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This is a post-peer-review, pre-copyedit version of an article published in Applied Microbiology and
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http://dx.doi.org/10.1007/s00253-020-10398-1

Sui, Y.-F., Ouyang, L.-M., Schütze, T., Cheng, S., Meyer, V., & Zhuang, Y.-P. (2020). Comparative
genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein
secretion capacity. Applied Microbiology and Biotechnology, 104(6), 2623–2637.
https://doi.org/10.1007/s00253-020-10398-1
Yu-Fei Sui, Li-Ming Ouyang, Tabea Schütze, Shu Cheng, Vera Meyer,
Ying-Ping Zhuang
Comparative genomics of the aconidial
Aspergillus niger strain LDM3 predicts
genes associated with its high protein
secretion capacity
Accepted manuscript (Postprint) Journal article |

Com parat ive gen omics of the ac oni di al A sperg illus niger st rain LD M3
pred icts gen es assoc iate d with it s high protein secret ion cap a ci ty

Yu - Fei Su i 1 , 2,† , Li - M ing Ouy ang 1,† , Tabea S chütze 2 , Shu Cheng 3 , Vera Me y e r 2 ,* , Ying -
Ping Z huan g 1, *

1 St ate Ke y Laborat or y o f Biore actor E ngin eerin g, Eas t Chi na Univ ersit y o f Science an d
Technology, Shanghai 200237, P. R. China;
2 Department of Applied and Molecula r Microbio logy , Institute of Biotechnology,
Techni sche Univers ität B erlin, Gustav - Me ye r -Allee 25, 13355 Berlin, Germany
3 BGI Institute of Applied Agriculture , BGI-Shenzhen, Shenz hen 518120, CHI N A
† Both authors contributed equally .
* Corresponding authors

Correspondence: Vera Meyer ( vera.m e y er@ tu -be rlin.de ); Yingping Zhuang
( y pz [email protected] n )

Abstract
Asper gillus niger is wide l y u s ed a s a c ell fa c tory for hom ologou s and het erologous protein produ ction. As
prev iou s s t u dies reported th at reduced s porulat ion favor s p rotein secretio n in A. niger , in t his st ud y we
conducted a comparative g e nomic analysis of t he non - sporula ting industriall y exploited A. niger strain LDM3
in C hina and th e m odel prot ein secreti on str ai n CBS 513.8 8 to predict the key gene s that mi ght de fine the
genetic basis o f LD M3 ’s h igh protei n produ cing potent ial in silico . Aft er se que nc i ng usi ng a hybri d approac h
com bining Illumina and PacBio sequencing platforms , a hig h - qu ality ge no m e se que nc e of LDM3 was
obtai ned w hich h arbors 11, 209 open re adin g frame s (OR Fs) and exhibits larg e chromosomal rearrangements
in co mpa ri son to CBS 513.88 . An alignm e nt of the two ge nom e s eq ue nces revealed that t he maj or ity of t he
45 7 ORFs u nique ly p res ent in LDM3 posse ssed predicted f un cti ons in re dox pa thw ays, protei n tran sport , and
protei n m odi f ication process e s . In addition, b ioinf o rmatic analys es revealed the presence of 65 6 ORFs in
LDM3 wit h no n - syno n ymou s mutati o ns en codin g for pr otein s relate d to pr otein translatio n, protein
m odification , protein secre tion , m etabol ism an d energy production . We stu died th e impact of tw o of t he se
on prot ein pr oducti on in t he est ablishe d lab m odel stra in N402. Both tupA and prpA gen es w ere selected
because availab le lite rature su ggested their invo l veme nt in a sex ual sp or ulatio n o f A. ni ger . Our co - expression
net work a na lysi s s uppo rt ivel y predicted the rol e of tupA in pro tein secretion and the role of prpA in ene rg y
generati o n, respectively . B y knockout experiments, w e s how ed that the Δ tupA muta nt d ispl ayed red uced
sporu lation (35%) accom pan ied by h i g her tot al prot ein s ecretion (65%) com p ared to its paren ta l strain. Suc h
an effect was, however, not observed in the Δ prpA muta nt.
Keywo rds: Comparative genomi c s; A sper gillus niger ; sporulation; pr otei n s ecretio n ; tupA ; ge ne c o -
exp ress ion net wor k

Intr oductio n
A spergillu s niger is a widel y u sed cell factor y in b ulk ma nufacturing of i ndustrial enz ymes and organ ic acids
(Cairns et al. 2 018 , 2019) . To d ate, fun gi ha ve been a crucial source of the m a jorit y of antibiotics (Liu et al.
2010 , 2012) . As it has rece ntly bee n reprog r ammed to produce secondary metabolites an d pharm ace ut ical
ingredie nts at a hi g h level , A. nige r is of general interes t as a mu l tipurpose cell factory (Boecker et al. 2018) .
Presently , a total o f 17 A. nige r g enomes have been s eq uenced si nce th e first A. nige r g eno m e beca m e
available in 20 07 (An dersen et al. 2011; B ak er 2006; G ong et al. 2016; P au l et al . 2017; Pel et al. 200 7; Vest h
et al . 2018; Yin et al. 20 14; Yi n et al . 2017) . Remark ab ly, all A. niger str ains ha ve hi gh geno me fle xibil it y
and shar e ab out 7,5 00 genes i n thei r co re geno me b ut d iffe r in hund re ds up to t hou sa nds of ge ne s , whic h
define the pan - g e nome and species - uniq ue ge nes , re spec tive l y (Vesth et al. 2018) .
A. nige r LD M3 is an indus tri al gluc oa myla se (GlaA) pr oducti on str ai n whic h feat ure s a ver y high G laA
produc tion leve l and is ph e n ot y pical ly characterized b y a n aconidial phenotype. This is of special interest
because an other high efficient Gla A - produc ing A. niger strain exploited in China (strain SH2 ) is no n -
spor ulating as w ell ( Yin et al. 2 014) . Most interesti ng l y, soli d - state fer m e ntations uncovere d that pro teins are
mainly secreted in the central an d peripheral regi ons of A. niger colo ny but not in the my c elial re gions
undergoi ng sporulatio n, indic ating that spo rulation inhib its p rotein secretio n (Kri j g sheld et al. 2013) . Sw ift
et al . (1998) have alread y proved about 20 y ears ago that the acon idial ph enoty pe of A. ni ger is beneficial to
protein biosyn t hesis an d /or secretion . Several mut a nts wi t h reduced sporulati on were isolated from
maltodextri n - limited c hemostat a nd pH auxostat c ultivation s of A . nige r strain B1 (carry ing 20 copi es of t he
Gla A e nco din g gene glaA ) . Tw o of th ese spon taneou s m orpholog ical mu ta n ts show ed alm ost y ello w and
white colon ies when cultivated on agar plates and presented a sign ifica ntl y imp ro ved G laA produ ction
com pared to th eir paren tal strain B 1, ev en though on e of the mut ants sho wed tha t mo re t ha n ha lf o f the glaA
cop ies w e re lost. Si milarly, Jorgens en e t al. (2011a) obtained two sporulatio n deficie nt A. nige r str ains sc l -1
and scl -2 thro ug h UV - mut age nesi s , in which several secondary metabolites were produced less but secreted
pro tein s we r e re m ar kably accumulat ed . Ho w ever, the molecular m ec h anisms linking protein s ecr etion a nd
ase xua l spor ul ation are n ot fully un derstood so far . I n gene ra l, sp or ulati on - de ficient Asperg illus strains are
kno wn to be d efec tive i n ma ny re gula tor s, inc lud in g the t ransc rip tio n fac tor s (TF s) B r lA an d F lb A. T he

fun ctions of both thes e 2 proteins have been wel l documen te d in A. niger , A spergillu s ni du lans , A spe rgillus
f umigat us , a nd A sperg illus ory zae (A dams et al. 1988; Krijgsh eld et a l. 2013; Le e and Adam s 1996; M ah and
Yu 2006; Pavezzi et al. 201 1; v an M unster et al. 2015; Yamada et al. 1 9 99) . BrlA is the centra l regulator of
conidiophore developm e nt which becomes activated by F lb A (K rijgshel d et al. 2013) . Notably , the deletio n
of flbA gene i n A. niger resul ts i n a fluff y phe not ype, accom p anied by a thinner cell wal l an d a m ore co m plex
secretome (Kr ijgs held et al. 2013) .
T o shed light o n the m o lec ul ar mec hani sms behi nd t he ac onid ial a nd hig h - secretion phenotype of L DM3
strain , we sequenced its gen o me by a hy brid ap proach co mbining PacBio RS and Illu mina Hi S eq 4000
techn o logies an d co m pared this geno me to the Gla A produ cing mode l strai n CBS 513.88. In addi tion, g ene
knoc ko ut exp er ime nts were per for med with t wo ge nes o f o ur inter es t , tupA and prp A , to in vestigate their
impact on protein secretion.
Ma terials and M etho ds
Stra ins and culture
A. niger strains used in this st udy are listed in Table 1. T he A. nige r strain LDM3 w it h aconidial phenot ype
w as kin dl y provi ded by L ongda Biotec hnol ogy (Sh andong, Chin a). C zapek– Dox s lop e and sub mer ged
medium were used to cultiv ate LDM3. The composition o f Czapek – Dox slope m edium is as fol lo w s: sucros e
3%, NaNO 3 0. 2%, MgSO 4 • 7H 2 O 0.05%, KCl 0.05%, F eSO 4 • 7H 2 O 0. 001%, K 2 HP O 4 0.1% , and a gar 1.7% ,
pH 5.5~6 .0. LDM S w as cu ltivat ed at 34 ° C for 5 day s. The co m p ositio n of th e Czapek – Do x sub merge d
medium is the same as the slope m edium s pecified a bove , b ut witho ut a gar. Culti vation was performed at
34 ° C, 180~ 200 rpm for 72 h and t he pelle ts were collec ted by filtratio n.
T he ot her A. niger s trains w e re cultured as follo w s : St rains were g ro w n at 30 °C us ing the co m p lete o r
mi nima l med ium ( Arentshorst et al. 2012) a nd supplemented with 1 mM uridine w here necessary . T o test the
yield of G laA am o ng di ffe re nt mut ants, 10 6 s pores/m L of st rains FW35. 1 (Wanka et al. 2016) , YS3 3. 10
( kusA ::DR - amdS - DR, tupA :: AopyrG , pyrG +) , and YS34.16 ( kusA::DR- am dS - DR, prpA::pyr G , p yrG + ) w ere
in oculat ed i n to 50 mL CM liqui d m edium w i th 3% w/v g l u c ose as the carbon source and cu ltivated at 30°C
and 250 rpm. Samples were taken at 24, 48, 72, 96 , and 120 h af ter i noculati on. Physiological parameters

(dry weight, total s ecr eted protein, residu a l glucose concentration an d enz ym e ac tivity o f GlaA in the m edia)
wer e measured. Experi m ents were perfor m ed in biological quadruplicates.
Geno m e DNA e xtracti on and seque ncing
A. niger transfo r m a tio n, ge no mic DN A ex trac tio n, and Sout her n hybr idi z ation were performed as previous ly
described (Arentshorst et al. 20 12) . Qua lit y anal ysis o f ge nomi c DN A, lib ra ry co nstr ucti o n, and seque nc ing
on PacBi o (RS II) and Ill umina (Hi Seq 4000) i nstrum ents were performed by BG I (S henz hen, China ).
Hybri d assem bly of the A. nige r LDM 3 geno m e se quence using Illu mina a nd PacB io sequenci ng
The dev elopm e n t of sev eral hy brid genom e assembly algorithm s all o w s t aki ng o f reads from multi ple read
sourc es (C hen et al . 2017; Rh oads and A u 2015) . Reads f rom the Illumina platform are sh o rt but accu rate,
while reads f ro m the P acBio ar e long b ut acco m panied by a high er ror rate. Hence, hy br id sequen c ing allows
the u se o f long reads for g eno me assem bl y (PacBio reads) w hile Illu mi na reads ca n be u sed for corrections.
A total of 296,149 s ubreads (2.37 Gb) w ere ge nerated on the P acBi o RS II platf or m w i th an average le ngth
of 8,006 bp, and 6.45 Gb of clean d ata (read leng th 150 bp, insert size 300 bp) w ere generat ed o n the Illumina
HiS eq 4000 platf orm. Th e subrea ds w ere self - corrected and then assembled using Falcon v 0.3.0
( https://git hub.co m/PacificBio sciences/ falco ). T he resu lti ng assem bl y was corrected thr ou gh Illumina reads
us i ng P roof read v 2.1 2 ( https://github.co m/BioInf - Wue rzb ur g/pr oo vrea d ).
Bioin for m atic an alyses
Gene m o dels were predicted using Augustus v 3.2.1 ( http://b ioinf.uni - gr ei fswa ld. de/a ugust us / ) , S NAP v
2010 - 07 - 28 ( http://korflab.uc davis.edu/ software. html ) and GeneMark - ES v 4.28 ( http://exon. gatech.edu/ )
wit h A. niger CBS 513.88 as the reference. G ene structure w as predicted using GeneWise v 2.20
( http://ww w . sanger. ac.uk/So ftware/Wise2 / ) . The predicted gene models w er e f unctionall y annotated by
ali gnin g the ir p ro tein se quenc es a gai nst t he KEGG (Min oru et al . 2015) , SwissPr ot
( http://ww w . gp m a w.com/ html/s w is s - prot.h t m l ) , GO (Ash b u rner et a l. 2000) , COG ( Clust er s of Or t holo go us
Grou ps ) (Galp erin et al. 2014 ) , KOG ( EuK ar yotic O rtho logo us G ro ups ) ( Huer ta - Cepas et al. 2015) , TrEMBL
(https:// www.unipro t.org/ ) , and No n - R edundan t protein databases ( http s:// www. ncb i. nlm. nih. gov/ ) wi t h
BLAS TP (E - val ue ≦ 1.0e - 5) . t RN ASc a n - SE v 1.3.1 ( http: //gt rnad b. ucsc. edu/ ) w a s u sed f or tRNA pre diction .

SNP, InDel and SV ana lys es
High qual ity filtere d short r eads fro m the Illu mina plat form were mapped to the re feren ce gen ome vi a B WA
(Burrow s - Wheel er A li gn er) ( L i and Du rbin 2010) . After filtering fo r Q < 20, th e p aired - end ( PE ) r eads w ere
aligned to all chromoso m es and t he ali gn ed P E reads w i th a distance of > 1000 bp w ere screened f or the
geno me as s e mb l y . GATK v1.6 - 13 ( http:// www.broad institute.o rg/gatk/ ) w as used to d etect s ingle nucleo tide
poly morphi sm ( SNP ) , insertio n and de letion (InDe l) b etw ee n A. nige r LDM3 and th e r ef erence CBS 513.88
base d on hi gh - qualit y align ment res ults. Ra w SNPs and I nDel s were fi l tered under a stringent cr iterio n of
GATK Unified Genot y pe r (Yi n et al . 2014) .
Synt eny an alysis
The assembled A. ni ger LDM3 gen o me sequence was fragmented into 1 kb le ngt h a nd was co m par ed to the
genome sequ e nce of A. ni ger CBS 513 .88 by BL ASTN using t he c uto ff val ue 1 × 10 -75 . The seq uenc e of t he
target fungu s was sorted according to th at of the referenc e f u ngus based on MUMm er alignm e nt result s
(Ku rtz et al . 2004) . Synt en y analysis was p erformed as previously described (An dersen et al. 201 1) .
Stra in - specific gene s in the L DM3 genom e com pared to CBS 513. 88
The imprint al gorithm used to d etermin e st rai n - specific gen e s in LD M3 w as per f o rmed as previ o us l y
described (A nders en et al. 2011) . Using BLASTP ( E- val ue ≤ 1.0e - 10, identity > 50 %, coverage > 80 % ) t he
CDS s of A. niger LDM3 was co m p ared to the amino acids sequence of A. niger CB S 513.88. The
corres pondin g amino acids se q uen ce from CBS 513.88 wa s named as ‘ I mp r i nt ’ . For the com parison o f CDS s
and Impr int, ge ne var iat io ns s uch a s InDel , synon ymous mutatio ns, frameshift mutatio ns, and partial hit f o r
each CDS p air w ere co llected in to gene mutation lis ts. T o e xtract the correspondin g f ull - l ength n ucle oti de
sequen ce of th e genes that w ere not ali gned t o CBS 513. 88 u nder th e above BL AS TP c riteria, genes with
100% alignmen t to t he ref erence w ere r em oved . In add ition, the remaining genes were manually analy zed
for f ramesh ift mu tation, s tart codon an d stop c odon los s, part ial m atch (c over age < 50%), earl y te r mi nation
and no hit s in LDM3 co m pared to CBS 513 .88.

Co - express ion netw ork of TupA and PrpA e ncoding gen e s
From 283 mi cr oarray experiments of A. ni ger hos ted by Fun giDB (Basen ko et a l. 2018) , the co - e xpre ssio n
ne two r k s of Tup A and P rp A enco ding gene s w ere retrieved according to a pr ev iou s study (Schape et al.
2019) . Gen e pairs passing a Spearman ’s correlation coefficient of |0.7| were used to construct co - e xpre ssio n
net work s. F or t he Tup A net wor k , 32 genes were n e gatively c orrelated , w hile for PrpA , th is number increased
to 57 6 ge nes . Bo th T upA and P rpA netwo rks wer e asse ssed for G O - en riched biological process relati ve to
A. niger geno me usi ng de faul t p ara m ete rs in Fun giDB , and the genes of interest were manu a lly filtered wh e n
Be nja m i ni – Hochberg FDR co rrected p - valu es were above 0.05.
Determination of biom ass dry weight, total secreted protein, residual glucose and enzy m e activity o f
Gla A
4 ml broth w as taken at the indicated time points f ro m sh ake fl as k cultures. Biomass an d culture supernatant
were separated by vacuum filtration f o llowed b y 3 tim e s washing w it h dei oni z ed w ater, frozen at − 80 °C,
and freeze - dri ed ove rnigh t for th e d eter m in a tion of biomass. T otal ex tracellular protein in the culture
supernat ant was determined vi a the Bradford as say (Bi oRad , Hercules, CA, USA ) a cco rd ing to t he
ma nufa cture rs’ protocols , and absorba nce (6 00 nm) w as measur ed usi ng t he GloMax® - M ulti Dete ctio n
S ys t e m ( P r o me g a , Madison , USA ). Quantificat ion of residual gl ucose in the cultiva tion mediu m was
perf ormed us ing the G lucose GOD /PAP kit ( H u ma n , Wiesbaden , Germany) according to the manufacturer’s
manual. En z yme activ it y i s expressed in AGI units, wh ic h is relate d to an o ff ic ially assig ned GlaA standard.
On e AGI un it is de f ined as t h e am ount of enzyme t ha t produces 1 µm ol g lucose per m in at 60 °C and pH 4. 3
from the soluble starch substrate. 20 µl sup er nata nt was mix ed w ith 230 µl p - NP G s ubstrate (2 g/l 4-
nitr op henyl α -D- gluc op y ra n oside aceta te buff er pH 4.3, pre - warmed for 5 min at 37 °C). After incu b ation at
37 °C for 20 min , 100 µl of 0.3 M Na 2 CO 3 w as a dded to s top the reac tion and the absorbance w a s i mmediately
measured at 405 nm using a plate read er. The stand ard GlaA fr o m A. nige r (E.C 3.2.1. 2 ; Sigma A ld rich ,
Darmstadt , Germany ) w a s use d to bu ild a st andard cu r ve w ith GlaA enzyme activity=
008 .
0
01 . 0 405 + OD

× dilution rate ( R 2 > 0.999).
Generation of Δ tupA and Δ prp A delet ion str ain s

T o impro ve the ho mologo us r eco mbi natio n ef ficie nc y, t he spl it mar ker metho d and the no n - ho molo go us en d
jo inin g (NHEJ ) deficient recip ient strain MA169 .4 (Carvalh o et al. 2010) wer e exploited . For gene deletions ,
deletio n cassette s contai ning h o m olo gous 5 ′ or 3′ flan ks (~1.5 k b) f or targ e ted integratio n and the selective
marker Aopyr G ( A. oryza e ) were constructed. T hes e were co - tra nsformed in to the pyr G - recipie nt strain
MA 169.4 , and t he tran sformant s were s creene d base d on uridi ne protot rophy . A. niger transf o rmatio ns w er e
carri ed out u sing the prot oplast t ransform ation me thod as described i n Ar en t s h orst et a l. (2012) . T he 5 ′ and
3′ flanks w er e a m plified by PCR with the pri m er s described in Supplementary Table S1 a nd S2. The details
about the co nstructio n of deletio n cassette s we re illustr ated in S upp leme ntar y Fig. S1 a nd S2 , and p ositive
tupA or prpA deletion strains were confi r med th rough diagnos tic PCR a nd S outhern a nal ys is ( Suppl e m entary
Fig. S3 , S4). T he auto no mou sly r epl icat ing plasmi d pMA171 (Carv alho e t al. 2010) was exp l oit ed i n t he
compleme ntation st udies. The ORF o f t upA inclu di n g approxim ately 0.6 k b promot er and 0.6 k b term inator
re gions wa s amplified takin g N40 2 gen o mi c DN A as template and cloned into Not I- linear is ed pMA171
(Sup pl eme ntar y Fig. S 5 ) . T hen the constructed p las mid pMA171 - t up A was tra nsfo rme d int o the Δ tupA
deletio n m utant. P ri mar y tra nsfo r m a nts c onta ini ng t he c o m p le ment at ion p las mid wer e purified o n M M
med ium conta ini n g 100 µg/ ml o f hygro myci n and fur the r an al y z ed b y d iagnostic P CR.
Data access
The complete chromosomal sequence of LDM3 is available at the GenBank under the assigned accession
num ber VTFN0000000 0.
Result s
Characteristics of A. nig er LDM3 genom e
In o rd er to id entif y the gene tic de ter minant s re spo nsi ble f or the u nique p he not ype of LD M3 , t he e ntire
genome of LDM3 was sequenced us i ng a hybrid appro ach t hat co m bi ned Pacific Biosci ences wi t h Illum i na
seq uenc ing , ob taini ng 6,447 Mb a nd 2,679 Mb data af ter fi lterin g fr o m the Illumina HiSe q 4 , 000 and PacBi o
RS II platform respectively. The hig h - qualit y reads were further used to asse m ble the genome of LDM3 a fte r
qualit y co ntrol , resul tin g in a 35 .2 8 Mb ge no m e seq uenc e w ith 1 1 s caffolds and a seq uenc ing de pt h of 1 77 ×
(Table 2). The assem b led genome base calls were corrected w ith Illumina h ig h- qu ality PE read s. A to tal of
11,209 O RFs w ere identified in LDM3 ( 94% of the gen es w ere auto mati call y ann otated based on protein

databa ses wi t h an average gene length of 1,691 bp ), displ ayin g a lo w er gene density (0.32 gene/kb) compared
to C BS 513.8 8 (0.42 g ene/k b) (Anders en et a l. 2011) ( Su pplem entary T able S3).
KOG analysis w as empl o y ed to id entify their b iological ro les . Ou t of the 11, 209 predict ed protei ns, 9,7 12
ORF s (87%) were ass igned to 2 4 KOG fun c tional cate gories in total ( S upp le m e nta ry Fi g. S 6 ). I n LDM 3,
slig htl y m o re gene s wer e allocated t o term G ( C arbohydra te trans port and m etaboli sm), A (RNA processi ng
and modi fication), L (Replicatio n, reco m b ination a nd repa ir), O (P osttranslational mod ification, prote in
turno ve r, c hap er ones ) and to t he unknown functi o nal gene clas s (S) com p ared to the c ontr ol . Both LDM3
and C BS 513.88 sh o we d a co m parable nu mber of predicted genes d istributed in each term, defin ing a hig h
geno me similarity. 264 tRNA g enes were identified in LD M3 , whic h we r e comparable to other en zyme -
produc in g stra i n s CBS 513.8 8 (269) an d SH2 ( 267), but m ore than oth er Aspe rgillus s tra ins inc lud in g A.
nidulans (188) a nd A. fumigat us (179) (Yin et al. 2014 ) .
Genome structure v ariation analysis
Stra ins L DM3 and C SB 513.88 s hare w idely dis tribut ed synt e ni c blocks, a ccounti ng for 96.56% of th eir
gen o m es. The dot pl ot depi cted in Supp le menta ry Fi g. S 7 s ho w s cons erved sy nteny between the t wo strain s,
reflecti ng a close p hylogenetic r elationship. Ho w ever, the synteny map illust rated re m arkable chromosomal
re arr ange m e nts fo r the 8 chr omoso mes (19 supe rco ntigs) of CBS 5 13. 88 (Fig. 1). T he L DM3 gen o m e was
ass e m bled int o 11 scaffolds , resembl i ng a hi gher qua lit y of assem bly tha n th e 19 supercon tigs of CBS 513.88.
T he entire length s of 4 out of 9 scaff olds in LDM 3 reached a long er or sim ilar le ngth com pared to thei r
corres pondin g c hrom osomes in CBS 513.88. S caff old s 1 an d 2 are la rger than a ny chr o m o so mes o f the
reference strain (6.0 Mb in maximum) reac hi n g 7.6 Mb and 7.5 Mb, respectively . This s u ggests a fusion with
other chromosomes, repr ese nt ing a noticeable st r uctural variation in LDM3. In addition, scaffold 7 and 8
com pose only t he t hird supe rco nt ig i n CBS 513.88.
Inte res ting ly, i n gene ral, 2 G laA enc od ing gene s can b e i dentified in most publis hed A. niger ge no mes,
n am ely glaA ( An 03g06550 ) and glaB ( An 12g03070, sh a rin g 25% identity with glaA ) , in w hic h glaA being
m ore strong ly expre ssed th an glaB (A nderse n et al. 2011; Sch ape et al. 2019) . This is also the ca se in
CBS513. 88 and SH2 , wher e onl y si ngle co p y of glaA and glaB are present in their ge no me s . H o w e ver , onl y
a single copy of glaB but no gla A is pre sent i n the LDM3 genom e , a nd is found w ithout any mutan ts compar ed
to that of t he co ntro l .

LDM 3 st rain - specific genes analysi s
A n ali gn ment o f t he t wo geno me seq ue nces of L DM3 and CBS513.88 show ed so me uniq ue re gio ns in
LDM 3 , incor pora ting 457 protein - e ncod ing ge ne s ( Sup ple ment ar y Table S 4). These include 19 6 O RFs with
frameshift mutatio ns, 17 OR Fs i n whi ch the start or stop codon w as lost, 81 ORFS with partial matc h
(coverage < 50%), 4 ORFs with e arl y ter mina tion a nd 157 O RFs which d id no t matc h the CB S 51 3. 88
sequen ce ( 75 we re annotated as hy potheti cal or of unknown functio n ) . To further characterize these strain -
specifi c genes in L DM3 , w e c om pared th eir sequences w it h pu b licly available genomes of other A. nige r and
Asp e rgillus str ains , de monstratin g tha t o rtho lo gs o f 49 out of 457 strai n - specif ic genes were id entified in a ll
the compared gen o m es ( Supplem entary T able S5) . GO anno tati ons f or 225 out of 457 OR Fs are available ,
wh ic h are e nrich ed i n catalytic activity , oxidoreductase activity, hydrolase activity , transferase activity,
pro tein binding, oxida tion - redu ction process, transporter activity , and localization G O ter ms ( Supplem entary
Fig. S 8 ). Am ong th e gen e s a nnotated in 2 signif ica ntl y enri ch ed GO term s o xidoreductas e activity (36 genes)
and oxidation - reduc tion proces s (30), 7 gen es could not be a lign ed to the ref ere nce g eno me, 7 ge nes s ho wed
frameshift mutations, and 1 gene wa s mappe d partiall y t o CBS 513. 88 gen o m e ( Supplementary T able S 6 ).
T he 7 unalign ed genes w er e mainly predicted to fun ctio n in a min o acid metabolic pathw a ys, including
degr adati on of prol ine, i soleucin e an d leucin e ( AN2_G LEAN_1000016 3, mmsB predicted as 3 -
hy droxybuty rate hy drogenase ), bios ynt hesi s of alanine, aspartic aci d, and g lutamic acid
( A N2_GLEA N_10000741, gabD predicted as succin ate - s e m ialdehy de dehy drogenase which supplem e n ts
succinic aci d for the T CA cycle (Y i n e t al . 2017) , and t he bi os ynthes is o f ar gi nine a nd pr oline
(A N2_G L E A N_10007014, pr oA pr e dicted as glutamate -5- se m ialdehy de dehy drogenas e).
SNP and I nDel anal ysis
Compared to those in the referen ce gen o m e of CBS 513.88, a tota l of 2,138 S NP and InD e l mutation s are
pr esent in the ge nome o f LD M3, in whic h no n - s ynon ymo us muta tio ns are distribute d in 656 O RFs
( Supp le menta ry T able S 7 ). KOG cl u stering a nalysis uncovered that the mutated genes are m ai nly cl ustered
in A ( RNA process i ng a nd m odifi cation ), C (Energy produ cti on and conv ersion ), E (Amino a cid
tran sportat ion and m etabolism ), G (Carbohy drate t ransport an d m etabol ism ), O (Postt ransl ati onal
m odification , protein turnov er, chaperon es), J ( Transl ation , r ibos omal struc tur e and bio gen esi s) and I ( Lipid

transpor t and metabolis m ) ( Fi g. 2) , all o f w hic h are ver y f und ament al fo r a hig h level of pr otei n expr essio n,
pro tein targeting , and secretion. Selected genes of interest are depicted in T abl e 3 and w il l be discu s sed in
detail in the next sectio n .
In vivo anal ysis of t wo select ed gen es putati vely invol ved in s porulati on
Give n the poss ible li nk betwe en the aconi dial an d high enzyme produ ction phenoty pe in A. ni ger , w e decided
to st udy t he func tion of tw o gen es of our i nterest ( tupA , An 15g00140 and prpA , A n18g 01170) am ong the
656 m utat ed genes i n LDM3 com pared t o CBS 513. 88. In doing so, w e se lect ed the lab s train M A 169.4 as
the p arent al strain , wh i ch is dev oid of th e NHEJ pa thw a y a nd th us ensure s a hi gh er h omol o gous
recombination rate (Ca rvalh o et al . 2010) . T up A (no n - syno ny mous) is a globa lly active tr anscriptio nal
repress or ( orth olog of t he repress or Rco - 1of Neuro spo ra c rassa a nd T u p1p o f S . cerevisiae ) . Notabl y , its
deletion has been shown to cause an aconi dial phe notype an d a r educed g ro w th rate in N. cra ssa (Ya m a shi ro y
et al . 1996) and A . n iger (Schach tschabel et al. 2013) . T hus , the p re sent m ut at ions in T upA prom pted u s to
inve sti gate the func tio n o f tupA in the uni que p heno type of LDM 3 . Prp A, a gene of un kno wn funct io n , is
absent in LDM3 . Its exp re ssio n is ind uc ed b y brlA and abaA - dependen t regul atory l oops i n A. niger a nd is
predicted to cause the aconidial phen o ty p e in the A. niger SH2 strain ( Yin et al. 20 14) . Bl ast result indicate d
that the protein sequence of TupA carrie s a 16 amin o acids i nsertion and on e a m i n o acid c hange from glyc ine
(G) to aspartic acid ( D ) ( Supplem entary Fi g. S 9 ).
To p redict the function of tupA and prpA in A. nige r , w e ha rnes sed o ur recen tl y publis hed gen o m e wide co -
expression database available on FungiDB to construct co - expres sion n etwork s for t upA a nd pr pA (Schape
et al . 2019) . T he co - expr essio n ne twor k o f tupA show ed an exclusively ne gat i ve co rrelation w i th t he ge nes
pred icted to function in p rotein secretio n, filame ntous gro wth, vesicle - mediated transpo rt, cellular protein
metaboli c process , and f unga l - ty pe cel l wall organizati on or b iogen esis (Fig. 3a), and this is consistent with
its functio n as a tra nscriptiona l repressor . Similarly, prpA sho w ed nearl y no positiv e co rrelations with other
gene s b ut surp ri sin gly a ne gat ive c or rela tio n with a high number of ge ne s (5 67 ). GO e nric hment a na lysis
reveal ed that the pr pA net wor k is m a inl y enr ic hed i n mitoc ho ndri on o rganiz a tion, pr otei n tar geti ng to t he
mitoch o ndrion and protein catabolic processes, n am el y asso ciated with e ne rgy ge nera tio n ( Fig. 3b). Notably,
whereas t he tupA n et w ork co nt ained overrepresented GO terms associated with g r owth or sporulation, th is
was not the cas e for the prpA co - expr ess ion ne t w or k ( Supplem entary T able S 8 , S 9 ).

To stu dy the impact of both ge ne s on s p orula tion and p rotein secretio n in A. nige r , s i ngle gene knockout
str ains were constru cted in MA169. 4 us i n g the spli t m ar ke r approach a s p ublis hed prev iously ( Fiedler et a l.
2018b) . As depicted i n Fig . 4 an d Fig. 5, the d eletion of tupA severely reduce d the mycelial g ro w th rate and
spor ulation efficienc y o f A. nige r , w h ich was not the case for th e prpA null muta nt. All Δ tu pA - c o mp l e me n t ed
transfor mants obtained gre w like the wild - t yp e, confi r min g that th e severe gr owth and spor ulation defect
of m u tant w as caused by the tupA deletion and t he pl as mid - based tupA ge ne is capable to near ly restore
the p heno typ e (Fig. 4). How ever, the Δ t upA - co m ple mented strain w a s s till witnessed a weak er grow t h rate,
sugge sti n g tha t the cell ula r a mo unt o f tupA is un der st ringent con trol . T he sporulat ion capacity of ∆ tupA wa s
redu ced by a bout 35% com pared to the referen c e st rain FW35.1, but Δ pr pA pr esen ted ne arly no difference
com pared to th e r ef er ence s train (Fi g. 5) . It has t hus be en co nfir med that k nocki ng out tupA in A. nige r
strongl y inhibits the gro wth rate of the stra in, which i s consi stent with a p revio us report (S chach t schabel et
al. 201 3) .
In order to determ ine whethe r th e sporulation def ect i n t he tupA nu ll mutant c ould impro ve the pro tein
produc tion capacit y of A. nige r , the reference s trai n FW35.1 , Δ prpA, and Δ tupA were cultured at the s hake
flask level. Pairwise co mparison of value s for ∆ prpA and parental s train gave comparable results in pr otein
secretion. While c ompared to the ref erence strain FW35.1 , the growth rate o f Δ tupA w as slo w er in th e ear ly
stage of f er mentation which was consisten t with the f i n dings of a previ ous report (Schachtschabel et al. 2013) ,
but dramatical l y increased after 24 h (Fig . 6b). Dur ing the fla sk - leve l fermentation, the reference strain
sh o w ed almos t n o color cha nge, but the brot h color of th e Δ tupA muta nt sud de nly t urne d yello wish a fter the
four th d a y a nd b ro wn on the f ifth da y (Fig. 6a). Hen ce, d uring t he submerged ferm e ntati o n process, there is
a variation i n g ro wth and physiological ch aracteristics bet w e en th e mutant strain an d th e reference.
As i t can be seen in t he Δ tupA f er m e ntation re sults, in t he firs t 3 d ays (e xpo nent ial a nd ear ly st atio nar y pha se
of fermentatio n) , Δ tupA secr eted much less protein especiall y Gla A whic h was i ndeed not detectable
com pared to the parental strain (Fig. 6c, d). How ever, af ter prolong ed cul tivation (day 4 - 5), th e amoun t of
extracellular protein wa s cons i dera bly accumulated in t he Δ tupA strai n and was sig nifi can tl y highe r t han t hat
in the c ontrol (i ncreased by about 67% at da y 5). In partic ular, the enz yme activit y of Gla A at 70 h post -
ino culat io n sho wed up to 54 - fol d i ncrease co mpared to that at 45 h , therefore the dramatic accumulation of
Gla A c ontributed m o stly to the increase of extracellular secreted protein.

Discu ssion s
In li ght o f the depen dence of su perior protei n capacity on de fective spo rulation , w e report ed in thi s st ud y a
hig h - qual it y asse mb led geno me o f an ind ust rial l y rele vant A. niger strain LDM 3 us ed for GlaA produ ction .
This was ach ieved b y a hybrid sequencing approach w hich u tilized Illu mina a nd P ac Bio seque ncing
tec hnolo gie s. Owing to its u ni que phenoty pe a nd high protein produ cing potential , i t is o f great intere st to
both th eo retical research a n d biotech i nd ustr y to explore the novel functional p roper ties of LDM 3 . Gene
functio nal annotatio n ( Su pple m entary Fig. S 10 ) co nfir m ed that the m ajorit y of genes w ere significantly
allocated in catal ysis , transpo rt, translatio n, carb ohydrate m e tabolis m , and ami no acid meta bolism, which
matched we l l wit h t he high - yield protein -p rod uci ng character is tic of LDM3. In additio n, a n incre as ing
numb er o f tR NA ge nes mig ht s ugge st t hat A. niger c ould re ly on a hig her t ransl ati on e ffic ie ncy i n
com parison t o A. nidulans and A. fumigat us .
Inte res ting ly, gl aB is witne ssed as the o nly s i ng le copy of t he GlaA enco di ng gene in L DM 3 , alb eit gl aA
behaves dominatingly in the majority of characterized Aspergillu s geno mes whil e being absen t in LDM3.
Pr evio usl y rep or ted that glaB displ a y ed div erse expres sion pattern s in A. o ryza e under disti nct culti vation
conditio ns, and wa s strikingl y expressed in solid - state cultures b ut s ho wed little or no e xpression in
sub mer ged cultivatio n (t e Biesebeke et al . 2005) . gl aB is regula te d at the tra nscriptional l evel, which could
be enhanced by star ch, low water activit y , hi gh t emp era tur e a nd li mited h yp h al e xtension (Kumar and
Saty anaray a n a 2009) . Similarly, it wa s also ind uced b y iso maltose i n A. nidula ns (Nakam ura et al. 2006) .
Giv e n t h e abundan t ex pression profile s of glaB , its performan ce in LDM3 requires further de scription in
sub mer ged c ultur es.
LDM3 and CBS 513.88 exhib it ed a clo se phylogenetic re lations hip , shar in g 97 % id enti ty. Ho we ver, the
uniq ue r egio ns b et ween t he two genome sequences mainly d efine th e geno me d iver si ty . Amon g the 872 TF s
predicted in A. niger (Par k et al. 2 008 ; Szkl arc zyk et a l. 2 017) , 9 T Fs thereof carr y InDel mutatio ns in LDM3
( Supp le menta ry T able S 10 ). A mong thos e is CpcA , w hic h is involv ed in th e degradati on of mi s f olded
protei ns an d p la ys a role in RNA processi ng and trans lation proces ses by th e endoplasm ic ret iculum protei n
respon se pat hw ay ( Jorgen sen et al . 20 09; Wan ke et al. 1997) . Cpc A is the f unctio na l o rtho log o f the
Saccharomyces cerevisiae transcriptional acti vator Gcn4p in Asperg illus and ind uces t he e xpr essio n o f
multiple ge nes associated with a mino acid b iosynthesis un der a mino acid star vation co nditions

(V ongsa ngna k et a l. 2011) . Previous reports indeed assumed th at a fra m e shifted cpcA ge ne mi ght be t he
caus e of e fficient expre ssion of the GlaA encodi ng gene gl aA and othe r enz ymes in A. niger (Andersen et al.
2011; Yin et al. 2014) . It is thu s very temptin g to speculate that this is al so the case wi t h LDM3.
Moreover, 41 out of 60 TFs c onta ini ng SNP displa y n on - synonym ous mutatio ns ( Supp le ment ar y Table S 10 ) .
A wid e ra nge of r egula to ry pa thwa ys, i ncl udin g star ch de gra da tion, cel l w a ll synt hesi s, nit ro gen a ssimi lat ion,
and ami no ac id s ynthes is p ath ways wer e affect ed. AmyR ( An 04g06910), for ex ample , is an essentia l m alto se -
dependent TF t hat regulates the expression of s tarch hy d rolase gen es s uch as extracellular hy dr olases
incl udi ng al pha - am yl ase A amA , al ph a - gl ucos id ases Agd A and AgdB , and GlaA. The consen s us sequence of
DNA bi nding of AmyR to the promoter re gions of its target gene s is well known ( CGGN8( C/A)GG ) (Yua n
et al . 2008) . S uch a sequence can indeed be found at position - 878 bp u pstream of glaB, sugge st ing t hat the
glaB gene i n LDM3 might be un d er tran scriptional co ntrol of A my R as in ot her Asperg ill us .
The majority of transporters carry i ng SNP mut atio ns belong to the MFS family of secon dar y active
transpor ters and facilita tors ( d e Vries et al. 2017) , su ch as glucose tran sporters An 15g04270 and An 11g09600
which m a y be beneficial for the uptake o f su b strates in LDM3. I n addition, there are 3 neutral amino acid
tra nsp orters A n07g 03690, An07g 03970 , a nd A n14g 07130 distrib uted non - syn onymous m utat ions. Ala , Leu ,
T hr , a nd Ser ar e the to p 4 amino acids of Gla A , a nd int erestingl y , all o f them are neutral amino acids
( Supp le menta ry T able S 11 ). It can thus b e po sited that the variation of neutral a m i no acid transpor ters coul d
be relev ant to the tra nsport of these 4 p r i ma r y am ino aci ds, to su pport the tra nslation of glaB mRN A.
Within t he 465 gen e s predicted to function in the secretory pathways of A. niger (Carvalh o et al. 2012;
Guillemette et a l. 2007 ; J orgensen et al. 200 9; Kw o n et al. 2 012) , 3 t a ke I nDel mu tations and 25 contain no n -
syno ny mous SNP s i n LDM3 ( Su ppl em ent ary T able S 12 ). M ost ge nes ar e invo lve d in pr otein transpor t,
unf olded protei n respon se, glycosy lati o n, and starch metabol ism , or ar e protease enc od ing gene s. T wo ge nes
wit h non - s y nonymous mutat ions a re worth me ntion ing: An07g 02190 ( S. cerevis iae sec7 orthol og) pl a ys a
predicted role in vesicles tra ffic in i ntra - Gol gi a nd E R - to - Golgi transport ( Wolf et al. 1998) and An08g 00290
( S. cerevisiae rud3 or tholo g) i s a matr ix p ro tein o f t he Go l gi and essential for its i ntegrity ( Gillingha m et al.
2004) .
Proteases are capable of hy dr oly zing protein peptide chains, w hich may be detri m e nt al to the accumulation
of secreted pro tei n. T he 3 mu t ated protease genes ( pepC , pepF and cpy 1p ) in L DM3 are all serine proteases.

Since the do m ina ting statu s of s er ine and thre o nine i n the compo si tion o f GlaA ( Sup ple me ntar y Table S 11) ,
the red uced degradation of serine enriched proteins cau sed b y t he m utations of t he serin e pr otease g enes ha s
aroused the in terest to m erit furthe r expl or ation in t he fu tur e .
The fungal cell w all determines the hyphal morp hology, cell ular integrit y , an d prote in secretion pro ductivity
dur ing the gro wt h and deve lop m e nt of A . niger (C airns et al. 2019) . Var iations in cell wall co m p osition and
my celial m orphology of A. nige r can t hus suppor t prote in pr oduc tion (Fiedl er et al. 20 18a, b) . For e xampl e,
gelB (An08g 07350) e nco ding a GP I - an chored glu cosyltr ansf erase im portant for β - 1,3 - gluc an s ynthe si s , i s
aff ec ted (no n - syn onymous) ( Supp le menta ry Tab le S 13 ). D eletion of gelB i n A. fum igatus caus e s the
re duc tio n of β - 1,3 - glucan cell w a ll levels accom p anied by abnor m al germination, decreased gro wt h a nd
def ici ent pigm ent biosy nthes is dur ing sporu lation (Mouyn a et al. 2005) . How ever, it has not been reported
so far whether mutations on the gluca n biosynthesis p athway lea d to the aconidi al phen otype of Aspe rgillus .
Ano ther i nte rest in g muta te d gene is a ch iti n synt hase en co ding ge ne An 02g02360 ( csmA ) with no n -
syno ny mous S NP s in LDM3 . Or tholo gs ar e kno wn for A. fumigatus ( ch sE) and A. nidul ans ( csmA ) a nd their
deletion phen o ty p es have been studied. Deletion o f the A. fumi gatus chsE gene p ro vok es a bnormalities in
hy p ha l m orphology , sporula tion, an d redu ce d spore survival rate (Aufauv re - Brown et al. 1997 ; Jim enez -
Orti gosa et al. 2012) , a nd deletion of the A. nidul ans csm A induc e s balloon - like s wo llen h ypha e a nd
intrahypha l hyphae for m atio n (T akes hita et al. 2002 , 2 00 5) . These observatio ns in d icated that chitin is
esse ntia l fo r m ai ntai ni ng co nid io pho re a nd sp ore integr it y ( J i me ne z - Ortigos a et a l. 2012) . H o w e ver , ot her
chit in s yntha se ge ne kno cko ut mut ant s ( chsA, chsB , ch sC, ch sD , chsG , a nd ch sF ) did not introdu ce abnormal
spore f or mation in A. fumi gatus (Mell ado et al. 1996a , b , 2 003; Rog g et al. 2011) . Interestingly, i n additio n
to th e aconidi al ph e n ot y pe, LDM3 als o under we nt a u nique m orpholog ical tra nsition pha se durin g the late
sta ges of su b me rged biorea ctor cul tivati ons, w hich is n ot comm o n t o other A. ni ger stra ins ( S uppl eme ntar y
Fig. S 11 ). In f ed - ba tc h culture s , LDM3 mycelia bega n to swell at their tip s during o xygen limitatio n, follo wed
b y m y celial fragme ntation and separatio n of dispersed mycelial struc tures into smaller entitie s (data not
sho wn). T his find i ng ass ume d that t h i s m orphologi cal res pons e improve d o xyge n tra ns fer . No tab ly, Gla A
dramatically acc umul ate d in the supernata nt, suggesti ng that hyphal s welling and fragmentatio n are
im portant for h igh GlaA pr oduction or release into the medium (data not shown) . It is thus temptin g to

speculate t hat the mutation o f csmA ma y b e rele va nt to the m orp h olog ical adapt ation of LD M3 during fe d -
batch fer mentation.
It is well kno w n t hat m ost spe cies o f Aspergilli us reprod uce asexually . A central reg ulatory pathway ( brlA
→ abaA → we tA ) is conserv ed in all Aspergillu s and Penicillium g eno mes and controls co ni d ial - speci fic
g ene expr ess ion a nd ase xua l sp or ulatio n (d e Vries et al. 2017) . BrlA is required to activate abaA a nd wetA
(Yin et al. 2014) . FlbA is the central regu lator , contro lling the bin ding of FlbB an d BrlA to the G - protein
coupled receptor , whic h re pr esses t he gr o wth o f veget ati ve m ycelium. T yp ic all y, a deletion in f lbA in
filame ntous fungi induce s ab normal conid iation (Lee and Adams 1994; P erez -de- Nanclares - A rregi and
Etx ebeste 2014; v an Munster et al. 201 5; Wieser et al. 199 4) . Moreover, d ou ble delet ion of brlA and flbA
re sult s in the fluffy phenoty pe of A. niger (v an Mu nster et al. 2015) . In LDM 3, o nly syno n ymo us muta tio ns
were distrib uted in the respective flbA h o m olog an d no mut ation in brlA or flbB wa s identi fied , th ere f ore
these 3 gen es are lik ely not rela ted to th e aconi dial phenotype of LDM3. An 12g02050 ( wA ) is i nvolved i n
th e process of c onidial developm ent in A. niger and is requi red for pigm e nt synthe s is . Deletion of wA leads
to A. niger co lonies w ith whi te or faw n colore d spores (Jorge nsen et al. 2011b; Zhang et al. 2016) . As th e wA
gene of LDM3 carries a no n - s yno nymo us SN P muta tion, it is wor th inv est igat ing in mor e d eta il in t he fut ure.
In vie w of t he a bo ve anal ysi s , w e fi nal ly fo cuse d o n two gene s, tupA and prpA to determine whet her the y
mediate both the aconi d ial and high - secr etio n p he not y pe s i n A. niger , and we select ed a conidial l ab strain as
the recipient for knock o ut studies. Our data sugges ted that t he knock out of pr pA barely affect ed sporulation
and protein secretion , while the knock o ut of t upA r educ ed gro w t h rate and spo rul atio n, t hat was par alleled
by increas ed secretion cap acities . Analysis of publicly avai lable transcripto m e data for Δ tupA in A. niger
(Schach tschabel e t al. 2013) uncovered th at the majority o f P rtT - dependent proteases were s ignificantly up -
re gulate d dur i ng t he early exponential phase, for instance, the express io n of pepA and pepB s howed an u p to
224 - and 99 - fo ld increase, res pectively . I n addition, s everal GO terms associated with amino acid
biosynthet ic o r met abolic processes were do w n - re gula ted , such a s br anc hed - c hai n famil y ami no a cid
biosynthet ic p rocesses , coenzym e b iosyntheti c p rocess, and pro tein secretion (Schachtschabel et al. 2 013) .
This expr ession data indicates that the low - leve l protein producti on during t he early fermentation phase of
Δ tupA co m pared to the reference st rain might be due t o the accumulation of extracellular proteases and
weaken ed amin o acid biosynt hesis . It has been re ported that a liquid cult ure of A. niger conidiat es abun dantly

at the cost of pr otein secretio n when it e nters carbo n starvation (Jor gensen et al. 2011a) . T he exp erime nta l
re sult s sho wn in Fig. 6 ma t c h es thi s ob serva tio n, fur the r su ggest ing t hat r ed uced coni d iation may increase
protei n product ion in A. niger in par allel . Ho wever, thorough transcriptome and secretome analyses from
biorea ctor s ampl es are n ecessary to prove or dis prove this a ssum ption.
In c oncl usio n, c o mpar ative ge no me ana lysi s in t his st udy re veal ed sev er al hundred s of uniqu e and m utated
gene s in LDM3 , so me of whi ch mi ght b e asso cia ted with i ts a conid ial pheno typ e a nd th us it s high Gla A
secretion capaciti es. The se d ata hi ghli ght that no vel hyp othe ses r egar din g the l in k bet wee n spo rulat ion a nd
pro tein secretion in A. nige r can be glean ed f r om com parative gen o mics. Other p otential lead i ng gene s ca n
be levera ged in the future for systematic o ptimizatio n of protein prod uction capacitie s in diff er ent A. niger
strains .

Notes
Ac k no wle dgements
Yufei Sui is grateful for a joint - PhD fello ws hip b y t he Chi ne se sc holar shi p co unc il.
Funding
T his work wa s funde d by the Basi c Rese arch Progr am of Sh enzh e n (JCYJ2015 0629165 423751) a nd the
Fund a m e ntal Rese arch Fu nds for t he Ce ntra l Uni ver siti es N o . 222 218 1 8014.
Autho rs' contri bution s
YFS, LM O a nd SC pe rfor med the ge no mic s a nalyses and execut ed in silico quali t y anal yse s. Y FS a nd T S
constru cted the co - expression networks. YFS and TS generated deletion strains and characterized them . YPZ
and VM initiated this stud y a nd coo rdinated the p roject. YFS , LMO a nd VM co - w ro te the final te xt. All
auth o rs read and a pprov ed the f inal m a nu script.
Ethics appr ova l and consent to partic ipate
This ar ticle does not co ntain an y studies with hu m a n parti cipants or animals perform ed b y any of the aut hors.
Consent for public ation
Not ap plicable
Competing interests
T he autho rs d eclare that they have no competing financi al i nteres ts.

Referen ces
A dams TH, Bol an MT, T im berlak e WE (198 8) BrlA is ne ce ss ary and s uffici e n t to di rect con idiophore
de velo pment i n Asper gillus nidulans . Cell 5 4 : 353 - 36 2. doi: 10.1016/0 092 - 8674( 88)90198 -5
Andersen MR, Salazar MP, Schaap PJ, van de Vondervoort P J, Cu lle y D, Thy kaer J, Frisvad JC, Nielsen
KF, Albang R, Al ber m a nn K, Berka R M, Braus GH , Braus - Stro m e y er S A , Corrochano LM, Dai
Z, va n Dij ck P W, Hof mann G, Las ure LL, M agn uso n JK , Me nke H, M eij er M, Meij er SL , Niels e n
JB , N ielse n ML, va n Oo yen AJ , Pe l HJ , Poul sen L, Sa mso n RA, S tam H , T sang A, va n de n Br ink
JM , Atki ns A, Aert s A, Shap ir o H, Pa ngilina n J , Sal amov A, Lo u Y, Lind quis t E, Luca s S,
Grimwood J, Grigoriev IV, Kubicek CP, Martinez D, van P ei j NN, Ro ubo s J A, Niel sen J , B aker
SE (2011 ) Com parative genomi cs of cit ric acid produ cing As pergill us niger A TCC 1015 v ersus
enz yme - produ cing C BS 513.88. Gen o m e Res 21 : 88 5 - 897. doi: 10.110 1/gr. 112169. 110
Are nts hors t M, Ra m AFJ, M eyer V (2 01 2) Us ing no n - ho mol ogo us end - jo ini ng - deficie nt strains for
func tio nal ge ne a nal yse s in f ila men tous fun gi. Me tho ds M ol B io l. 83 5 : 133– 15 0. doi: doi:
10.1007/ 978 -1- 61779 - 50 1 - 5_9.
Ash b u rner M, Bal l CA, Bla ke JA, Bots tein D , Cherry J M (2000) G ene on tology : Tool f or th e unific atio n of
biolo gy. Nature Genetics 2 5 : 25 - 29. d oi: 10. 1038/ 75556
Auf auvre - Brow n A, Mel lado E, Go w N, Hol den D (1997) Aspe rgillus fumigatus ch sE : A gene related to
CHS3 of S accharomyces cerevisi a e an d i m portan t for hy p hal g ro w th and c oni diophore
development but not pa thogenicit y . Fun gal Genet Bio l 21 : 141 - 152. doi : 10 .1006/f gbi.199 7.0959
Baker SE (2006) Aspergillu s niger gen o mics: past, present a nd into th e future. Med My co l 44 Suppl 1 :
S17 - 21. doi: 10.1 080/1369 3780600921 037
Ba senko EY, Pul man J A, Sha n mugas un dr am A, Harb OS, Crouch K, Starns D, W arrenfeltz S,
Aurrecoechea C, S toeckert CJJ, Kissinger JC, Roos DS, Hertz - Fowler C (2018) Fung iDB: An
integrated bioinf or matic resource for fun gi and oo my cete s. J Fung i (B asel) 4 : 39 - 67. doi:
10.3390/ jof401 0039
Boecker S, Grätz S, Kerwat D , Adam L, Schirmer D, Richter L, Schüt ze T , Petras D, Süssmuth RD, Meyer
V (201 8) Aspergi llus nige r i s a s uperior e xpress ion host for t he produ ction of bioacti ve fung al
cyc lodepsipe ptide s. Fung al Biol Biote chnol 5 : 4- 17. d oi: doi : 10.1 186/ s40694 - 018 - 0048 - 3.
eCol lection 2018
Cai rns TC, Nai C, M eye r V (2018) How a fungu s shape s biotechn ology: 100 years of As pe r gill us niger
research. Fungal Biol Biotechn ol 5 : 13 - 27. doi: 10.11 86/s40 694 - 018 - 005 4 -5
Cair ns T C, Zheng X, Z heng P , S un J , Me y e r V (2019) Moul ding t h e m o u ld: under stan ding and
re pro gra mming fila ment ou s funga l gr o wth and mor pho gene sis for ne xt ge ner atio n cel l fa ct ori es.
Biote chnol Biofuels 12 : 77 - 96 . doi: 1 0.1186/ s13068 - 019 - 14 00 -4
Carvalho ND, Arentsh o rst M, Kwon MJ, Meyer V, Ram AF ( 2 010 ) Exp and ing the ku70 to olbox f or
filame ntous fungi: estab lishm ent of compleme ntation vecto rs and r ecipient strai ns for adva nced
gene analyses . Appl Microbiol Biotechnol 87 : 14 63 - 1473. d oi: 10.1007/ s00253 - 01 0 - 2588 -1
Car val ho ND , Jø rge nsen T R, Are ntsho rs t M, Nitsche BM, van den Hondel CA, Archer DB, Ra m A F
(2012) G enom e - wid e e xpr essi on a nal ysis upon c on stit uti ve a ctiva tio n of t he H ac A bZIP
transcriptio n factor in Aspergill us niger r eveal s a coo rdinated cell ular respons e to counteract ER
stre ss. BM C Geno mics 1 3 : 350 - 367. d oi: 10. 1186/147 1 - 216 4 - 13 - 350
Che n CC, Gha ffar i N, Q ia n X, Y oon B J ( 201 7) Opti m a l hyb r id s eque nci ng and asse mbl y: Fe asib ilit y
conditio ns for accurate genome r econstructio n and cost minimizatio n strate gy. Comput B iol Chem
69 : 153 - 163. doi: 1 0.1016/ j.com pbiolchem .2017.03.016
de Vr ies RP, Riley R, Wieb enga A, Aguilar - Osorio G, Amillis S, Uc hima C A , Ander luh G, Asadolla hi M,
Askin M, Barry K, B attaglia E, Bayram O, Benocci T, Braus - S tro meyer SA, Caldana C , Canovas
D, C erq ueir a GC, Che n F, C he n W , Cho i C, Clum A, D os Sa ntos R A, Dama sio AR, Dial li nas G,
Emri T, Fekete E, Flipphi M , Frey berg S, Gallo A, Gourn a s C, Habgood R, Hainaut M, Harispe
ML, Henriss at B, Hilden KS, Hope R, Hossain A , Karabika E, Karaffa L, Karanyi Z, Krasevec N,
Kuo A, Ku sch H , La Butti K, Lage ndij k E L, Lap idu s A, Lev asse ur A, Lind qui st E, Lip zen A,
Logrieco AF, MacCabe A , Makela MR, Malavazi I, Melin P, Meyer V, Mieln ic huk N, Mi skei M,
Molnar A P, Mule G, Ngan CY, Orej as M, Orosz E, Ouedrao g o JP, Overkamp KM, Park HS,
Perro n e G, Piu m i F, Pu nt PJ , Ram AF, Ramo n A, Ra uscher S, Recor d E, Riano - Pachon DM,
Rob er t V, Ro hrig J , Rul le r R, S ala mov A, Sali h NS , Sa mso n R A, Sand or E, S angui nett i M,

Schutze T, Sepcic K, Sheles t E, Sherlock G, S o phianopoulou V, Squ i na FM, Sun H, Susca A,
Todd RB, Tsa ng A, Unk les S E, van d e Wi ele N , van Rosse n - Uffin k D, Oliv e ira JV, Vesth TC,
Visser J, Yu JH, Z ho u M, An der sen MR, A r cher DB, Baker SE, Benoit I, Brakh age AA , B raus
GH, Fischer R, Fri svad JC, Goldman GH, Houbrak en J, Oakley B, Pocsi I, Scazzocchio C ,
Sei bot h B, va nK uyk P A, W or tman J , D y er PS, Gri gori ev IV (2 017 ) Co m p ar ative ge no mics
reveals high biological diversity and specific adaptation s i n the i ndustrially and medically
im portant f ungal g e nus Asp ergillu s . Genome Biol 1 8 : 28 - 72. doi: 10.11 86/s130 5 9 - 017 - 1151 -0
Fiedler MRM, Cairns TC, Koch O, Ku b is ch C, Me y er V (2 018a) Conditional ex pr ess ion of th e s mal l
GTPas e A rfA impa cts secre tion, m orphology , growth , and act in ring positi on in As pergi l lus niger .
Front Micr obiol 9 : 8 78 - 894 . doi: 10. 3389/ fmi cb.2018 . 00878
Fiedler MRM, Barthel L , K ubisch C, Nai C, Meyer V (2018b) Constru ctio n of an im pr oved Asp ergil lus
niger p latf or m for en h anced glucoa m ylase secretion. Microb Cell Fact 17 : 95 - 106. doi :
10.1186/ s12934 - 018 - 0941 -8
Galperin MY, Makarov a KS, Wo lf YI, Koonin EV (2014) E xpande d m icrobial genome co ve rage and
improved protein f a mily annotation in t he COG database. Nucleic A cid s Res ear ch 43 : D2 61 - 269.
Gil lingham AK , Tong AHY, B oon e C, Mu nro S (2004) T he GTPase A rf1p a nd the E R t o Golgi c argo
receptor Erv14 p coop erate to recruit the golg in Rud3p to the cis - Golgi. J Cell B iol 167 : 28 1 - 292.
doi: 10. 1083/jc b.20040708 8
Go ng W, C hen g Z, Z ha ng H, Liu L, G ao P , W ang L (20 16 ) Draft ge no me se que nce of Asp erg illus n ige r
str ain An76. G enome Announc 4 : e 017 00 - 01715. doi : 10.11 28/gen o m eA.01700 - 15
Guil leme tte T , van Pe ij N, G oosen T , Lantha ler K, Ro bso n GD , van d e n Hond el C A, Sta m H, Arc her DB
(2007) Genomic analys i s of the secretion s tres s respons e i n the enzyme - p roducing cell factor y
Asper gillus niger . BMC Genomics 8 : 15 8. doi: 10.1186/ 1471 - 2164 -8- 158
Huer ta - Ce pas J , Szkl ar czyk D, Forslun d K, Cook H, H eller D, Walter MC, Rattei T, Mende DR, Sunag a wa
S, K uhn M, Je nsen LJ vo n Me ri ng C, B or k P (2015) e ggNOG 4.5: a hiera rchica l orthol ogy
fram ework wi t h i m proved func tional annotat ions for eukaryoti c, prokary otic and viral sequen ces.
Nucleic A cids Research 44 : D286 - 293. doi: 1 0.1093/n ar/g kv1248
J i me ne z - Ortigosa C, Aimanianda V, Muszkieta L, Mouyna I, Als tee ns D , Pire S, Beau R, Krapp m a nn S,
Beauvais A, Dufrene YF, Roncero C, Lat ge JP (2 012 ) Chiti n s yntha ses with a myo sin mot or - like
domain cont r ol the resis tance o f Aspergil lus fum igatus to ec hinoca nd ins. Ant imi cro b Age nts
Che mot her 5 6 : 6121 - 623 1. doi : 10.1128/A A C.00752 - 12
Jorgen sen T R, G oosen T, Hon del CA , R am AF, Iv ersen J J ( 2009 ) Transcriptom ic co m p arison of
Asper gillus niger growing on t wo diff er ent sugars r eveals coordinated regulati o n of th e secretory
pathway. BMC G eno mi cs 10 : 44. doi : 10.118 6/1471 - 2164 - 10 - 44
Jo rge nsen T R, Ni else n KF, Ar ents hor st M, Pa rk J , van d en H ond el C A, Fri s v ad JC, Ram AF (2011a)
Su b mer ged coni diation a n d product f or m ation by Asp ergillu s niger at low specific g ro wth rates
are aff ected in aerial developmental mut a nts. Appl Environ Microbiol 77 : 5 270 - 52 77. doi:
10.1128/ AEM.0011 8 - 11
Jo rge nsen T R, P ark J , Ar ents ho rst M , van W elz en AM, La me rs G, Vank uyk P A, D amve ld RA, va n d en
Hondel CA, Nielsen K F, Frisvad JC, Ram AF (2011b) The m o lecular and genetic basi s o f conidial
pigmentatio n in Asper gillus niger . Fungal Genet B iol 48 : 544 - 553. d oi: 10.101 6/j.fg b.2011. 01. 00 5
Krijgshel d P, Nitsche BM, Post H, L e vin AM, Muller WH, Heck AJ, Ram AF, Altel aar AF, Wo sten H A
(2013) D eletion of flbA results in i ncreased secretome complexity a nd reduced secretion
hete ro gene ity i n col onie s o f Asper gillus niger . J Proteome Res 12 : 18 08 - 1819. d oi:
10.1021/ pr30115 4 w
Kumar P, Saty a narayana T (2009) Microbial glu coamylases: characteristics and application s. Crit Re v
Biote chnol 29 : 225 - 255. doi : 10.1080/ 0738 855090313 6076
Kurtz S, P hillippy A, Delc her AL, S moot M, Sh umway M, Antone scu C, Salzberg SL (2004) Versati le and
open software f or comparing large g eno m es. Geno m e Biol 5 : R12. doi: 10.1186/ gb - 200 4 -5-2- r12
Kwon MJ, Jørgensen TR, Nitsche BM, A ren tshorst M, Park J, Ram A F, Meyer V (2012) The
transcripto mic fingerpr int of glucoa mylase ove r - e xpre ssio n i n Aspe rgillus niger . BMC G eno mics
13 : 701 - 718. doi: 1 0.1186/1 471 - 2164 - 13 - 701
Le e BN, Adam s TH (199 4) Ove rexpres sion of flbA , an early r egu lator of Aspe rgillus asexual sporu lation ,
leads to activation of b rlA and pr em ature initiation of develo pm ent. M ol Microbiol 14 : 323 - 334.
doi: 10. 1111/j .1365 - 2958 .1994.t b01293.x

Le e BN, Adam s TH (199 6) F lu G a nd flbA function interd ependently to initiate conidio pho re develop m e nt
in Asper gill us nidulans through brlA beta activ atio n. EMBO J 15 : 299 - 309. doi: 10.1002/ j.1460 -
2075.199 6.tb0036 0.x
Li H, Durbin R (2010) Fas t and accu rate lon g - read alignmen t w it h Burro ws - Wheeler transform.
Bioinfor matics 26 : 589 - 595. d oi: 10.1093/ bioin form atics/bt p698
Liu X , As hfor th E , Ren B , So ng F, Dai H, Li u M, W an g J, X ie Q, Zhang L (2010) Biopr os pecting
microbia l natural pr oduct libra ries from the m ar ine e nv ir onme nt f o r drug di scover y . J Antibio t 63 :
415 - 422. doi: 10. 1038/ ja.2010 .56
Liu X , B olla K, As hfo rth EJ , Z huo Y, Gao H, H uang P , S ta nley S A, Hu ng D T, Zhang L (2012)
Syste m a tics - gui ded bioprospe cting for bioact ive mi crobial nat ural produ cts. A nt o ni e Van
Le e uw enhoek 101 : 55 - 66. d oi: 10. 1007/ s10482 - 011 - 9671 -1
Mah J H, Yu JH (2006) Upst ream and down s tr eam regulati on of asexual developm ent in Asperg illu s
fumi gatus . E ukar yot Ce ll 5 : 1585 - 1595. doi : 10.112 8/EC.00 192 - 06
Mellado E, Aufauv re - Bro w n A , Go w N, Holden D (1996a) The As pergill us fumigat us ch sC and chsG
genes en code class III chitin syn t hases with diff er ent functions. Mol Microbiol 20 : 667 – 679. doi:
10.1007/ BF004 005 54
Mell ado E, Spech t CA, R obbins P W, Hol den DW (19 96b) C loning an d chara cterizat io n of chsD , a c hiti n
synt hase - li ke ge ne o f As pergillus fumigatus . FEMS Micro biol Lett 143 : 69 - 76. doi:
10.1111/ j.1574 - 69 68.1996 .tb08463.x
Mellado E, Dubreucq G, Mol P, Sarf ati J, Paris S, Diaq uin M, Hold en DW, Rod riguez - Tudela JL, L at ge JP
(20 03) Cell wall biogenesi s in a double chitin synt hase mutant ( c hsG -/ c hsE - ) o f A sperg illus
fumi gatus . Fungal Ge net Biol 38 : 98 - 109. doi: 10.1016/ s1087 - 1845(02)0 0516 -9
Mino ru K , Yo ko S, Ma sayuki K, Miho F, Mao T ( 2015) KEGG as a reference resource for gene and
pro tein annotation. Nucle ic A cids Res 44 : D457 - 462. doi: 10.109 3/nar/ gkv1070
Mouyna I, Morelle W, Vai M, Monod M, Lech enne B, Fontaine T , Beauv ais A, Sarf at i J, Prevost MC ,
H en r y C, L atge JP (2005) D elet ion of GEL2 encoding for a beta(1 - 3)glucanosy ltransferase affects
morp ho gene sis and vir ule nc e i n As pergill us fumigat us . Mo l Microb iol 56 : 1675 - 1688. doi :
10.1111/ j.1365 - 29 58.2005 .04654.x
Nakamura T, Maeda Y, Tanoue N, Makit a T, Kato M, K obayas hi T (2 006 ) Expr essi on p ro fil e of
a mylol ytic gene s in Aspergi llus nidulans . Biosci Biote chnol Bioche m 70 : 2363 - 2370. doi:
10.1271/ bbb.5069 4
Pa rk J , Pa rk J, J ang S, Ki m S , K ong S, Cho i J, Ahn K , Ki m J, Lee S, Ki m S, P ar k B, J ung K , Ki m S, Ka ng
S, Lee YH (2008) F T FD : an in format ics pipel i n e supportin g phyl ogenomic ana l y s is of funga l
transcriptio n factors. B ioinfor matics 24 : 1024 - 1025. doi: 10 .1093/bioi nform atics/ btn058
Pa ul S, L ude na Y, V ille na G K, Yu F, S her ma n DH, G uti err ez - Correa M (2017) H igh - qual i t y dra ft ge nome
sequence o f a biofilm for ming lignocell ulolytic Asp ergillu s niger stra in ATCC 10864. Sta nd
Genomi c Sci 12 : 37 - 45. doi: 10.118 6/s407 93 - 017 - 0254 - 2
Pavezzi FC, C ar neiro AA, Bocchini - Mart ins DA, Alves - Prado HF, Ferreira H, Martins PM, Gom es E, d a
Sil va R (2011) I nflue nce of di ff erent su bstrat e s on the produc tion of a m utant th er m ostable
gluco a mylas e i n sub mer ged fe r menta tio n. Ap pl B ioc he m B i otec hnol 1 63 : 14 - 24. doi :
10.1007/ s12010 - 010 - 8963 -7
Pel HJ, de Winde JH, Arch er DB, Dyer PS, Hofmann G, S chaap PJ , Turn er G, de Vries R P , Al bang R,
Albermann K, An d ersen MR, Bendtsen JD, Benen J A , va n den B erg M, Breestraat S, C ad dick
MX, Contreras R , Cornell M, Coutinho PM, Danchin EG, Debets A J, Dek ker P, van Dij ck PW,
van D ij k A, Dij kh uize n L, D ri esse n AJ , d'Enf ert C, Ge y sens S, Goos en C, G root GS, de G root
PW, Guillemett e T, Henrissat B, Herweijer M, van den Hombergh JP, van den Ho n del CA, van
der Heijden RT, van der Kaaij R M, Klis FM, Kools HJ, Kubicek CP, van Kuyk PA, Lauber J, Lu
X, van der Maarel MJ, Me ulenb erg R , Me nke H, Mo rti mer MA, Nie lse n J , Ol iver S G, O lstho or n
M, Pal K, van Peij NN, Ram AF, Rinas U, Roubos JA, Sagt CM, Schmoll M, Sun J, Uss er y D,
Var ga J , Ve rvec ken W, van d e V onde rvo ort PJ, Wedl er H , Wo sten H A, Ze ng AP, va n Oo yen AJ,
Visser J, S ta m H (2007) Genome sequencing and analysis of th e versatile cell factor y Asp e rgillu s
niger CBS 513.8 8. Nat B iot ech nol 25 : 221 - 231. doi: 1 0.1038/ nbt1282
Perez - de - Nan clares - A rregi E, Et xebe ste O (2014) Photo - convertible taggi ng for localiz ation and d ynamic
an alyse s of lo w - exp re ssio n pr ote ins i n fila ment ous fun gi. F un gal Ge net Bio l 70 : 33 - 41. doi :
10.1016/ j.fg b.2014.06. 006

Rh oads A, Au K F (2015) Pac Bio sequ encing a n d its appl ication s. Genom Proteom Bioinf 13 : 278 - 289. doi:
10.1016/ j.gpb.2 015.08. 002
Rog g LE, F ort we ndel JR, Juv vadi PR, Lille y A, S tei nbac h W J (2 011 ) The chiti n syn tha se ge nes chsA and
chsC ar e not requi r ed for cell w all stress responses in the human pathogen As pergill us fumigatus .
Bioch e m Biophys Res Commu n 411 : 549 - 554 . doi: 10.1016 /j.b brc. 2011. 06.180
Schachtschabel D, Aren t shorst M, Nitsch e B M, Morris S, Niels en KF, va n den Hondel CA, Klis FM, Ram
AF (2013) The t ranscr ipti onal repres sor TupA in Asper gillus nige r is involved in controllin g gene
expression re lated to ce ll wall bio syn the sis, develop ment, an d nitrogen so urce availabili ty. PLoS
One 8 : e78102. doi: 10. 1371/jou rnal .pone .0078102
Schape P, Kwon MJ, Bau m a nn B, Gut schmann B, Jung S, Lenz S, Ni tsc he B, Paege N, Sch utze T , Cairn s
TC, Mey er V (2019) U pdatin g gen o me annot ation for t he microb ial cell factory Aspergil lus niger
usi ng ge ne co - exp ressio n net work s. Nucle ic Acids Re s 47 : 559 - 569. doi: 1 0.1093/n ar/g ky1183
Swi ft RJ, Wiebe MG, R obson GD , T rin ci A P J (1 998) R ecom b in a nt gl ucoamyla se product ion by
Asper gillus niger B1 in chemo stat and pH auxo stat culture s. Fungal Genet Bio l 25 : 100– 109. doi :
10.1006/ fgbi .1998.1089
Szkl arc zyk D , Mo rri s JH , Coo k H, K uh n M, W yder S, Si mo novic M, S anto s A, Donc he va NT , Ro th A,
Bork P, Jensen LJ, v o n Me ring C (2017) The STRIN G dat aba se in 2017: qu ality - co n trolled
pro tein - protein association networks, made broadly acces sible. Nucleic Acids Res 45 : D3 62 -
D368. d oi: 10.109 3/nar/ gkw 937
T akeshi ta N, Oht a A, H or iuch i H (2 002 ) csm A , a gene enc o ding a clas s V c hiti n s yntha se wit h a myos in
mo t o r - like do main o f Asp er gillus ni dulans , is translated as a sin gle polypeptid e and regula ted in
respon se to osm otic conditi o ns. Bi ochem Biophy s R e s C o mm un 298 : 103 - 10 9. doi:
10.1016/ s0006 - 291x (02)02 418 -x
Takeshita N, Ohta A, Horiu ch i H (2005) CsmA , a class V chitin synthase with a m yosin motor - like
domain, is loc alized thro ugh direct inter action with t he actin cytoskeleton i n As pergill us nidulans .
Mol B iol Cell 16 : 1961 - 1970. doi: 10.1091/ mbc. e04 - 09 - 0761
te B iese be ke R, va n Bi eze n N, de V os W , van d en Ho nde l C, Punt P J (20 05) D iff erent control mechanisms
regulat e glucoamylase and protease gene transcription in Aspe rgillu s oryza e in solid - sta te and
su b me rged fe r me ntat ion. Appl Mic robiol Bi otech nol 67 : 75 - 82. doi: 10. 1007/s 00253 - 004 - 1807 - z
van M uns ter J M, Nits che BM, Akero y d M, Dij khu izen L , va n der Maarel MJ, Ram AF (2015) Sy ste ms
approach es to pre dict t he fun ctions of glycos ide hy drolases duri ng the life cycl e of Aspe rg illus
niger usi ng d eve lop ment al muta nts brlA and flbA . PL oS One 10 : e 0116269. d oi:
10.1371/ journ al.pone. 0116269
Ves th T C, N y bo JL, The ob ald S, Frisva d J C, Lar se n TO , Ni elsen K F, Hoo f JB , Br andl J, Sal amo v A, Ri le y
R, Gladden JM, Phatale P, Niel sen MT, Lyhne EK, Kogle ME, Strasser K, McDon nell E, Barry K,
Clum A, Che n C, LaButti K , Harid as S, Nolan M, Sandor L, Kuo A , Lipzen A, Hainaut M, Drula
E, Tsang A, Magnuson JK, Henrissat B, Wiebenga A , Simmons BA, Makela MR , de Vries RP,
Gr igor iev IV , Mo rte nse n UH, Ba ker S E, A nder sen M R (2 018 ) I nvesti gatio n o f inter - and
intraspecies variation t hr ou gh geno me seque nci n g of Asperg illus sectio n Nigri . Nat Ge net 50 :
1688 - 16 95. doi: 10.10 38/s415 88 - 018 - 0246 - 1
Von gsangn ak W, Ha nsen K, Niels e n J (20 11) In tegrat ed analy sis of the gl obal trans criptiona l res ponse t o
alp ha - amylase over - produc tion by A spergillu s o ryza e . Biote chn ol Bioeng 108 : 1130 - 113 9. doi:
10.1002/ bit.2303 3
Wanka F, C a irns T, Boecker S, Berens C, Happel A , Zheng X, Sun J, Krappmann S , Meyer V (2016) Tet -
on, or Tet - off, t hat is t he q ue sti on: Adva nced cond iti onal ge ne exp res sio n in A spergillus . Funga l
Genet Bio l 89 : 72 - 83. doi: 10. 1016/j.f gb.2015. 11.00 3
Wa nke C, E c kert S , Alb re cht G , van H art in gsvel dt W , P unt P J , van de n Hond el CAMJ J, Bra us GH ( 19 97)
T he Asp ergil lus ni ger GCN4 hom ologue, cpcA , is transcriptionally regulated and encodes an
unus ual le ucine zipper. Mol Microbiol 23 : 23 - 33. doi: 10.1 046/j.1 365 - 2958.1 997.174154 9.x
Wieser J, Lee BN, Fondon J, 3rd, Adams T H (1994) Genetic requirem ent s f or initi ating asexual
de velo pment i n Asper gillus nidulans . Curr G enet 2 7 : 62 - 69. doi: 10.1007/ bf003265 80
Wo lf J , Nic ks M, D eitz S, T uinen E v, Franz uso ff A (19 98 ) An N - end rule de stabiliza tion mutant re veals
pre - Gol gi requ iremen ts f or Sec7p in y east mem brane traf fic. Bioch e m Biophs Res Comm u n 243 :
191 - 198. doi: 10. 1006/ bbrc.1 998 .80 84

Yamada O, L ee BR, Gomi K, Iimura Y (1999) Cloning an d functional an al ys is of the Asp erg illus o ryzae
conidiatio n regulator ge ne brZ A by its di srupti on and mis scheduled e xpre ssion . J Biosci Bi oeng
87 : 424 - 429. doi: 1 0.1016/S 1389 - 172 3(99)80089 - 9
Y a ma s hi ro y CT, Ebbole DJ, Lee BU, Brow n RE, B ourlan d C, Madi L , Yano f sky C (1996) Characterization
of rco -1 of Neu rosp ora c rassa , a pleiotropic gen e affectin g gro w th and development that encodes
a hom olog of T u p1 of Saccharomyces cerevisiae . Mol Cell B iol 16 : 621 8– 622 8. doi:
10.1128/ mcb. 16.11.6218
Yin C, Wang B, He P, L in Y, Pan L (2014) Gen omi c analy sis of t he aconidi al and h igh - perfor m a nce
pro tein prod ucer, industrially re levant Asper gillus niger SH2 str ain. Ge ne 54 1 : 107 - 114. doi:
10.1016/ j.gen e.2014.03.0 11
Yin X, S hi n HD, Li J, D u G, Liu L, Che n J ( 20 17) Compar a tive ge no mics a nd tra nscri pto me a nal ysis o f
Asper gillus niger and m etabo lic engineering for citrate product ion. Sci Rep 7 : 41040 - 41057. doi :
10.1038/ srep4104 0
Yua n XL, van d er K aaij RM, van de n H onde l CA , P unt PJ, v an der Maar el MJ, Dijkhu ize n L , Ram AF
(2008) As pergill us niger ge nome - wide an al ysis reveals a larg e n um ber o f novel alp ha - gluc an
acting en zymes with unexpected expression profiles. Mol Genet Gen o mics 279 : 545 - 561. doi:
10.1007/ s00438 - 008 - 0332 -7
Zha ng C, M eng X , W ei X, Lu L (20 16 ) Hi ghly ef ficie nt C RIS PR mutage ne sis b y micr oho mo log y - mediated
end jo ining i n Aspe rgillus fumigatus . Fungal Genet Bio l 86 : 47 - 57. d oi: 10.101 6/j.fg b.2015. 12.007

Figures

Fig. 1 Synteny m ap of the s c af folds o f A. niger LD M3 t o the sup erc onti gs o f A. nige r C BS 513.88. T he
co lor ing of t he sc af fold s sho ws s ynte nic r egi ons i n A. nige r C BS 513.88. A rabic n umeral s sh o w th e number
of the sup erc onti g in A. niger C BS 513.88. G r e y areas sh o w r egio ns not fo und in the CBS 51 3.8 8 geno me
sequen ce. The black line un derneath a sect ion of the scaffolds indicates invers io n sequence. The blue
rectangular s hado w acro ss t he scaff old s i ndicates the t ransposition betw ee n th e t wo s equence f rag ments
separa ted by a red lin e in the shado w

Fig. 2 Distributio n of mutated genes in all KOG ter m s. T he hei ght o f the hist ogra m sho ws the t ota l number
of ge ne s o f A. niger LDM3 in v arious KOG clusters. The grid p art represents the n um ber of mutated genes
in each K OG category, and the whit e p art means the num be r of unmut ated gen es

Fig. 3 Co - exp re ssio n net wor k for T upA and Prp A encod ing gene s. Pro tein names are repres ented by circles ,
and the q uer y pr ote ins are giv en in d ia mond bo xes. O nl y ne gati ve c or rela tio ns fo r bo th Tup A ( a ) an d P rpA
(b ) are sho w n a s b oth o f the m o nly exp re ss si gni fican t ne gati ve a ssoci at ion with o ther gene s. If ge ne na me s
were not a vailable in A. niger , th e na me f or either t he A. nidulans or S . cerevis iae o rtho lo g w a s used. Gene
pa irs w i thi n the c o - e xpre ssio n sub - networks al l pas sed the | 0.7 | Spearman correlation coeffici ent cut - of f.
Overrepresented GO term s with functio ns in biological pro cesses a bove a Benjami n i – Hochberg fals e
discovery rate corrected p valu es of 0.05 w ere assessed, and genes of interest manually filtered

Fig. 4 P henot yp ic ana lys is of Δ t upA , Δ prpA mut a nt s , a nd Δ tupA complemented stra in on so lid agar plate s.
Spores (5* 10 4 ) w ere spot - ino cul ated on t he diff eren t ty p es of m edia an d i n cubated a t 30°C for 3 da ys. St rain
FW35.1 was used as the reference strain . T o ful ly repress any growth , 150 µ g/ ml h ygro mycin was adopted .
hyg h ygro myci n

Fig. 5 S porul ati on qua nti fica ti on of Δ t upA , Δ prpA muta nts a nd Δ tupA comple mented str ain on sol id agar
plates. ( a ) 100 0 spores of each strain w ere inocu lat ed on the di fferent ty pe s of m edia , respectively , a nd
in cubated a t 30° C f or 6 day s. (b ) T he colony size of differe nt strains after 6 days of cultur e on C M ( stri pe)
and MM (spot) m ed ium, res pectively. (c) The n um ber of spor es per square centimet er of interested strains .
All experi m ents were conducte d in biological tr iplicates. Significance values were calculated w ith the 2 -
taile d t - test with independe nt variab les (* p <0.05, * * p < 0.01, *** p <0.001)

Fig. 6 Characteris t ics of Δ tu pA and Δ prpA mutant s dur ing s ub merge d c ulti vatio n. (a ) Co l o r ch a ng e o f Δ tupA
co m p ar ed t o FW 35 .1 d uring s hake fla sk c ulti vati on i n CM med ium. ( b ) B iomass a cc um ulati on in cul tur es
of F W35.1 ( sq uare ), Δ pr pA ( diam ond ), and Δ tupA strain (triangle). ( c ) Acc umulation of total secrete d pro tein
(norm alize d b y per gram bi o ma ss ) i n FW35.1 ( black ) , Δ prpA (grey ), and Δ tupA (spot). ( d ) Enz yme ac ti vity
of Gla A (normalized by per gram biomass) in F W 35.1 ( black ) , Δ prpA (g rey) and Δ tupA ( spot) . All
experiments were conducted in biological quadrupli cates. Significance values were calculated w ith 2 - tailed
t - test wit h in d epende nt variabl es (* p <0.05, * * p < 0.01, *** p <0.001)

Tab les
Tab le 1 Asper gillus niger s trai ns use d in t his stud y
Strain
na me

Ba ckgro und
strain

Relevant genot ype/descrip tion References
LDM 3

Aco nid ial p henot yp e
Long da Biotech nology ,
Sha ngha i

MA 169.4

AB 4. 1

cspA 1 - ,kusA::DR - am dS - D R, py rG −

(Carvalho et al. 2010)

FW35.1

AB 4. 1

cspA 1 - , p yrG +

(W anka et al. 2016)

YS33.10 MA169. 4
kusA::DR - amdS - DR, tupA:: Ao py rG,
pyrG

+
, singl e copy

T his stud y
YS34.16 MA169. 4
kusA::DR - amdS - DR, prpA:: Ao pyrG pyrG + ,
single cop y

T his stud y
YS3 9 .1 MA 169.4
kusA::DR - amdS - DR, tu p A:: Ao py rG pyrG + ,
pMA 171 - tupA

T his stud y

Tab le 2 Gen om e characteristics of select ed A. ni ger strains with industr ial relevance
Str ain na me

（ NCBI ）
ASM285v 2 NR RL3 ATCC 1015 SH 2 LDM 3
Na me syno n ym CBS 513.88 ATCC 9029 NRRL 328

Acce ssio n

Num ber
GCA _00000285

5.1 unp ublis hed GCA _000230395.2 GCA_000633 045.1 VTFN000 00000
Inst itute a nd
co untry

DSM,
Net herl and
Integrat ed

G en o mi c s ,
USA
DOE/JGI, USA SCU T , Chi na

ECU ST , Chi na
Project

Ch ronology 2000 - 20 07 2000 2005 - 2011 2013 - 2014 2016 - 2017
Geno me le n gth

34.02 M b

33.7 M b

34.85 M b

34.63 M b

35.28 M b

Seq uenci ng

tec hnolo g y BAC tiling Shot gun Shot gun Illumina H i S eq
Illumina H i S eq +

PacBio
Cove rage

~7.5 ×

~6×

~8.9 ×

~120 ×

~177 ×

Genomi c library

insert size < 150 kb 1- 2 kb 3 kb 8 k b 40 kb 500 bp
270 bp

(Illu m i na)
Num ber of

cont igs or
scaffolds
19 Scaf folds 9510 C ontig s 24 Cont igs 349 Scaf fol ds 11 Sca ffold s
Num ber of

predicted genes 14,16 5 14,00 0 11,20 0 11,517 11,209

Tab le 3 Selected list of genes present in bo th LDM3 a nd CBS 513.88 geno mes wit h SNP o r INDEL
mutation s
Gene ID

( CBS 513. 88
no mencl atur e)

Gene

na me
SNP o r InD el

Predicted gene fun ction

Reference

Tr anscription factor s

An 01g07900

cpcA

Insertion

Tr anscription factor

im portant f or a m ino acid
bi os y nt he sis under a m i no
acid star vation conditio ns

(Jorgensen et al. 2009; P el

et a l. 2 007 ; Vongs ang na k
et al. 2011; Wanke et al.
1997; Yi n et al . 2014)

An 04g06910

amyR

No nsyno n ymous

Tr anscription factor for
starch hydr ola se ge nes

(Yu an et al . 2008)

An 04g06920

agdA

No nsyno n ymous

Secreted α - gluc osid ase

An 15g00140

tupA

No nsyno n ymous

Transcriptional repressor

important for ce ll w all
re m o delling

(Schachtschabel et al.

2013)
Transporters

An 15g04270

No nsyno n ymous

Sugar trans p orter

An 11g09600

No nsyno n ymous

MFS m o nosaccharide
transpor ter

An 07g03690

No nsyno n ymous

Neutral a mino acid
transpor ter

An 07g03970

No nsyno n ymous

Neutral a mino acid
transpor ter

An 14g07130

No nsyno n ymous

Neutral a mino acid
transpor ter

Protein secretion an d degradation

An 07g02190

sec7

No nsyno n ymous

G ua n yl - nucle otide

exchange f actor important
for intra - Go lgi a nd ER - to -
Golg i trans port

(Wolf et al. 199 8)

An 08g00290

rud3

No nsyno n ymous

Matrix pr otein of Golgi

(Gilling ham et al. 200 4)

An 07g03880

pepC

No nsyno n ymous

Subtilisi n - lik e serine
protease

An 07g08030

pepF

No nsyno n ymous

Ser ine - typ e
carbox ypept idase

An 11g06350

cpy1

No nsyno n ymous

Serine carboxy p eptidases

Cell wall bio synthesis

An 08g07350

gelB

No nsyno n ymous

Glucosyltransferase

(Mouy na e t al. 2005)

An 02g02360

csmA

No nsyno n ymous

Chi tin s ynthas e

(A ufau vre - Br own et al.

1997; Jim enez - Ortigosa et
al. 20 12; Takeshita et al.

Strain app lication
Gla A

produc tion
Gluconat e
produc tion
Citric acid
produc tion
Gla A

produc tion
Gla A production
Reference (P el et al. 2007)
(Bake r 2006;

Vesth et al.
2018)
(Andersen et al.
2011) (Yin et al . 2014)
T his stud y

200 2; Takeshita et al.
2005)

Spor ulation

An 02g03160

flbA

Syno ny mous

Regu lator of G - pr otein

signallin g
(Le e and Adams 1994;

Perez -de- Nanclares - A rregi
a nd Et xebest e 2014; v an
Muns ter et a l. 2015;
Wieser et al. 1994; Y in et
al. 201 4)

An 12g02050

wA

No nsyno n ymous

Po lyket ide synt hase

im portant f or pigm ent
bi os y nt he sis

(Jorge ns en et a l. 2011b;

Zha ng et al . 2016)
An 18g01170

prpA

Ab se nt

Role in asex ual sporulatio n

(Yin et al. 2014)

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