24±18.4 3 39.88±5.73 51.69±5. 87
Asp 2 3.85±0.70 3 3.88±6.69 26.36±7.53 32 .58±5.52
Ala 14.38±0.7 4 15.41±3 .13 12.17±2.41 25.08±5 .58
Ser 12.45±1.36 1 1.44±1.12 10.20±1. 84 14.95±3. 74
Orn 5.37±0.12 7.52±0.79 5.48±0.92 11.48±3.36
Thr 6.17 ±0.3 7 3.81±0.34 5.47±0.2 7 8.46±0.7 9
Lys 3.38±0.23 4.32±0.8 6 3.12±0.44 5.02±0.66
Asn 3.00±0 .04 5.3 0±1.08 3.09±0.67 4. 61±1.34
Gly 2.50±0.81 2.29±0.5 1 1.91±0.29 3.28±0.6 0
Val 1.11±0.0 5 1.45±0.3 1 0.97±0.18 1.80±0.3 4
Pro 0.78±0.041 0.97±0.19 0.6 3±0.03 1.21±0.37
His 0 .72±0.051 2.86±0.53 1.13±0.26 n.a*
Cys 0 .67±0.037 0.38±0.026 0.54±0.038 1.12±0.15
Ile 0.4 3±0.058 0.69±0.26 0.35±0.031 0.61±0.14
Phe 0.38±0.053 0.45±0.15 0.27±0.033 0.43 ±0.063
Tyr 0.28±0.037 0.38 ±0.095 0.25±0.034 0.45±0.077
Met 0 .09±0.044 0.20±0.13 0.07±0.021 0.10 ±0.021
Total 367.68 450.73 353.8 6 477.03
n.a The his abundance in OE maeA was detec ted incorrectly, thus it didn’t show here.
Genetic perturbation led to a significan t flux redistribution on the central metabo lic network.
Metabolic flux anal ysis is the most authoritative met hod for me asuring in vi vo fluxes (Lawson et
al. , 2019). In t his study, we used ou r previ ously up dated A. niger GSMM iHL1210 to predict in
silico the flux distribution of the central carbon metabolic network duri ng steady-state conditions
of three strain s overexpressing gndA , gsdA a nd maeA , r e s p e c t i v e l y ( F i g u r e 6 . 1 0 ) ( L u et al. , 2016).
As depicte d in Figure 6.9, the upper glycolytic p athway interm e diates (G6P and F6P) were
distinctly lower in two PPP engineered strains ( p < 0.05), resu lted from the i mproved carbon flux
towards t he PPP diverted at t he branch node G6P af ter the engi n eering of gsdA a nd gndA .
Additionall y, the further blocked flux from G6P to F6P in OE gsdA al so links to the f eedback
inhibition of the 6PG on the activity of phosphohex ose i someras e (PGI) (Sachla and Helmann,
2019) and the inhibitory effect of PEP on the pr eparatory phase in glycolysis (Ogawa et al. , 200 7),
as both 6PG and PEP pools wer e a ccumu lated in this engineered s train. T his could be reflected by
the lowest carbon uptake rate in OE gsdA ( Table 6.3). On the lower glycolytic pathway, t he


C h a p t e r 6
105


accumula tion of intermed iates PEP and PYR r espectivel y in OE
gsdA
and OE
gndA
l e d t o a
reduced fl ux thr ough 3PG (Figure 6.10), accompanied by a reduct ion of the serine family pool in
these two optim ized strains. Thi s data could be well explain ed by the close association bet ween
the abu ndance of me tabolite precursors on the central metaboli c network and the pool size of t he
correlate d amino acid families as already reported elsewhere (J ordà
et al.
, 2014). Similarly, the
pool sizes of aromatic ami no acids are coupled with the abundan ce of PEP.

Figure 6 .9 T he pool si ze of a par t of the m etabo lit es on c entr al carbon metabolis m pathways for the A. niger
refere nce st rain B3 6 and thr ee overexpression stra ins under st e ady state. Blocks in the heat map r epresent B36,
OE gsdA , OE gndA and OE maeA from left to right. Red a rrows i ndic ate the enzyme s overe xpres sed.


Ch a p t e r 6 106 

7 32 83 0
30
26
1
35
14
18
62
-3
B36
OE gsdA
OE gndA
OE mae A
G6P
6PGL 6PG RU5 P
PEP
Glu cose
PYR
AC-CoA. m
ICIT.m
OAA.m
MAL.m
AKG.m
SUC-CoA .m
FUM.m
gsdA gndA
CIT.m
Mito chondr ion
F6P
FBP
DHAP GAP
3PG
SUC.m
R5P
S7P
E4P X5P
CIT
OAA
MA L
mae A
Glucos e.ex
GA P
26 32 97 0
0 0 0 53
GL X
100/ 100/ 100/ 10 0
78/ 98/ 86/ 98
52/ 47/ 0/ 78
56/ 54/ 32/ 64
113/ 111/ 91/ 121
105/ 104/ 82/ 113
10 1/ 99/ 78/ 108
39/ 35 / 11 / 43
12/ 13 / 35/ 3
GLC NT
22 2 14 2
19/ 0/ 14/ 0
G1P
19/ 19/ 19/ 19

Figure 6.10 In vi vo flux d istribut ion of E MP , PPP , and TCA cycle in B36 and three engineer ed strains at steady
state predicted by iHL1210
Importantly, the PPP is not only key for NADPH generation but p rovides also t wo important
precursors for amino acid bi osynthesis, i.e. ribo se 5-phosphate ( R 5 P ) a n d e r y t h r o s e 4 - p h o s p h a t e
(E4P), whi ch i n turn are required f or Hi s and aromatic amin o ac i d biosynthesis. Overproduction
of gn dA channeled more carbon flux from 6PG to Ru5P, r esulting in a low er abundance of 6PG as
expect ed ( p <0.05). Surprisingly, the R5P pool s declined in both strains o verexpressing gsdA and
gndA , respectively. However, taking into accou nt the i ncreased biomass formation and higher His
pools in t hese two str ains, the increas ed carbon f lux towards R 5P was possi bly channeled to wards
nucleotide biosynthesis. Besides, different fr om the performanc e of PPP pools in other protein
overproducin g strains (Lu et al. , 2018), the reductive PPP i nterm ediates R5P, S7P, G3P were
present in lower abundances. Ho we ver, the flux remarkably accum ulat ed i n E4P in OE gsdA an d
OE gndA ( p < 0.05), so as to excess carbon from PPP back to the glycolyti c path way. In agreement
with previous s tudies on high pr otein secretion conditions (Dri ouch et al. , 20 12; Lu et al. , 2015)
and our in silico metabolic fl ux simulations, the flux t hrough the TCA cy cle was also reduced
induced by the engi neering of the PPP (overexpressing gsdA o r gn dA ) here . However, the picture
for the TCA cycle in termediates is inhomog eneous. The TCA cy cle int ermediate s AKG, FUM,


Ch a p t e r 6 107 

and OAA were less ab undant ( p < 0.05), while only MAL was observed to accumulate ( p < 0.0 5) ,
demonstrating a higher flux from FUM to MAL in OE gsdA . In the ca se fo r gndA overex pression ,
the experimental data showed only weak differences for the pool s izes of TCA intermediates when
compared to strain B36 (Figure 6.9 and 6.10).
Last but not least, ov erexpression of maeA facil itated not onl y amino acid biosynthesis but also
improved the carbon flux towards the EMP and TCA cycle (Figure 6.8 and 6.9) and increased the
pools of t he key intermedi ates F6P, G3P, PEP, OAA and F UM but n ot PYR. Howe ver, PYR did
not accumulat e notably in OE maeA , which is associated with th e role of pyruvate as a met abolic
node to th e TCA cycl e and as a precursor of two amino acid fami lies. Oxal ic a cid is a m ajor by-
product secreted during A. niger fermentati on and converted fr o m OAA by oxaloacetate hydrolase
(Kubicek et al. , 1988) . Whereas th e oxalic acid pool did not diff er in OE gsdA a n d O E gndA
compared to B36, but it was ex clusively increased by about 30% in OE maeA, suggesting that the
elevat ed pool size of OAA cou ld b e re sponsible for the accumu la tion of this by-product (Fig ure
6.6B).
In sum, above simulation and experimental results i ndicated tha t the rela tive flux throu gh the EMP
and TCA cycle decreases after redirecting a higher flux through th e P PP wh en gndA o r gsdA w as
o v e r e x p r e s s e d , a s i t u a t i o n w h i c h w a s r e v e r s e p r e d i c t e d w h e n maeA was overexpressed. When
compared with other autono mously evolved m etabolic networks fro m forced protein
overproduction (Ohn ishi et al. , 2005; Driouch et al. , 2012; Lu et al. , 2015), it illustrates a
comparable flux pattern t hrough the PPP and T CA cycle as observ ed here, suggesting similarities
of core carbon metabolism in the central me tabolic network in d iv erse high-yi eld protein
overproducin g A. n iger cell fact ories.
6.3.5.4 How does NADPH engine ering regulate protein production ?
Nota bly, ov erexpress ion o f the gsd A g e n e improved biomass accumulation and only slightly
increased NADPH productio n, wh ich was parallel ed by reduced pro tein production capacity by
about 40%. This observation is i n general co nsistent with previ ous reports for A. nige r and ot her
filamentous fungal cell factories. For example, a hi gh specific p r o t e i n p r o d u c t i o n r a t e h a s b e e n
shown t o correlate with relatively low growth rates in Trichoderma reesei (Castillo et al. , 2016),
due to a reduction of proteome allocated for central metabolism (Nielsen, 2019). Also, a recent
multi-omics analysis from our g roup that integrated transcripto mics, metabo lomics and GSMM
simulations proposed that an i ncreased Gl aA product ion is proba bly achieved t hrough reduced
growth, which likel y i s r egulated at several met abolic m echanis ms: (i) An increased carbon
catabolism that generates m ore amino acid precursors for protei n production; (ii) A reduced fatty
acid a nd ribosome biogenesis and, thu s, red uced growth; and (ii i) An increased flux throug h t he
glyoxylate bypass to redu ce NADH form ation from the citric acid c y c l e a n d t o m a i n t a i n t h e c e l l u l a r
redox balance (Lu et al. , 2018). In the current study, at least for the two strain s ove rexpressing

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