F iedler etal. Microb C ell F act (2018) 17:95
https://doi.org/10.1186/s12934-018-0941-8
RESEARCH
C onstruc tion ofanimpr o v ed Aspergillus
niger platf orm f orenhanced gluc oam ylase
secr etion
Mar kus R. M. F iedler , Lars Bar thel, Christin Kubisch, Corrado Nai and V era M eyer *
Abstr ac t
Backgr ound: The lif estyle of filamentous fungi depends on the secretion of h ydr olytic enz ymes into the surround-
ing medium, which degrade polymeric substances into monomers that ar e then taken up to sustain metabolism.
This f eature has been exploited in biot echnology to establish platf orm strains with high secretor y capacity including
Aspergillus niger . The accepted paradig m is that prot eins become mainly secret ed at the tips of fungal hyphae. Ho w-
ever , it is still a matter of debat e if the amount of gr owing hyphal tips in filament ous fungi correlates with an incr ease
in secretion, with pr evious studies showing either a positive or no correlation.
Results: Here , we f ollowed a syst ematic approach to study pr otein secretion in A. niger . F irst, we put the glaA gene
encoding for glucoam ylase (GlaA), the most abundant secreted pr otein of A. niger , under control of the tunable T et-
on system. Regulation of glaA gene expression b y omitting or adding the inducer doxyc ycline t o cultivation media
allowed us t o study the effect of glaA under- or ov erexpr ession in the same isolate . By inducing glaA expression in a
fluorescently tagged v-SNARE r epor ter strain expr essing GFP -SncA, we could demonstrate that the amount of post -
Golgi carriers indeed depends on and cor r elates with glaA gene expression. By deleting the r acA gene, encoding the
Rho -GTPase RacA in this isolate , we generated a strain which is identical t o the parental strain with r espect to bio -
mass formation but pr oduces about 20% more h yphal tips. This h yper branching phenotype caused a more compact
macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by r epeated
addition of doxycycline, this hyperbranching strain secret ed up to f our times more GlaA int o the culture medium
compared t o its parental strain.
Conclusion: The data obtained in this study strongly indicat e that A. niger responds to f orced transcription of secre -
tor y enz ymes with increased f ormation of post-G olgi carriers to efficiently accommodate the incoming car go load.
This physiolog ical adaptation can be rationally exploited to generat e hypersecr etion platforms based on a h yper-
branching phenotype. W e propose that a r acA deletion back gr ound ser ves as an excellent chassis f or such hyperse -
cretion strains .
Keywor ds: Aspergillus niger , T et- on, P rot ein secretion, GTP ase RacA, v-SNARE, H yperbranching, P ost- Golgi carrier ,
Glucoamylase
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Open Ac c ess
Microbial Cell Factories

*Correspondence: [email protected]
Depar tment Applied and Molecular Microbiology , Institute
of Biotechnology , T echnische Universität Berlin, Gustav-Meyer-Allee 25,
13355 Berlin, Germany

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F iedler etal. Microb C ell F act (2018) 17:95
Backgr ound
F ilamentous fung i follow a f oraging fe eding b eha v iour .
Dur ing growth, the y actively search for food through their
intricate h yphal ne twork and n utrients are acquired by
extrac ellular digestion of comple x poly mers such as plant
poly sacc harides [ 1 ]. The tip of a g rowing fungal hy pha is
suppo sed to b e the most active region of protein se cre -
tion and a strong corre lation be tween sustaine d pol arise d
grow th and pr otein se cretion at t he hyphal tip is gener -
ally accepted [ 2 – 5 ]. H owe ver , investigations on the fungal
cell factories Asper gillus niger and A. or y zae g a ve con -
tradictor y result s while asse ssing a direct link b etween
amount of active fungal hy phae and sec retion, with pre -
vious studies show ing either a positive correlation [ 6 , 7 ]
or none [ 7 ]. Similarly , despite f our dec ades of studying
protein secretion in fil amen tous fung i, our understand -
ing about protein trafficking and sec retion is still limited,
thus ham per ing the ra tional establishment of hypers ecre -
tion platform strains for biotechnologic al purp oses [ 8 , 9 ].
Given the outstanding s ecretor y c apacities of filamentous
spe cies such a s A. niger and their pr omise s as sup erior
platform strains cultivable on rene wable ligno ce llulosic
fee dstock s , it is of great inter est to fully underst and and
exploit t he link be tween p olarise d grow th and sec retion.
Hyphal grow th is characterise d by apical ex tension.
This is ensure d by polar ised exoc y tosis of biosy nthetic
enzy mes and their substrates eventually expanding the
cell membrane and c ell wall [ 10 – 13 ]. A central h ub of the
con ventional sec retor y pat hway is the nonstacked fun -
gal G olgi b ec a use it s ort s prot ein cargoe s either t o the
plasma membrane, the endov asc ular system or the ex tra -
cellular space (f or a compr ehensive rev iew , the reader is
directe d to an e xc ellent r ecent paper [ 13 ]). In brief, coat
protein c omplex II (COPII)-coated vesicles bud off from
the endoplasmic reticulum (E R) and coalesce with early
G olgi cisternae, which ensure protein glycosylation.
G olgi cisternae prog ressively change t heir protein and
lipid cont ent th u s be coming enriched in cargo and e ven -
tually mat uring to late Golg i cisternae (als o called t ran s -
G olgi network , TGN). In A . nidul ans , it wa s shown that
the T GN ci sternae finally mat urate t o po st -G olgi car ri -
ers by recr uiting RabE and engag ing motor pr oteins [ 14 ,
15 ]. The latt er assi st in movement of po st -G olgi se cre -
tor y vesicles toward the hyphal apex along microtubules
(long-distance transpor t via k inesin-1 or kinesin-3) and
actin filaments (myosin-5-me diated final transpor t to
the plasma membrane) [ 16 ]. Howe ver , b efor e se cre -
tor y vesicles undergo fu sion with the plasma membrane,
they accumulate a t the hyphal tip and be come visible
in a structure calle d Spitzenkörper [ 17 ]. Most re cen tly ,
it wa s discovered that secretor y vesicles accumula te at
the Spitzenkörper in a pulsator y way , mediating a step -
wis e ex tension of the h yphal tip [ 16 ]. Thi s obser v ation is
congruent with a pulsator y calc ium influx which contr ols
actin p olymeri zation and ex o c y tosis [ 18 ]. Hence, secre -
tor y vesicles are retaine d in the Spitzenk örp er before
being tethered to the pla sma membrane. By molecu -
lar interactions betwe en SNARE (soluble N -e th ylma-
leimide-sensitive f actor at tachment protein r eceptor)
proteins, the p ost -G olgi c argoes b ecome release d into
the ex tracellular space or embedde d into t he grow -
ing plasma membrane, thus either se creting enzy mes
or providing the enzy mes require d for c ell wall expan -
sion [ 13 ]. Se veral SNARE proteins were repor ted to be
involved in fu sion steps involving retrograde and antero -
grade ve sicular transpor t be tween the ER and the G olgi
as well as in f usion steps involving endo somal or vacu -
olar transp ort [ 19 , 20 ]. D uring vesicle fu sion, the α -helix
of a monomeric vesic ular - SNARE (v - SNARE) in p ost -
G olgi se cretor y vesicles interacts w ith thr ee α-helices of
an oligomeric target - SNAR E (t - SNAR E) in the plasma
membrane, forming the trans- SNAR E com plex [ 21 – 23 ].
This triggers f usion of the vesicle with the target mem -
brane, forming the cis - SNARE complex, followed by
A TP-dependent SNARE complex dissoc iation [ 24 ]. Cal -
cium f unctions as a reg ulator of vesicle fusion; howe ver ,
not all SNARE-me diated fu sion steps in the sec retor y
path way are calcium-dep endent [ 25 ]. In S. cerev isiae , the
v- SNAR Es Snc1p and it s paralog Snc2p lo cate t o po st -
G olgi se cretor y vesicles [ 21 , 26 ] conferring fu sion of the
po st -G olgi car rier with the pla sma membrane via the
interaction with the membrane-locali zed t - SNARE s pro -
teins Sso1p and its p aralog Sso2p [ 27 , 28 ]. Studies tha t
analyse d the locali zation of ortholog s of Snc1p in vari -
ous filamentous fung i including A. niger reveale d a highly
pol arise d accumulation of A oSnc1 ( A . or y zae ), SncA ( A .
ni ger ), SynA ( A . ni dulan s ) and SYN-1 ( Neu rospora cr a ssa )
at the tip of growing hyphae [ 2 , 29 – 31 ]. In all likelihood,
they are a component of post -G olgi se cretor y vesicles in
filamentous fung i and be come a transien t com ponent of
the plasma membrane when exoc ytosi s occurs , but are
thereafter rec ycle d by the sub-a pical endo c ytic r ing . I t i s
thought tha t this involves p ost -G olg i sorting endo somes
which ensure S ynA/Snc1-containing membranes to be
transpor ted along microtubules back to the T GN where
they e ventually fus e with new c argo-loaded p ost -G olgi
carr iers [ 2 ]. Hence, endoc ytotic rec ycling process es are
essential for maintaining hyphal pol arity in filamentous
fung i [ 13 ].
In A. niger , we c ould demonstrat e that a pical domi -
nance in young and mat ure h yphae of A . niger is also
mainly contr olled by the Rho GTPa se RacA , though t
to mediate actin poly meri zation and depoly merisa -
tion at the h y phal apex [ 32 ]. The A . ni ger R ho GTPases
RacA and Cf tA (Cdc42p) can substitute each other
with resp ect to actin p olymeri zation at the h yphal tip,

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F iedler etal. Microb C ell F act (2018) 17:95
but actin dep olymeri sation is sec ured by RacA and not
by CftA . Henc e, a Δ racA strain is im paired in ac tin dis -
ass embly and in consequence frequently lose s a pical
dominance th us provoking a hyp erbranching phenotype
[ 32 ]. Notably , this h yp erbranching phenotyp e wa s paral -
leled by reduce d GFP - SncA accumulation a t hyphal tips ,
although physiological profiles g at hered f rom c ontrolled
bioreactor cultiv ations of t he Δ racA and its wildty pe
strain uncovered that t heir grow th cur ves , ma ximum
spe cific grow th rat es and sp ecific protein se cretion rates
were nearly identical. W e thus h yp othesize d that t he
same amount of secretor y ve sicles is merely distributed
to more tips in the Δ r acA strain, resulting in le ss sec re -
tor y vesicles p er individual tip, and that the capacity of
the h y phal tip growing apparatus to ac commodate vesi -
cles is therefore—a t le ast in the Δ racA strain—not fully
exploit ed [ 35 ]. T o refute or verif y this hy pothesis , we
challenged in the current study the Δ r acA strain to over -
expr ess one of it s homologous and abundantly secrete d
proteins by put ting it under conditional transcriptional
contr ol of the T et -on s ystem [ 33 ]. W e sele cted glucoa -
mylase ( glucan 1,4-α-glucosida se, GlaA) as mo del pro-
tein as this is the major se creted protein of A. niger (up
to 30 g/L [ 34 ]) with important implications for the f oo d
and biofuel industr y [ 8 ]. By using a GFP - SncA labelle d
repor ter strain as background strain, we show here that
(i) more post -G olgi car riers accumulate a t hy phal tips in
both Δ racA and its parental strain upon T et -on dr iven
overex pression of the glaA gene, and that (ii) this spec ifi -
cally lead s to an increase d glucoamylas e secretion in the
hyperbranching Δ racA strain. Our study thus validates
the h y pothesis that the amoun t of grow ing h yphal tip s
po sitively aff ec ts protein sec retion, and has impor tant
repercu ssions for industrial biotechnolog y .
Results anddiscussion
Apical distribution ofsecret or y vesicles a thyphal
tips isdriven b ysecretory cargo inbothwildtype
andhyperbr anching Δ racA str ain
In order to st udy protein secretion in A . niger in a sys-
tematic manner , we selecte d our prev iously descr ibe d
repor ter strain FG7 [ 30 ] (T able 1 ), which e xpresses the
fluorescently tagged v- SNARE SncA (GFP- SncA) in an
other wis e wildty pe backg round as ancestor strain. In this
strain, we deleted the chromosomal glaA gene, g iving
strain MF7.4. W ester n blot analysis of the culture super -
natants of FG7 and MF7.4 cultivated in minimal me dium
(MM) supplemented with 5% w/v glucose confirme d
that no gluc oamylas e was de tectable in MF7.4 (Addi -
tional file 1 : F ig . S1). Subse quently , we re-introduced the
glaA gene into the pyrG lo cus but being this time under
contr ol of the do xyc ycline-inducible T et -on expr ession
system [ 33 ]. Corre ct integration of a single copy of T et -
on- glaA at pyrG in the resulting strain MF19.5 wa s con -
firme d by Southern analysi s (Additional file 2 : F ig . S2).
This sy stem ena bled u s to pr ec isely con trol in a grow th-
indep endent manner glaA gene expression upon addition
of doxycycline [ 33 ]. F inally , we deleted the endogenous
racA gene in MF19.5 giving strain MF22.4. Resp ec -
tive cloning steps are descr ibe d in detail in the “ Meth -
ods ” se ction. F or bre vity , we will f urther refer to FG7 as
wildty pe strain, MF7.4 a s Δ glaA strain, MF22.4 a s Δ r acA
strain and MF19.5 as the parental strain of MF22.4. As
desc ribe d above, all strains contain a single eg fp::sncA
gene copy and the distribution of po st -G olgi car riers at
hyphal tips c an th us be dire ctly com pared among the
strains .
All four strains were cultivated on minimal medium
(MM) plates in the pr es enc e of glucose (k nown to induce
glucoamylase expression) with or without 20 µg/mL
doxycycline (D O X) for 2 days at 22 °C , and GFP - SncA
fluorescence along 20 µm from the tip wa s quantified
by confocal micros copy in a t lea st 20 individual hyphae
per strain (Fig . 1 ). As we repor ted b efore [ 35 ], t he po st -
G olgi marker SncA shows a distribution with highest
fluorescence a t the near -apical region. Most interest -
ingly , vesicle amount decrea sed sig nificantly upon dele-
tion of the chr omosomal gl aA gene, but , upon T e t -on
driven gl aA overe xpression in strain MF19.5, a distribu -
tion of se cretor y vesicles identical to the wildty pe w as
obser ved (F ig . 1 a). These ob ser vations have four im por -
tant implications. First, to the best of our k nowledge it
demonstrates for t he first time that t he transcr iptional
le vel of secretor y c argoes inde ed drive s the amoun t and
T able 1 Aspergillus niger strains used inthis work
Name Genotype Referenc e
FG7 ∆ kusA, pyrG + , egfp::sncA (derivative of MA70.15) [ 30 ]
SS1.1 ∆ kusA, pyrG − , egfp::sncA (derivative of FG7) This study
MF7.4 ∆ kusA, pyrG + , egfp::sncA, ∆ glaA::DR-Aop yrG - DR (derivative of SS1.1) This study
MF9.1 ∆ kusA, pyrG − , egfp::sncA, ∆ glaA (derivative of MF7.4) This study
MF19.5 ∆ kusA, pyrG + , egfp::sncA, ∆ glaA, T et - on::glaA (derivative of MF9.1) This study
MF22.4 ∆ kusA, pyrG + , egfp::sncA, ∆g laA, T et - on::glaA, ∆racA::hy gR (derivative of MF19.5) This study

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F iedler etal. Microb C ell F act (2018) 17:95
distribution of s ecretor y ve sicles at h y phal tips —i.e.,
if less protein molecule s are destined for se cretion, less
se cretor y vesicles accumulate a t hyphal tips . S econd,
it allows for the first time to obtain a rough estimation
for the amoun t of po st -G olgi se cretor y vesicles c arr y ing
protein cargoes mainly impor tant for h y phal extension
(73% = GFP- SncA fluorescence in the a bsence of D O X) as
oppo sed to those de stined for se cretion. The y acc ount t o
a flexible capacity of up to 27%, which can be pro duced in
respons e to increase d transcr iption of prot ein cargo such
as GlaA (Fig . 2 ). Third, it shows tha t transcr iption of glaA
under contr ol of the T et -on systems or its ow n native
promoter (t he strongest know n promot er in A . ni ger
ensuring se cretion of about 30 g/L g lucoam yla se int o
the en vironment [ 34 ]) gives comparable accumula tion of
se cretor y vesicles at hyphal tips . F our th, it suggests that
there is a maximum amount of secretor y ve sicles an indi -
vidual hy phal tip can accommodate, as the GFP - SncA
fluorescent profiles of h yphae f rom the wildtyp e strain
(FG7) and the T et -on- glaA , Δ gl aA strain (MF19.5) upon
presence of 20µg/mL D O Xp erfec tly over lappe d.
W e thus investigated the distribution of se cretor y vesi -
cles in the hyperbranching Δ racA strain MF22.4 either
with or without D O X (F ig . 1 b) and could validate the
obser vations f ormulated above: In the absence of glaA
expr ession ( − D O X condition), less s ecretor y ve sicles
were visible at the h y phal tip. U pon gl aA overex pression
( + D O X condition), more sec retor y vesicles accumu -
lated at h y phal tips . Notably , the latt er vesicle distr ibu-
tion per fectly overlappe d with data obtaine d previou sly
upon native glaA expression in the Δ r acA strain [ 35 ];
F ig . 1 b), which not only reflects the repro ducibility of this
approach but again strongly im plies that there is a maxi -
mum amoun t of po st -G olgi c arriers which can b e acc om-
mod a ted by a g rowing hypha at the a pex .
Fig . 1 Distribution of secretory vesicles in both wildtype and hyperbranching (∆ racA ) backgrounds . Quantification of fluorescence intensity
(arbitrar y units) by CLSM microscopy of the post -G olgi v esicle marker SncA fused with GFP ( a ) in the wildt ype (FG7), ∆ glaA (MF7.4) and
GlaA-overexpression ( T et-on- glaA, ∆ glaA ; MF19.5) strains with 20 µg/mL doxyc ycline ( + DO X); ( b ) in the hyperbranching GlaA- overexpr ession strain
( T et-on- glaA, ∆ glaA , ∆ racA ; MF22.4) with or without induction of glaA with DOX. All strains expr ess the GFP-SncA fusion. Fluor escence of vesicles
along the hyphae (up to 20 µm fr om the apex) was quantified from at least 20 h yphae. c Representative pictures (z-stacks) ar e shown (scale bar ca.
20 μm)

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F iedler etal. Microb C ell F act (2018) 17:95
Remarkably , the amount of post -Golg i vesicles is
reduced in the Δ racA mutan t when compared to its
parental strain (F ig . 1 ). The gradient is sharpene d upon
both native glaA expression [ 35 ] and T et -on driven gl aA
overex pression (this study). As shown in Fig. 2 , GFP-
SncA fluorescence cur ves of the Δ racA m utant and its
parental strain show a con vex and concave sha pe, resp ec -
tively , indicating that both amount and distribution of
se cretor y vesicles differ in the two strains . W e calcul at ed
the amounts of sec retor y vesicles along 20 µm hyphal
tips by appro ximating t he mea sured GFP- SncA fluores -
cence with a t hird order polynomial c ur ve (for calcula-
tions , se e Methods). This c alculation indicated that t he
hyperbranching Δ racA strain has , along the first 20 µm
of hyphal tips , on average ca . 23% less vesicles . W e prev i -
ously repor ted that deletion of r acA results in ab out 30%
higher branching frequenc y , and enumeration of h yphal
apices in individual myce lia har ve sted from contr olled
bioreactor cultiv ations in Δ r acA and p aren tal strains
resulted in 17 ± 6 for t he wildty pe (N402) and 22 ± 6 for
the Δ r acA mutant (T able 1 in [ 35 ]); that is , the wildty pe
has 77% of hyphal tip s in com pari son to the Δ r acA
mutant. This is in p erfe ct agre ement with t he ratio calcu -
lated in this study .
The obser vations regarding the amoun t and di stribu -
tion of se cretor y vesicles along hyphal tip s again suggest
that t he total po ol of po st -G olgi se cretor y vesicles i s simi -
lar in b oth strains but sim ply distribute d to mor e hyphal
tips in the Δ racA strain as we have pr e viously propo sed
[ 35 ]. F ur thermore, it implies tha t tip-directe d sec retion
is somehow differen tly orchestrat ed in the Δ racA strain
compared to the wildtyp e. This might be me chanistically
explained by the prev iously obser ve d shift of the endoc y -
totic ring towards the hyphal apex (about 1–2 μm) of A .
ni ger as v isualise d by the marker pr oteins AbpA (actin-
binding protein in volved in invag ination, scission and
release of endo c ytotic vesicle s) and SlaB (adapter pr otein
linking actin to endo c ytosis and involved in e arly endo -
c ytic site initiation; [ 35 , 36 ]).
T et‑ on‑driven glaA ov erexpr ession inΔ racA r esults
inincreased GlaA secretion
Strain MF19.5 (parental strain with T et -on- glaA , Δ gl aA )
and MF22.4 ( T et -on- glaA , Δ gl aA, Δ racA ) were cultivated
in 50mL MM and c omplete medium (CM) for 18h in the
presence of microtalc par ticles to con trol mycelial mac -
romorpholog ies [ 37 ]. Induction of gl aA transcription w as
achieve d by the addition of 20 µg/mL D O X, and physi -
ologic al parameters were mea sured at 0, 24, 48 and 72 h
po st induction. Biomass y ield and t otal protein se cretion
wa s overall similar in both strains af ter 24, 48 and 72 h
(F ig . 3 a, b). In ag reement , total GFP - SncA fluorescence
signals in f reeze drie d biomass samples of b oth strains
did not differ at these time p oints (Additional file 3 : Fig.
S3). The exponential growth pha se of both strains wa s
concluded alr eady af ter 48 h p ost induction, a s glucose
wa s com pletely consumed at this time point (F ig . 3 c ).
W e note d, however , tha t b oth strains diff ered in bioma ss
accumulation and gluc ose consumption after 18 h of pre-
cultivation (Fig. 3 a , c), whic h suggeste d tha t the Δ r acA
strain consumes glucose more slowly . A s a consequence,
both strains might have en tered the po st -exponential
grow th phase at differen t time points . Micros copic anal -
yse s reveale d disp ersed mac romorphologie s for both
strains which is due to the addition of microtalc parti -
cles (F ig . 3 d). However , smaller and a bit more com pact
myc elial clumps which branc hed more f requent were, as
expecte d, obser ved for the ∆ r acA strain. As their diam -
eter wa s less than 200 μm in size (Fig. 3 d), which is the
critic al transpor t distance for o xygen p enetrating A . ni ger
aggregates [ 38 ], any differenc es in mass transfer limita -
tions are ver y unlikely .
As it is k nown that DOX stability is dependent on the
pH and, on the other hand, growth and morpholo gy of
A . ni ger i s unaffected by the addition of 125 μg/mL DOX
[ 33 ], we dec ided to repe at t he experiment descr ibe d
above but to add D O X rep eatedly . In doing s o, we cul -
tivated biolog ical quadr uplicates of the paren tal and the
∆ racA strain in CM plus microtalc par ticles in 50 mL
liquid shake flask cultures with pulse s of induction with
20 μg/mL D O X (after 18 h pr e-incubation, considered as
time point 0 h, a s well as after additional 24 and 48 h).
W e ran the experiments in C M only to obtain higher
biomass y ields . W e determine d biomass y ield, glu -
cose consumption, t otal protein se cretion and sec reted
GlaA in the super natan t by W e stern analysis (dot blot),
each 3 h p ost induction (Fig . 4 ). Bioma ss yield and total
Fig . 2 P olynomial cur ve approximation of distribution of secret or y
vesicles in both wildtype and hyperbranching (∆ racA ) backgr ounds.
Approximation of v esicle distribution for ∆ r acA ( T et-on- glaA, ∆ glaA ,
∆ racA ; MF22.4) and its par ental strain ( T et- on- glaA, ∆ glaA ; MF19.5)
under glaA over expression conditions ( + DO X) with a polynomial
cur ve of the third or der using an Excel trendline function. Both strains
express the GFP -SncA fusion. Cur ves are taken from F ig. 1

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F iedler etal. Microb C ell F act (2018) 17:95
protein secretion of b oth strains gave c omparable results
( ex cept for higher values in total protein se cretion for the
∆ racA strain a t time point 0 + 3 h). Remarkably , differ -
ences in glucose consumption be came again apparent as
already obser ved in the prev ious experiment. A s shown
in F ig . 4 b, the ∆ r acA hyperbranching strain s eeme d to
consume glucose fa ster prior to time point 0 + 3 h, but
slower upon f urther cultivation (24 + 3 h). No detect -
able sec reted GlaA 3h af ter the first induction pulse with
D O X demonstrates , as expe cted, that the T et -on s ystem
is tight [ 33 ], and that longer incubation times are nee ded
to achieve dete ctable le vels of ex trace llular GlaA . Pair -
wis e com pari son of values for ∆ racA and parental strain
showed an up to 4-fold increas e in glucoamylase se cre -
tion in the h y perbranching strain at time point 24 + 3 h
and 48 + 3 h ( p < 0.05). Le ss glucoamylase at time point
48 + 3 h in com pari son with 24 + 3 h suggest s extrac el -
lular deg radation of the enzyme (the cultures must have
enter ed p ost -ex ponential g rowth phas e already prior to
48 h as glucos e is fully consume d in both strains), and
might also b e due to diff erences in glucose consumption
in both strains (Figs. 3 c , 4 c) .
Ba se d on the da ta presented here, we concluded that
when a se cretor y cargo, which is not import ant f or sus -
taine d h y phal tip ex tension, is increas ed by T et -on driven
overex pression, a ∆ racA h y p erbranching phenotype i s
advantage ous to re lea se more cargo, i.e. GlaA , into the
medium. The trans criptomic fingerpr int of r acA lo ss-of-
func tion uncover ed that 139 out of 14,165 A . ni ger gene s
were differ entially expr esse d [ 35 ], which like ly form the
fund amen tal ba sis for this obser vation. Beside s genes
predicte d to enc ode proteins func tioning in protein traf -
ficking , actin lo calisation, (phospho)lipid met abolism and
calcium sig nalling , also four genes supp ose dly rela ted
to carbon c atabolism were differen tially expr esse d:
An06g00560 ( ortholog of the Saccharomyces cere v isiae
Hxt13p hex os e transpor ter) and An12g00160 ( or tholog
of the Saccharom yce s cerev isi ae Mae1 malic enzyme)
were both up-regul a ted, wherea s An16g01770 (predicte d
xylose re ducta se) and An07g01340 (predicte d phospho -
enolpyr uvate carboxylas e) were both down-regul at ed
in the ∆ r acA strain in comparison to the wildty pe. It
be comes theref ore import ant t o elucidate in future st ud -
ies , whether the T et -on driven p ositive effec t on GlaA
Fig . 3 Gro wth profiles and prot ein secretion of both wildtype and hyperbranching (∆ r acA ) backgrounds obtained from shake flask cultivations.
The ∆ r acA ( T et-on- glaA, ∆ glaA , ∆ racA; MF22.4) and its parental strain ( T et-on- glaA, ∆ glaA ; MF19.5) were used in this experiment. F or each
strain, 5 × 10 6 spores/mL wer e inoculated in 50 mL medium in Erlenmeyer flasks, cultivated f or 18 h at 30 °C and 250 rpm. Glucoamylase production
was induced with 20 µg/mL DO X (time point 0 h). 0, 24, 48 and 72 h post-induction, physiological parameters w ere determined and micr oscopic
pictures taken. a Biomass yield (dr y weight), b total secret ed protein, and c r esidual glucose concentration in the media was determined. Results
are av erage and error of three biolog ical replicates . Significance values wer e calculated with 2-tailed t-test with independent variables (* p < 0.05,
** p < 0.01). d Microscopic pictures showing repr esentative pictures of mycelial macr omor phologies at 0 h post -induction (scale bar 100 µm)

P age 7 of 12
F iedler etal. Microb C ell F act (2018) 17:95
se cretion is attributed to the h yp erbranching phenotyp e
only or additionally also linke d to diff erent metabolic
activitie s in the ∆ r acA and its parental strain.
Enhanced sp ecific protein yields in the ∆ racA hyp e r -
branching strain can have enormous repercu ssion not
only for GlaA production but als o for ot her enzy mes pro -
duced by A . ni ger in industri al biotechnolog y [ 8 , 39 ]. The
strain ∆ racA is espe cially suitable for industri al ex ploi -
tation since it does not show any a pparent differenc e in
maximum sp ecific g rowth rate c ompared to the wildtyp e
strain [ 35 ]. Remarkable phenotyp es of the ∆ racA strain
are, however , a hyper pol arisation of actin at the h yphal
ap ex [ 32 ], a shif t of the endoc y tic ring of 1–2μm towards
the h y phal a pex [ 35 ], a conve x instead of a concave distri -
bution of po st -G olgi se cretor y vesicles at the hyphal tip,
e ven under T et -on forc ed trans cription of the glaA gene
([ 35 ] and this work) and a maximum le vel of post -G olg i
se cretor y vesicles at an individual hy phal tip (this work).
P ossible explanations t o bring these ob ser v ations in to a
consisten t frame work could be that (i) t he f usion kinetics
of v- SNAR E-labelle d post -G olgi vesicles w ith the plasma
membrane occurs f aster in the ∆ r acA strain ( con vex
Fig . 4 Gro wth profiles and prot ein secretion of both wildtype and hyperbranching (∆ r acA ) backgrounds obtained from shake flask cultivations
after repeated DO X induc tion. The ∆ r acA ( T et-on- glaA, ∆ glaA , ∆ racA ; MF22.4) and its parental strain ( T et-on- glaA, ∆ glaA ; MF19.5) were pre- gr own for
18 h as described in F ig. 3 ; glucoam ylase production was then induced with 20 µg/mL DOX, as w ell as after additional 24 and 48 h; samples were
collected 3 h post-induction with DOX t o obtain time points 3 h, 24 + 3 h and 48 + 3 h, after which ( a ) biomass yield (dr y weight), ( b ) total pr otein
secretion and r esidual glucose ( c ) were determined as r epor ted in F ig. 3 . d , e Glucoamylase secr etion was quantified by dot blot analysis using a
monoclonal antibody . Each sample was spotted t wice (technical r eplicate) and signal int ensities quantified with ImageJ. Low est row of the dot blot
includes a dilution of glucoamylase as standar d (0.01–1.00 mg/mL glucoamylase). Note the clonal variance between the four biolog ical replicates ,
which is a general phenomenon in culture samples taken from shake flask cultivations. W e decided to discarded clone 4 fr om both strains since
we obser ved an in verse trend in comparison with the other thr ee clones with regard t o glucoamylase secretion (i.e . for clones 1–3 less extracellular
GlaA was obser ved at time point 48 + 3 h than at 24 + 3 h, whereas for clone 4 the opposite was true). Results are thus calculat ed from the first
three biological r eplicates . Average sig nal intensity of the technical duplicates was determined, and values w ere normalized by biomass yield and
total prot ein secretion bef ore calculating median values and quartiles, and plotting on box plots. L owest median value (i.e . median for par ental
strain at 48 + 3 h) was set ar bitrarily as 1. Significance values wer e calculated with 2-tailed (bar charts) or 1-tailed (box plots) t-test with independent
variables (* p < 0.05, ** p < 0.01)

P age 8 of 12
F iedler etal. Microb C ell F act (2018) 17:95
GFP- SncA distr ibution), hence less post-Golgi ve sicles
accumulat e at the tip and/or t hat (ii) post -G olgi vesicles
which carr ied GlaA b ecome much faster endo c ytose d,
i.e. rec ycled due to the for ward shif t of the endoc ytic
ring . In this conte xt it is worth mentioning tha t differ -
ent subpopulations of po st -G olgi se cretor y vesicles have
be en desc ribe d in N. cr a ssa , where t he Spitzenkörp er
consists of micro- and macrovesicles containing either
chitin synthase s , or glucan sy nt has es , respe ctively [ 13 ,
40 ]. In A . ni dulan s , it w as re cen tly shown by super reso -
lution microscopy that secretor y ve sicles con taining the
chitin synthase C hsB can be transp orte d by kinesin-1 on
microtubules ver y fa st (7–10 μm/s) towards the tip and
on early endosome s mediated by k inesin-3 much s lower
(2–7μm/s) towards the hyphal tip and away from it (sup -
po sedly to the T GN; [ 16 ]). Hence, t he v- SNAR E SncA in
A . ni ger could supp ose dly also lo calise to multiple vesi -
cles and early endos omes and could also move with dif-
feren t velocitie s towards or a way from the hyphal apex.
Inter estingly , the overall amount of secrete d prot eins
is identical in the wildty pe and hyp erbranching ∆ r acA
strain (F igs . 3 b, 4 b), although the latter secrete d about
4 times more GlaA (F ig . 4 ). Here, we spec ulate t hat this
is likely balanced by the home ostatic RESS (repression
under se cretion stress) con trol system, a phenomenon
well known for A . ni ger , which ensures a selective dow n-
regulation of genes co ding for e xtrace llular enz ymes
when others are str ongly up-reg ulated [ 41 ]. Still, one
puz zling question remains to be ans wered in future
experiments . Although the hyphal tip remains the main
route of pr otein se cretion in A . ni ger , how much of GlaA
be comes release d into t he medium v ia sept al sec retion?
In A . or yz ae , septum-direc ted se cretion of α-am ylas e
(AmyB) has be en shown [ 42 ], while N. cr a ssa int eg ra tes
the vacuolar pump PM A-1 in to the plasma membrane
subapically without pa ssing the Spitzenkörper [ 43 ]. B oth
obser vations indicat e alternative routes of secretion at
non-apical hyphal regions . W e most recently obtained
suppor ting indications for GlaA accumula tion at septal
regions in A . niger [ 44 ]. I t will b e therefor e inter esting
to study in t he future, whether there is an y higher GlaA
se cretion via s epta in the ∆ racA strain due to T et -on
driven overexpr ession of the glaA gene or not .
C onclusions
In this study , we successfully valid at ed the hyp othesis
that c hallenging the ∆ racA strain to over expr ess the glaA
gene increa ses the amount of post -G olgi se cretor y vesi -
cles at h y phal tips , and eventually results in up to 4- times
higher se creted GlaA . Therefor e, a po sitive corre lation
be tween the amount of growing hyphae and se cretion
exists in A . ni ger , given that transcr iption of the secre -
tor y protein is con tinuously for ced by the T et -on system.
Given the enormous impor tance of A. niger as industr ial
cell factor y for the pr oduc tion of proteins, enz yme and
metab olites, this study ha s prof ound implications for bio -
technolog y . B ase d on the data obtaine d in this study , we
propos e a r acA deletion background as a def ault, hyp er -
se cretion strain for enhanced ex tracellular product yield.
Methods
Strains andgener al cloning procedur es
Strains use d in this study are summarize d in T able 1 , pl as -
mids and primers in Additional file 4 : T able S1. Molecular
techniques for E . coli followed protocols desc ribe d earlier
[ 45 ]. A . ni ger transformation and genomic DNA ex trac -
tion from selec ted transformants w as done accor ding to
[ 46 ]. Strains were grown at 30 °C in minimal me dium
(MM) [ 47 ] or complet e me dium (CM), consisting of MM
supplemented with 1% w/v yea st ex tract and 0.5% w/v
ca samino acids . When require d, 100 µg/mL hygromycin,
10 mM ur idine or 10 mM histidine were adde d to the
medium.
T o obtain py rG − strains , 2 × 10 7 spores were plat ed
on MM plates con taining 75 mg/mL 5-fluoro orotic acid
(FOA), 10 mM ur idine and 10 mM proline. Pla tes were
incubated at 30 °C for 1–2 we eks until single colonies
were visible. FOA-r esist ant m utants were purifie d on
MM + FOA pla tes once and tested for their uridine auxo -
troph y on MM plates or MM plates con taining 10 mM
uridine, resp ectively . Cloning and relat ed mole cular te ch -
niques were per formed according to standard pr oce dures
[ 45 ], whereas A . niger transf ormation, genomic DNA
extraction and Southern blot were per formed a s prev i -
ously desc ribe d [ 46 ].
Strain FG7 was u sed a s ancestor strain in which the
pyrG gene w as counterselected w ith FO A to obtain
the py rG − strain SS1.1. T o construc t a pyrG -rec yclable
glaA deletion c assette, promoter (P) and terminat or ( T)
regions of the glaA gene were amplified u sing primers
listed in Additional file 4 : T able S1. Using a combined
fu sion PC R and ligation appr oach, a PglaA - AopyrG -
PglaA - T glaA c asse tt e wa s constructe d and cloned in
pJET1.2 giving r ise to pSS3.34. This pla smid was trans -
formed into strain SS1.1 to delete t he glaA gene, g iving
strain MF7.4, which was s electe d via S outhern analysis .
The A opyrG marker wa s coun terselecte d in this strain
via FOA selec tion and strain MF9.1 was generated,
being ag ain uracil-auxotr oph ( pyrG − ). T o construct
a Te t - on ::gl aA ex pression plasmid, the gl aA sequence
wa s amplified using primers li sted in Additional file 4 :
T a ble S1 and ligated into t he unique P meI restriction
site of the plasmid p VG2.2 [ 33 ] harb ouring the T et-on
system and the A . ni ger py rG* a s selection marker , g iv -
ing ris e to pMF19.1. This construct wa s targeted to the
pyrG lo cus in MF9.1 and strain MF19.5 w as sele cted

P age 9 of 12
F iedler etal. Microb C ell F act (2018) 17:95
on transformation plat es lack ing uridine and verifie d
via S outhern analysis . T o c onstruc t a racA::hygR dele -
tion ca ssette for knock -out of the endogenou s r acA gene
in MF19.5, the split mark er approach was us ed [ 48 ]. In
brief, the 5 ′ and 3 ′ se quences of r acA and the h ygromy -
cin resistance gene were amplified using pr imers listed in
Additional file 4 : T able S1, fus ed v ia PC R , and lig at ed into
the plasmid pJET1.2 giv ing rise to pMF14.3 ( P racA - hyg R )
and pMF15.1 ( hygR ::T racA ), respe ctively . B oth fragment s
were transformed into strain MF19.5 and a transformant
with a deleted racA gene wa s selecte d via S outhern anal -
ysis (strain MF22.4).
Conf ocal microscopy ofindividual A. niger h yphae
Microscopy wa s per formed a s prev iously descr ibe d
[ 30 , 35 ]. Briefly , for conf oc al las er scanning micros copy
(C LSM) conidia were spotted on MM plates , supple -
mented with differen t concen trations of do xyc ycline a s
indicated and incub at ed at 22 °C for 2 days , following
ex c ision of the colon y and pl acing it upside dow n in to a
gla ss-b ott om P etri di sh. Liquid MM medium (if ne ede d,
supplemented with the same conc entration of do xyc y -
cline tha t wa s present in the MM plat e) wa s added and
cells were incubat ed at 22 °C until the c ells resumed
grow th. Ce lls were analyse d using an inverted TCS SP8
confocal micros cope system (Leic a, G ermany). Images
were captur ed u sing a HC PL AP O CS2 20 × /0.75 IMM
obje ctive with a pinhole at air y unit 1 (48.8 µm) at an
image resolution of 1024 × 1024 pi xe ls at 700 Hz . F or
GFP detec tion, 3% laser (488 nm) int ensity wa s use d
coupled with an emission de tection of 495–545 nm at
a gain of 800. 10 z-st acks were taken using the system-
optimize d calculation of z- stacks . The GFP- SncA fluore s -
cence of single z-stacks w as qu an tifie d with the provided
sof tware L A S X (Leica , G ermany) using the tool “Draw
Line” in the tab “ Quantif y ” . A line wa s drawn by hand
along the h y phae, starting from the tip, resulting in a
value for the int ensity of fluorescence (in arbitrar y units ,
whereas 256 i s the maximum value below overexposure)
appro ximately ever y 0.46 µm. The fluorescence signal
wa s measure d over a length of 20µm.
Calculation ofGFP ‑SncA fluorescence a thyphal tips
W e calc ulated the amounts of sec retor y vesicles along
20 µm hyphal tips ba se d on the measured GFP- SncA
fluorescence. Fluorescen t signal c ur ves depic ted in F ig . 2
were appr oxima ted using E x ce l (Microsof t Office Pack -
age 2010) with the following poly nomial func tions and
coefficients of de termination
R 2

(

parental + DOX ) y = 0.0311 x
3

− 0.5712 x
2

− 9.0959
x

+
253.15 ( with R

2 =
0.9902 )

Solv ing the polynomi al functions over the whole 20µm
hyphal length w ith t he integral
give s the f ollowing v alues ( I = appro ximated amounts of
vesicles along 20µm hyphal tip s)
and the ratios
Shake flask cultiva tions of A. niger
F or produc tion of glucoamylase, 5 × 10 6  spores/mL of
strains MF19.5 ( T et -on- glaA , ∆ gl aA ) or MF22.4 (T et -
on- glaA , ∆ gl aA , ∆ r acA ) were inocul a ted in 50 mL liquid
medium and strains were cultiv at e d a t 30 °C , 250 r pm
in shake flask cultures containing MM or CM with 5%
w/v glucose a s carbon s ourc e and with 10 g/L mic ro
talc par ticles to con trol m ycelial macromorpholog ies as
desc ribe d in [ 37 ]. F or me dium com po sition pleas e ref er
to [ 46 ]. Af ter 18 h incubation (c onsidered a s time point
0), T et -on driven expression of GlaA wa s induced with
20 µg/mL do xyc ycline (D O X) and f urther incubated for
0, 24, 48 and 72 h before analysis of physiolog ical param -
eters ( biomass dr y weight a s well as total protein secre-
tion and residual glucose concentra tion in the media ;
se e below). F or rep eated induction of gl aA ex pression
in 50 mL liquid shake flask cultures of CM w ith 10 g/L
micro talc par ticles , 20µg/mL DOX was adde d 18h post -
inoc ulation ( considered a s time point 0) as well a s after
fur ther 24 and 48 h of inc ubation. Samples were taken
(�

racA + DOX ) y =− 0.0432 x
3

+ 1.9981 x
2

− 32.4820
x

+ 259.68
(

with
R

2 = 0.9859
)

(�

racA − DOX ) y =− 0.0499 x
3

+ 2.0248 x
2

− 26.9690
x

+ 183.65
(

with
R

2 = 0.9713
)

I

=
20 µ m


0 µ m

 ax 3 + bx 2 + cx + d 
dx

( parental

+
DOX ) I

=
2964.62

(� racA

+
DOX ) I

=
2297.47

(� racA

−
DOX ) I

=
1682.67

 racA + DOX
parental

+
DOX

=
2297.47
2964.62

× 100% ∼
=
77%

 racA

−
DOX
 racA

+
DOX

=
1682.67
2297.47

× 100% ∼
=
73%

P age 10 of 12
F iedler etal. Microb C ell F act (2018) 17:95
3 h post -induction with D O X to determine physiological
parameters and extrac ellular GlaA . F or microscopic pic -
tures , a small amoun t of culture ( ca . 0, 1 mL) wa s sam-
pled; image s were taken using a SA8APO e quippe d with
a MC120HD camera (L eica , USA). Exper iments were
per formed a s biologic al triplicates/quadr uplicates .
Determination ofbiomass dry weight, t otal protein
secretion, residual gluc ose andtotal GFP‑SncA
fluorescenc e
F rom shak e fla sk cultur es , 4 mL of samples were taken at
the indicated time point . Biomass and culture super na -
tant were separated by suction filtration under vac uum.
Biomass w as collec ted, frozen at − 80 °C, and f reeze drie d
overnight to determine bioma ss yield ( dr y weight). T otal
protein secretion in the culture super natan t wa s deter -
mined v ia the Bradford assay (BioRad) acc ording to t he
manufacturers’ prot ocols and abs orbance (600 nm) wa s
mea sured using a GloMax ® -Multi Detec tion S ystem (Pro -
mega). Q uantification of residual glucose in the cultiv ation
medium w as p erforme d with the Glucose G OP/P A P Liqui -
color kit (H uman, Ger many) ac cording t o the manufactur -
er ’ s manual. T otal GFP - SncA fluorescence wa s determine d
in freeze dr ied bioma ss . 50mg dried bioma ss were grinde d
and resusp ended in 1 mL 50 mM N aP O 4 buff er pH 7.0.
F ollow ing ultrasonific a tion for 10 min, fluorescence signal
in super natan t wa s determine d using a GloMax ® -Multi
Dete ction System (Promega) equipp ed w ith a blue filter
( ex citation: 490nm, emission: 510–570nm).
Analysis ofe xtracellular glucoam ylase (GlaA) b y W estern
analysis/dot blot
Supernatant har ve sted by suction filtration (see ab ove) was
analyse d with regard to GlaA cont ent by dot blot using
the Minifold I Spot -Blot sy stem (Whatman S chleicher &
Schuell). Briefly , 100 µL samples were mi x ed w ith 150 µL
phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM
KCl, 1 mM Na 2 HPO 4 , 0.2 mM K H 2 PO 4 ), heated at 100 °C
for 10 min and coole d down. 200 μL were then spotted
in the slots of the dot blot dev ice provided w ith a nitr o -
cellulose membrane (PROTR AN, Schleicher & Schuell)
prev iously soake d on PBS under vacuum to allow pro -
tein binding . After suction, 200 μL PB S were a pplied to
wa sh the membrane under vacuum. A standard dilution
of GlaA (0.01–1.00 mg/mL g lucoam ylas e) wa s done in
PBS and blotted a s descr ibe d above. The membrane wa s
then removed from the dot blot de vice and soaked 1 h at
25 °C in 30 mL Protein Blocking Buffer (PBB: 100 mg/mL
milk powder in PB S + 0.1% v/v T ween 20) under shaking .
Monoclonal anti-GlaA antibody (k indly provided by Peter
Punt, TN O , The Ne therlands) was then adde d (10 µL , i.e.
final dilution 1:3000), and the membrane was f urther incu -
bated overnig h t at 4 °C under shaking . PBB w as dis carded
and membrane wa shed three times for 5 min a t 25 °C with
PBS + 0.1% v/v T we en 20. HRP-con jugated, se condar y anti-
mouse antibo dy (Agilent T e chnologie s, USA) w as then
added (6.7 µL in 20 mL PBB, i.e. final dilution 1:3000), and
the membrane was inc ubated for 1 h at 25 °C under shak -
ing . PBB wa s discarde d, and membrane wa shed (three
times with PBS + 0.1% v/v T we en 20, and then onc e with
PBS for 5 min e ach a t 25 °C ). Chemilumines cenc e reaction
wa s perfor med by using an EC L Prime W estern Blotting
Dete ction Kit (GE Healthcare), and signal de tected w ith
ChemiDo c ™ MP Imaging System using the Image Lab sof t -
ware ( both from BioR ad). Signal intensity was quantifie d
with the open source sof tware ImageJ using a standard pro -
tocol ( htt ps ://image j.nih.gov/ij/do cs/exam p les/dot -blot/ ).
F or W estern blot analysis (Additional file 4 : F ig . S1),
10 µL culture super natant of FG7 (wildtyp e) and MF7.4
(∆ glaA ) g rown in MM in 20 mL shake fla sk cultur e for
90 h at 30 °C , 250 r pm (inocul a tion 5 × 10 6 spores/mL)
were loaded to a 12% w/v SDS -P AGE and transferred to
a PV DF membrane (Roth, Germany) after the proteins
have been s eparated. De tection w as p erformed w ith the
same primar y and s econdar y antibo dies a s descr ibe d
above. 10 µL of c ultur e super natant wer e directly ana -
lyse d via W estern blot using an anti-glucoamylas e anti -
bo dy . Incub ations wer e per formed in PB S + 0.1% v/v
T we en 20 supplemen ted w ith 5% w/v dr y milk . The pri -
mar y antibo dy incubation wa s per formed at 4°C for 16 h,
while the blot was inc ubated with the se condar y anti -
bo dy at room tem p era ture for 1 h. Chemilumine scence
reaction w as p erformed by u sing an EC L Prime W ester n
Blotting Dete ction Kit (GE Healthcare).
Additional files
Additional file1: F ig. S1. W estern blot analysis of wildtype (FG7) and
∆ glaA (MF7.4). 5 × 10 6 spores/mL were inoculated in 20 mL MM medium
in Erlenmeyer flasks, and cultivated f or 18 h at 30 °C and 250 rpm. 10 µL
of culture supernatant were dir ec tly analysed via Western blot using an
anti-glucoamylase antibody .
Additional file2: F ig. S2. Southern blot analysis of wildt ype (N402) and
T et- on- glaA (MF19.5). The glaA gene under control of the doxycycline -
inducible T et-on expression system was re-introduced into the pyrG locus
of MF9.1, resulting in the T et- on- glaA strain MF19.5. Correct integration of
a single copy at pyrG was confirmed by Southern blotting (A). Genomic
DNA of MF19.5 and N402 was digested using NcoI and BsrGI . and h ybrid-
ised with a 600 bp probe, homologous t o par ts of the pyrG gene. The
expected band sizes were 9005 bp + 4231 bp for MF19.5 and 3126 bp f or
N402, respectively (B).
Additional file3: F ig. S3. T otal GFP-SncA fluor escence in freeze dried bio-
mass of both wildtype and hyperbranching (∆ racA ) backgrounds obtained
from shake flask cultivations. The ∆ r acA ( T et-on- glaA, ∆ glaA , ∆ racA; MF22.4)
and its parental strain ( T et-on- glaA, ∆ glaA ; MF19.5) were used in this
experiment. Each 5 × 10 6 spores/mL were inoculated in 50 mL medium in
Erlenmeyer flasks, cultivated f or 18 h at 30 °C and 250 rpm. Glucoamylase
production was induced with 20 µg/mL DO X (time point 0 h). 0, 24, 48

P age 11 of 12
F iedler etal. Microb C ell F act (2018) 17:95
Authors ’ contributions
MRMF and VM designed the study , MRMF and CK conducted the plasmid
construction and generation of the A. niger repor ter strains , MRMF per formed
the CLSM analyses, MRMF , LB and CN per formed g rowth assays and analysed
secreted pr oteins and glucoamylase . MRMF , CN and VM prepared the manu-
script. All authors read and approv ed the final manuscript.
Acknowledgements
W e ack nowledge support by the German R esear ch F oundation and the Open
Access P ublication F unds of TU Berlin.
Competing int erests
The authors declare that they hav e no competing interests .
A vailability of data and materials
The datasets used and/or analysed during the current study are available fr om
the corresponding author on reasonable r equest.
Consent f or publication
Not applicable.
Ethics approv al and consent to participate
Not applicable.
F unding
This work was par tly supported by a grant from the Deutsche F orschungsge -
meinschaft (DFG) to VM (Grant No . ME 2041/5-1) being par t of the Schwer -
punktprogramm DiSPBio T ech (SPP 1934).
Publisher’ s Note
Springer Nature remains neutral with r egard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 8 March 2018 A ccepted: 8 June 2018
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