Isolation of four pepsin-like protease genes from Aspergillus niger ...

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Fungal Genetics and Biology 45 (2008) 17–27
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Isolation of four pepsin-like protease genes from Aspergillus niger
and analysis of the effect of disruptions
on heterologous laccase expression
Yongchao Wang a , Wei Xue a , Andrew H. Sims b , Chuntian Zhao a , Aoquan Wang a, *,
Guomin Tang a , Junchuan Qin c , Huaming Wang b
a Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, PR China
b Genencor, A Danisco Division, Palo Alto, CA 94304, USA
c School of Life Science, Nanjing University, 210093 Nanjing, PR China
Received 10 February 2007; accepted 24 September 2007
Available online 29 September 2007
Abstract
Four new aspartic protease genes pepAa, pepAb, pepAc and pepAd from Aspergillus niger were identified using a comparative genomic
approach. All four gene products have highly conserved attributes that are characteristic of aspartic proteases; however, each one has
novel sequence features. The PEPAa protease appears to represent an ortholog of a pepsin-type aspartic protease previously identified
from Talaromyces emersonii and Scleotinia sclerotiorum. The PEPAb protease appears to be an ortholog of an aspartic protease previously
identified from BcAP1 of Botryotinia fuckeliana. The PEPAc protease also appears to be an ortholog of BcAP5 from B. fuckeliana.
These four genes appear to be conserved in many species of filamentous fungi, all except PEPAb contain a predicted signal peptide. Transcriptome
analysis revealed that transcripts of the pepAa gene of Aspergillus nidulans were significantly up-regulated due to recombinant
chymosin secretion, suggesting that silencing these genes may lead to improved yields of secreted proteins. To establish the effects of
reduced protease activity on the stabilities of secreted proteins, three of the four genes were individually disrupted by double crossover,
although we were unable to disrupt the pepAc gene. The secretion level of heterologous laccase in the pepAa, pepAb and pepAd disruption
mutants were increased by about 21%, 42% and 30%, respectively. And their total glucogenic enzymes secretion were also increased by
about 18.7%, 37.0% and 5.20%, respectively.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Aspergillus niger; Pepsin-like protease; Disruption; Heterologous laccase expression
1. Introduction
* Corresponding author. Fax: +86 010 64807505.
E-mail address: wangaq@sun.im.ac.cn (A. Wang).
Filamentous fungi represent excellent hosts for secretion
of homologous and heterologous proteins (Conesa
et al., 2001). However, proteolytic degradation is a major
problem during protein production even in a well-established
organism such as Aspergillus niger (Archer et al.,
1992). Proteolytic degradation affects mainly heterologous
proteins evidenced by improvement of heterologous protein
production upon deletion of the protease genes
(Archer et al., 1992; van den Hombergh et al., 1997b).
Four extracellular proteases with acid pH optima (an
aspartic protease, PEPA, a glutamic protease, PEPB and
two serine carboxypeptidases, PEPF and PEPG) have previously
been characterized (Berka et al., 1990; Inoue
et al., 1991; Krishnan and Vijayalakshimi, 1985; Mattern
et al., 1992; van den Hombergh et al., 1994). A fifth protease
gene, encoding an extracellular subtilisin-type serine
protease, PEPD, has been cloned based on conserved
amino acid sequences within subtilisins (Jarai et al.,
1994a). Furthermore, three proteases that are homologous
1087-1845/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2007.09.012

18 Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27
to yeast vacuolar proteases have been cloned from A.
niger; these are PEPE, a pepsin-type aspartyl endoprotease
that is the homologue of the vacuolar pep4 gene
product in yeast (Jarai et al., 1994b), PEPC, a subtilisintype
serine endoprotease (Frederick et al., 1993) and
CPY, a serine carboxypeptidase (Yaver et al., 1995). Following
the whole-genome sequencing of A. niger (Pel
et al., 2007), approximately 200 predicted proteases have
been identified, with similar numbers also seen following
whole-genome sequencing of Aspergillus nidulans (Galagan
et al., 2005), Aspergillus oryzae (Machida et al.,
2005) and Aspergillus fumigatus (Nierman et al., 2005).
Pepsin-like enzymes such as aspartic proteases are
members of the A1 family of peptidases (Rawlings
et al., 2004). This family comprises proteins with a
three-dimensional structure close to that of pepsin. Generally,
these enzymes form two domains with different
amino acid sequences, but basically similar folds. The
catalytic site of the pepsin-like enzymes is formed at
the junction of the two domains. It contains two aspartic
acid residues, Asp32 and Asp215 (human pepsin numbering),
one in each domain (Blundell et al., 1998). In accordance
with the accepted mechanism of the pepsin-like
enzyme function (James, 2004), the Asp215 has to be
charged, whereas Asp32 has to be protonated. A remarkable
property of the catalytic center is its adaptation for
the action in a wide range of pH, from 4.0 to 7.0. Aspartic
proteinases have been identified from Botrystis cinerea
(ten Have et al., 2004) and A. oryzae (Machida et al.,
2005).
Berka et al. showed that deletion of the pepA gene
increased bovine prochymosin production by more than
66% (Berka et al., 1990). Van den Hombergh et al. disrupted
three different acid-protease genes in A. niger,
pepA, pepB and pepE, to determine the contribution of
each protease to the overall protease spectrum using
bovine serum albumin as a broad substrate. They concluded
that the PEPA and PEPB proteases appeared to
constitute 84% and 6% of the extracellular acidic proteolytic
activity, respectively, whereas the PEPE protease is
responsible for 68% of the intracellular acidic protease
activity (van den Hombergh et al., 1997a). Moralejo
et al. also showed that a defect in the pepA gene reduced
degradation of overexpressed thaumatin in A. niger
(Moralejo et al., 2000).
Here, we report the isolation and characterization of
four ‘new’ genes (pepAa, pepAb, pepAc and pepAd)
encoding pepsin-like aspartic proteases from A. niger
and demonstrate that these genes are conserved within
the Aspergillus genus. The proteins possess different characteristics
and may have distinct localizations and functions.
Transcription of the pepAa gene during
recombinant protein production was observed with our
A. nidulans cDNA microarray (Sims et al., 2005). Therefore,
we examined the effect of disrupting these proteases
on the secretion of the homologous and the heterologous
proteins in A. niger.
2. Materials and methods
2.1. Strains and growth conditions
A. niger GICC2773 (Valkonen et al., 2003) was used for
the disruption experiments of the putative aspartic protease
genes. This strain contains the disruption mutant of the
pepA gene and integrated heterologous gene (lcc1 of
theTrametes versicolor laccase gene) expressed as fusion
to the glucoamylase. Escherichia coli DH5a served as host
for routine DNA manipulations (Sambrook et al., 1989).
Aspergillus niger GICC2773 strains were maintained on
solid sporulation medium, GMP (2% glucose, 2% maltose
extract, 0.1% peptone, 1% agar) supplemented with 0.3%
casein when necessary. Disruption mutants were grown
on GMP supplemented with 200 lg hygromycin B per
ml. Seed cultures of the A. niger strains in 30 ml of S3Y2
medium (3% soluble starch, 2% yeast extracts, 0.5%
KH 2 PO 4 , 0.5% corn powder) were incubated at 30 °C with
200 rpm agitation in a rotary G10 incubator (New Brunswick
Scientific, NJ) for 14 h. For laccase production study,
A. niger strains were grown in 30 ml of modified Promosoy
medium (Ward et al., 2004), where the promosoy was
substituted with tryptic soy broth (Difco, Detroit, MI).
2.2. Protease identification and sequence analysis
The predicted pepsin-like proteases were identified as
described previously (Sims et al., 2004a,b). Briefly, files
containing all the predicted open reading frames (ORFs)
from species of filamentous fungi with recently completed
whole-genome sequences (Table 2) were downloaded
from their respective databases and filtered for sequences
of 100 or more amino acids beginning with a methionine
start codon. These were then searched for putative aspartic
protease domains (PF00026) using the protein families
and HMM database (Pfam) release 11.0 (Bateman et al.,
2004). The results were compared to known protease
families in the peptidase database, MEROPS release 6.4
(Rawlings et al., 2004). The coding sequences for predicted
proteases were manually compared using the ClustalW
program (Thompson et al., 1994) with default
settings. Additional sequences of genes from other species
that have previously been cloned and characterized
were added to demonstrate that the ORFs represent
putative orthologs of known genes. The Treeview program
was used to draw and visualize hierarchical trees
based upon amino acid sequence alignments (Page,
1996). The SignalP (Nielsen et al., 1997) and ProP
(Duckert et al., 2004) programs were used to identify
prepro signal sequences and propeptide cleavage sites,
respectively. The big-PI (http://mendel.imp.univie.ac.at/
sat/gpi/fungi_server.html) fungal predictor was also used
to identify likely GPI modification sites (Eisenhaber
et al., 2004). The disulfide bonds were predicted using
DIpro 2.0 (http://contact.ics.uci.edu/bridge.html) (Cheng
et al., 2006).

Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27 19
2.3. Microarray analysis
Transcriptome analysis was performed as described previously
(Sims et al., 2005, 2004b,c). Briefly, a pyrG- singlecopy
recombinant chymosin-producing A. nidulans strain
(Cullen et al., 1987) was compared to its parental strain
transformed with an empty vector. Shake flask cultures
were grown on 500 ml of SCM media (Ward et al., 1990)
in 2 l flasks. BLASTx was used to identify ESTs (expressed
sequence tags) representing predicted cDNA (ORF)
sequences from the A. nidulans genome sequence
(www.broad.mit.edu). Differential gene expression was calculated
after normalisation in MaxDView (www.bioinf.man.ac.uk/microarray/)
and significance values
determined as described previously (Sims et al., 2004c).
2.4. Plasmid construction
For construction of gene disruption plasmid, the four
pepAx genes (x represents a, b, c, or d) were amplified from
genomic DNA of A. niger using primers P Ax1 and P Ax2
listed in Table 1, which were designed according to the
sequence of the pepAx genes of A. niger. At the 5 0 end of
each primer, a restriction enzyme recognition site of HpaI
was introduced. The PCR was carried out with Pfu DNA
polymerase and consisted of one cycle at 94 °C for 4 min
and 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C
for 2.5 min, and then a final extension at 72 °C for
10 min. Following the PCR, amplified DNA was cloned
into a pBluescript KS(+) vector to yield the plasmid
pBS-TMx. A 1.5 kb BamHI/SalI fragment from pMW1,
containing the PpcbC-hph expression cassette, was filled
in with Klenow fragment and then inserted into the
intended disruption site of pBS-TMx to construct the final
disruption plasmid pAxS-T.
2.5. Transformation of A. niger
Protoplasts of A. niger strains were prepared with reference
to Campbell et al. (1989) and transformed by the polyethylene
glycol method (Yelton et al., 1984). For the
disruption of the pepAx genes in A. niger (strain
GICC2773), the plasmid pAxS-T was digested with HpaI.
The resulting DNA disruption fragment and the 3.0 kb
Table 1
List of primers used in the disruption experiments a
5 0 Primer 3 0 Primer
P Aa1 5 0 -GCACTTCTTTCCCCTTTTTGTTTAC-3 0 P Aa2 5 0 -AGGTTAACTTGAATTGTAGATACAGCCAC-3 0
P Ab1 5 0 -ACGTTAACCATATCACAGCTATATCCCC-3 0 P Ab2 5 0 -ACGTTAACGCCAGGTCCTCCTTCTGC-3 0
P Ac1 5 0 -TGGTTAACGAGGGATTGCTCTATTG-3 0 P Ac2 5 0 -TGGTTAACTGTGCTATGCTATTGGTG-3 0
P Ad1 5 0 -TGGTTAACTCGTAAGTAGGTAGGCTG-3 0 P Ad2 5 0 -ATGTTAACCCGAGGTGCTGCTTG-3 0
P hph 5 0 -GAGGGCAAAGGAATAGAGTAGATG-3 0
P outAa n.d. P outAa 5 0 -TCATGGATTAGGGTTAGAAAGAGTG-3 0
P outAb 5 0 -GGAGAGATAGGACGTAAACTTCATG-3 0 P outAb n.d.
P outAc n.d. P outAc 5 0 -TCATGTGAATATGTGCACGAAGGTG-3 0
P outAd n.d. P outAd 5 0 -AGAGCAGAGAAGAAATACTGAGGAG-3 0
P Aa3 5 0 -TCCTCCAGTCCCTCATTGTTGCC-3 0 P Aa4 5 0 -AACTCGTACTCGCCCACTCCGTC-3 0
P Ab3 Same as P Ab1 P Ab4 Same as P Ab2
P Ac3 Same as P Ac1 P Ac4 5 0 -TCTGCTCGTCGGTGGTTGTG-3 0
P Ad3 Same as P Ad1 P Ad4 Same as P Ad2
a The introduced restriction sites of HpaI are indicated in bold.
Table 2
Predicted aspartic proteases in the species of filamentous ascomycetes
Species
No. predicted
ORFs
Sequenced by
Website
Aspergillus fumigatus 7 Consortium (Nierman et al., 2005) http://www.cadre.man.ac.uk/
8 NRRL 3—Integrated genomics/genencor http://www.jgi.doe.gov/
Aspergillus niger 8 ATCC 1015—Joint Genome Institute http://www.jgi.doe.gov/
8 CBS 513.88—DSM (Pel et al., 2007) http://www.jgi.doe.gov/
Aspegillus oryzae 12 National Institute of Technology and Evaluation (Machida
et al., 2005)
http://www.bio.nite.go.jp/ngac/e/rib40-
e.html
Aspergillus nidulans 9 Broad Institute (Galagan et al., 2005) http://www.broad.mit.edu/
Magnaporthe grisea 16 Broad Institute http://www.broad.mit.edu/
Neurospora crassa 16 Broad Institute http://www.broad.mit.edu/
Fusarium
15 Broad Institute http://www.broad.mit.edu/
graninearum
Trichoderma reesei 16 Joint Genome Institute http://www.jgi.doe.gov/
Open-reading frames were found to contain the Pfam domain PF00026, suggesting that they are members of the peptidase A1 family (pepsin family).

20 Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27
vector fragment were used to transform the A. niger strain
GICC2773. Hygromycin B resistant transformants were
selected in GMP with 0.8 M sucrose as osmotic stabilizer
supplemented with 200 lg/ml hygromycin B.
2.6. DNA isolation
Genomic DNA of the A. niger strains was obtained by a
modification of the benzyl chloride extraction method (Zhu
et al., 1993) as follows: the fungi were grown in S3Y2 medium
for 16 h at 30 °C and harvested by filtration on Nytal
30 filters. To each sample, 500 ll extraction buffer (100 mM
Tris–HCl, pH 9.0, 40 mM EDTA), 100 ll 10% SDS and
300 ll benzyl chloride were added. The tube was vortexed
and incubated in 50 °C for 1 h with shaking or repeatedly
vortexing at 10 min interval to keep the two phases thoroughly
mixed. Then 300 ll 3 M NaOAc, pH 5.0, was added
to the tube and kept on ice for 15 min. After centrifugation
at 12,000 rpm, 4 °C for 10 min, the supernatant was collected,
and DNA was precipitated with isopropanol.
0.1 M HOAc buffer at pH 4.6) as substrate and incubated
at 37 °C for 30 min. The reaction was stopped by boiling.
The glucose liberated was quantified by the glucose oxidase/peroxidase
method using a glucose assay kit
(Zhongsheng Biotechnology and Science Inc. product).
The optical density at 525 nm was measured and the
amount of glucose produced was calculated. The expression
level of total glucogenic enzymes was indicated in
the units of total enzymes activity secreted per gram dry
2.7. PCR screening of transformants for homologous
recombination
The organization of the targeted gene and its neighbouring
regions were examined by PCR for the disruption of
pepAx genes using oligonucleotide primer pairs as follows:
(i) primers annealing to the hph gene and the sequence outside
homologous fragment: P hph and P outAx (see Table 1);
(ii) primers annealed outside the disrupted site of pepAx
genes: P Ax3 and P Ax4 (see Table 1). PCR consisted of one
cycle at 94 °C for 4 min and 30 cycles at 94 °C for 1 min,
55 °C for 1 min, 72 °C for 2.5 min, and then a final extension
at 72 °C for 10 min.
2.8. Laccase expression assay
Aspergillus niger strains GICC2773, DpepAa, DpepAb
and DpepAd were grown in modified Promosoy medium
at 28 °C and 200 rpm for 144 h. Culture filtrate and mycelium
were collected by centrifugation. For laccase assay a
30 ll amount of appropriately diluted sample solution
was mixed with 2.9 ml 0.1 M HOAc buffer (pH 4.6) and
70 ll 20 mM ABTS substrate and incubated at 37 °C for
30 min. The optical density at 420 nm was measured. One
unit of laccase activity is defined as the amount of enzyme
oxidizing 1 lmol of ABTS per min. For determination of
mycelium dry weight the collected mycelium was washed,
squeezed, blotted dry and dried at 80 °C for 3 h before
weighing. The expression level of laccase was indicated in
the laccase activity (international units) produced per gram
dry weight of mycelium ( IU/g ).
2.9. Assay of total glucogenic enzymes activity
A60ll amount of culture filtrate collected above was
mixed with 140 ll H 2 O and 2.8 ml 2% soluble starch (in
Fig. 1. Hierarchical clustering of sequence alignments of the ORFs
predicted to represent genes of the A1 aspartic protease family (pepsin
family) in the Aspergillus genus. Clustering performed using the ClustalW
(Thompson et al., 1994) program with default settings. The tree was drawn
with Treeview program (Page, 1996) and is rooted with human pepsin A
sequence. Previously isolated fungal aspartic proteases in the MEROPS
database (Rawlings et al., 2004) are in bold. GenBank Accession numbers
are presented in brackets, Aspergillus hypothetical proteins are defined by
locus ID accession numbers, for details of genome sequences (see Table 2).

Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27 21
weight of mycelium (U/g). One unit of glucogenic enzyme
activity was defined as the amount of enzymes that liberated
1 lmol glucose per min from soluble starch.
3. Results
3.1. Identification and characterization of four pepsin-like
proteases
In a survey of all the predicted proteases encoded by 22
species of fungi that have recently undergone whole-genome
sequencing (Sims et al., 2004a), a number of putative
novel pepsin-like aspartic proteases were identified. Within
the filamentous ascomycetes, between 7 and 16 open-reading
frames for each species were predicted to represent
aspartic proteases based upon their conserved sequence
to the Pfam domain PF00026 (Bateman et al., 2004) for
members of the peptidase A1 (pepsin) family of proteases
(Table 2). The Eurotiales class (including three species of
Aspergillus) contained approximately half as many predicted
aspartic proteases as the Pezizales and Sordariales
classes (including Magnaporthe grisea, Neurospora crassa,
Fig. 2. Alignment of the predicted amino acid sequences of the four A. niger aspartic proteases (PEPAa–PEPAd) with that of aspergillopepsin A (PEPA) using
ClustalW (Thompson et al., 1994). Signal peptides for all sequences (except PEPAb) are boldfaced. The possible KexB cleavage sites found in pepAa (RR),
pepAc (KR) and pepAd (KR) were marked with an arrow. Hallmark motifs in aspartic proteases are indicated by black shading blocks. The active site motifs
were shown in italics. A serine-rich region at the C-terminus was boxed. The predicted GPI-modification site was double underlined. Locations of the cysteine
residues for the predicted disulfide bonds are shown with grey shading blocks. The insertion sites for PEPAa, PEPAb and PEPAd were shown in ‘‘.’’.

22 Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27
Fig. 3. The A. nidulans putative ortholog (AN2517.1) of the A. niger
pepAa pepsin-like protease is significantly differentially expressed during
increasing heterologous chymosin secretion. Expression in chymosinproducing
strain is relative to the parental strain. Error bars are equal to
one standard deviation of the mean (three repeats) and the values above
and below the dotted lines were previously shown to represent significant
changes in gene expression (Sims et al., 2005).
Fusarium graninearum and Trichoderma reesei). Eight predicted
ORFs from the NRRL3 strain of A. niger sequenced
by Genencor/Integrated were found to contain the Pfam
domain PF00026 for members of the peptidase A1 (pepsin).
All eight ORFs with 100% amino acid sequence
matches were found in the ‘best protein models’ download
of the whole-genome sequencing of the ATCC 1015 strain
of A. niger by the Joint Genome Institute. Eight predicted
genes were also identified to contain this Pfam domain in
the whole-genome sequencing of the CBS 513.88 strain of
A. niger by DSM (Pel et al., 2007). Four of these genes
(referred to from here on as pepAa, pepAb, pepAc and
pepAd) encode pepsin-like proteases. Putative orthologs
for all four genes were also identified in A. nidulans, A. oryzae
and A. fumigatus, suggesting that these genes are well
conserved within the Aspergillus genus (Fig. 1). However,
the sequence of A. nidulans AN6686.1 appears to be distinctly
different from the putative A. niger and A. fumigatus
pepAb orthologs. It may be due to incorrect gene designation
which has been a problem with the A. nidulans wholegenome
sequence (Sims et al., 2005). Comparison of the
predicted aspartic protease sequences listed in the peptidase
database (MEROPS) using ClustalW (Thompson et al.,
1994) demonstrated that the predicted pepAx sequences
were likely to be orthologs of previously isolated genes
from different species of filamentous fungi. The first,
denoted PEPAa protease seems to represent an ortholog
of the pepsin-type aspartic protease previously identified
from Talaromyces emersonii (AF439995, unpublished)
and Scleotinia sclerotiorum (Poussereau et al., 2001). Further
conserved sequence, PEPAb and PEPAc appear to
be orthologs of aspartic proteases (BcAP1 and BcAP5) previously
identified from Botryotinia fuckeliana (ten Have
et al., 2004). The closest known ortholog to PEPAd is
aspartyl protease 4 from Coccidioides posadasii
(ABA54909, unpublished). The pepAa orthologs have an
extra A. oryzae one, as previously demonstrated for the
pepA and pepE ‘clusters’ (Machida et al., 2005).
The pepAx genes encode proteins of 394, 426, 453 and
480 amino acids, respectively, which are in agreement with
other aspartic proteinases of fungal origin. PEPAa, PEPAc
and PEPAd appear to contain a signal peptide (boldfaced
in Fig. 2). Possible KexB cleavage sites (Arginine and
Lysine) were detected in PEPAa, PEPAc and PEPAd (see
Fig. 2). Alignment of the predicted amino acid sequences
of the four A. niger proteins (PEPAx) with the previously
isolated aspartic protease, aspergillopepsin A (PEPA) demonstrates
that functional regions of these proteins are
highly conserved (Fig. 2), such as the active site motif
Asp101-Thr102-Gly103 of aspergillopepsin A numbering
in Fig. 2 (James, 2004). Asp residue (Asp156 in aspergillopepsin
A) was not conserved since the PEPAc (see
Fig. 2) has a Glu residue at this position as seen in other
aspartic protease (Capasso et al., 1998). PEPAd has a long
C-terminal extension consisting of about 70 residues. This
extension contains lengthy stretches of hydrophilic, predominantly
serine residues, terminating with roughly 20
hydrophobic residues, which may facilitate extracellular
attachment. An algorithm for identifying fungal glycosylphosphatidylinositol
(GPI) modification motifs (Eisenhaber
et al., 2004) predicted that GPI modification in PEPAd
occurs at Gly456 with high probability scores of S >15
(see Fig. 2). A disulfide bond predictor (DIpro 2.0) (Cheng
et al., 2006) suggested that PEPAa had one disulfide bond
between residues 382–389, in the same region as one predicted
for PEPA. In addition to conserved predicted disulfide
bonds in this region (between residues 365–402 and
339–370, respectively), PEPAc and PEPAd were also predicted
to have an N-terminal disulfide bond (between residues
130–144 and 137–142, respectively), however, PEPAb
was predicted to have none (see Fig. 2).
3.2. Microarray results
One of the unknown transcripts found to be significantly
up-regulated during recombinant chymosin secretion (25–
50 h) in A. nidulans shake flask cultures (Sims et al.,
2005) was putatively identified by sequence similarity to
represent a pepsin-like peptidase (Fig. 3). BLASTx was
used to establish that this transcript represents the hypothetical
ORF AN2157.1, which appears to encode the A.
nidulans ortholog of the PEPAa protease (Fig. 1).
3.3. Disruption of pepAa, pepAb, pepAc and pepAd genes
We disrupted pepAa, pepAb and pepAd genes by homologous
recombination. Fig. 4a shows disruption strategies
for construction of the disruption plasmids and for identification
of disruption strains. In all three cases, the corresponding
genes were amplified by PCR (data not shown)
and the hygromycin resistant gene was inserted into the

Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27 23
gene at a restriction site so the hygromycin resistant gene respectively (Section 2). Screening of fungal transformants
was flanked by homologous AB fragment at the 5 0 end by PCR using the primer pair of P hph and P outAx (Table 1)
and homologous BC fragment at the 3 0 end (Fig. 4a). identified transformant #23 as a disruption strain for the
Aspergillus. niger GICC2773 protoplasts were transformed pepAa gene (Fig. 4b), transformant #17 as a disruption
with HpaI fragment of pAaS-T, pAbS-T and pAdS-T, strain for the pepAb gene (Fig. 4c) and transformant #29
Fig. 4. (a) Disruption strategy for the pepAx (pepAa, pepAb, pepAc and pepAd) gene of A. niger. The expected pattern of PCR products is indicated for the
parental strain (fragment I) and for the disruption mutants (fragment II) using the indicated primer pairs of P Ax3 and P Ax4 . No PCR product is expected
for the parental strain. Fragment III is expected for the disruption mutants using the indicated primer pairs of P hph and P outAb . Fragment IV is expected
for the disruption mutants using the indicated primer pairs of P hph and P outAa ,P outAc and P outAd . (b) PCR analysis and identification of the pepAa
disruption strain. From left to right, lane 1 is the DNA molecule weight marker, lanes 2 and 3 used template DNA from the parental strain (GICC2773),
lanes 4 and 5 used template from a transformant with ectopic integration (AN#6), and lanes 6 and 7 are the disruption mutant (A. NDpepAa #23). The
PCR primers used for lanes 2, 4 and 6 are P Aa3 and P Aa4 . The PCR primers used for lanes 3, 5 and 7 are P hph and P outAa. (c) PCR analysis and
identification of the pepAb disruption strain. From left to right, lane 1 is the DNA molecule weight marker, lanes 2 and 3 used template DNA from the
parental strain (GICC2773), lanes 4 and 5 used template from a transformant with ectopic integration (AN#1), and lanes 6 and 7 are the disruption mutant
(A.NDpepAb#17). The PCR primers used for lanes 2, 4 and 6 are P Ab3 and P Ab4 . The PCR primers used for lanes 3, 5 and 7 are P hph and P outAb. (d) PCR
analysis and identification of the pepAd disruption strain. From left to right, lane 1 is the DNA molecule weight marker, lanes 2 and 3 used template DNA
from the parental strain (GICC2773), lanes 4 and 5 used template from a transformant with ectopic integration (AN#8), and lanes 6 and 7 are the
disruption mutant (A.NDpepAa#29). The PCR primers used for lanes 2, 4 and 6 are P Ad3 and P Ad4 . The PCR primers used for lanes 3, 5 and 7 are P hph and
P outAd.

24 Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27
as a disruption strain for the pepAd gene (Fig. 4d). The specific
bands (1.14 kb PCR product from the pepAa disruption
strain, 1.19 kb PCR product from the pepAb
disruption strain and 1.08 kb PCR product from the pepAd
disruption strain) resulted from homologous recombination
were detected only in the disruption strains, not in
the wild type strain (GICC2773) or the epitopic integration
strain (AN#6 of Fig. 4b, AN#1 of Fig. 4c and AN#8 of
Fig. 4d). The specific PCR fragments were also sequenced
to confirm their identity (data not shown). In addition,
using primer pair of P Ax3 and P Ax4 (Table 1), the amplified
PCR fragments showed differences of 1.5 kb when comparing
the disruption strains to the wild type strain or the epitopic
integration strain. In the pepAa disruption strain a
2.32 kb PCR fragment was detected compared to a
0.82 kb fragment in the wild type strain (Fig. 4b). Similarly,
the pepAb disruption strain had a 3.67 kb rather than a
2.17 kb PCR fragment (Fig. 4c) and the pepAd disruption
strain had a 3.66 kb rather than the 2.16 kb PCR fragment
in wild type strain (Fig. 4d). In an attempt to disrupt the
pepAc gene in A. niger GICC2773 strain, we generated
286 transformants in four experiments and screened all
transformants by PCR with primers P hph and P outAc . However,
no transformant could be identified containing the
pepAc disruption pattern of a 1520 bp specific band resulting
from homologous recombination.
3.4. Effect of gene disruptions on laccase production
We tested laccase expression of all disruption strains by
growing them in shake flasks for 144 h in modified Promosoy
medium and then assaying the laccase activity in the
supernatants. We observed a 42% average increase of the
laccase production in the strain A.NDpepAb#17 with
respect to the parental strain GICC2773 in two parallel
experiments. Analysis of secretion level of the laccase also
revealed that a 21% average increase of the laccase expression
in the strain A.NDpepAa#23 and a 30% average
increase in the strain A.NDpepAd#29 (Table 3).
3.5. Effect of gene disruptions on production of native protein
The secretion of total glucogenic enzymes in culture filtrate
was analyzed in the three disruption mutants. The
strain A.NDpepAa#23 and A.NDpepAb#17 showed significant
increase in the expression level of their glucogenic
enzymes by approximately 19% and 37%, respectively.
However, there was a only slight increase (5.2%) in the
strain containing the disruption of the pepAd gene (Table 3).
4. Discussion
Four pepsin-like protease genes were identified and
characterized from A. niger, the products of which have
characteristics that are novel among aspartic proteases
from filamentous ascomycetes. Sequence analysis demonstrated
that these genes appear to be highly conserved
within the Aspergillus genus. Both the sequence alignment/clustering
program, ClustalW and Pfam/MEROPS
classification system are dependent upon the level of
sequence similarity. Therefore, the clusters that are generated
correspond to discrete members of the same MER-
OPS and Pfam families. At the most conserved level,
within individual protein families, clustering by sequence
similarity can identify orthologs and paralogs (Sims
et al., 2004a). Whole-genome sequencing of closely related
species has resulted in the identification of predicted proteases
that represent orthologs of previously isolated genes
from different species. Using this approach, it is also possible
to identify orthologs which have not been isolated previously
and may also lead to the re-classification of some
proteases. For example, A. niger PEPA is denoted
A01.016 in MEROPS peptidase database (Rawlings et al.,
2004) and yet it is sandwiched between A01.026 peptidase
F from A. oryzae and A. fumigatus (see Fig. 1).
The Aspergillus PEPAd sequences appear to have a conserved
C-terminal modification that may facilitate their
association with the cell membrane by means of GPI
anchor (Hamada et al., 1998). The Saccharomyces cerevisiae
aspartic proteases yapsin 1, 2 and 3 have all been shown
to be GPI-anchored proteins (Ash et al., 1995; Cawley
et al., 1995) and B. cinerea BcAP3 and 4 contains similar
domain (ten Have et al., 2004). Moreover, PEPAd contains
serine-rich stretches just upstream of the GPI modification
site (Fig. 2). These stretches might be targets for O-glycosylation,
which may facilitate adherence to the extracellular
glucan matrix of A. niger. An aspartic protease that is
attached to the cell membrane by a GPI anchor, or embedded
in the hyphal matrix, might support various functions
such as the maturation of other fungal hydrolytic enzymes,
the proteolysis of host cell wall protein in the vicinity of the
hyphal tip.
Perhaps the most distinctive features of these four
A. niger sequences are those of PEPAb. While it does contain
all the residues necessary to function as an active proteolytic
enzyme, the sequence of PEPAb is highly atypical; it
is not predicted to contain any disulfide bonds. In particular,
the C-terminal disulfide bond conserved in virtually all
other eukaryotic aspartic protease sequences is absent, the
precursors are commonly synthesized on membrane-bound
ribosomes and the nascent polypeptide chains are directed
into the endoplasmic reticulum where the disulfide bonds
are introduced by post-translational modification to
enhance stability. Predicted orthologs of PEPAb from a
number of filamentous fungal species were also predicted
to lack disulfide bonds and a conventional signal peptide.
These enzymes have sequences with very acidic isoelectric
points; the predicted pI value for the putative N. crassa
ortholog of PEPAb is almost as acidic as that of pig pepsin
which operates in a very acidic environment (pH < 2) in the
stomach. Thus, whatever the mechanism of trafficking of
these proteins, the cellular compartment in which they
finally reside can be predicted to be very acidic, otherwise
each polypeptide would unfold and denature. Although a

Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27 25
signal peptide sequence could not be identified for PEPAb,
the absence of a ‘typical’ signal peptide sequence in a
secreted protein is not unprecedented. The superoxide dismutase
BcSOD1 in B. cinerea also lacks a signal peptide
sequence but was unequivocally demonstrated to be a
secreted protein (Rolke et al., 2004).
Real-time quantitative RT-PCR was performed to
establish whether the pepAx genes are stimulated by heterologous
protein production. All four aspartic protease
genes were transcribed in the laccase expressing strain
(Fig. 5), although there was no significant difference in
the level of gene expression compared with parental strain
(data not shown), suggesting that transcription of the proteases
does not directly respond to the expression of heterologous
laccase. Perhaps these proteins are constitutively
expressed under given certain culture conditions or only
induced by certain heterologous proteins. The expression
of a transcript representing the A. nidulans ortholog of pep-
Aa was observed to up-regulated in a chymosin-producing
strain compared to its parent (Fig. 3).
Fig. 5. Detection of pepAx transcripts by RT-PCR. 215, 100, 236 and
176 bp amplified products were detected for pepAa, pepAb, pepAc and
pepAd genes, respectively. RNA was extracted from GICC2773 strain.
The effect of silencing pepAa, pepAb and pepAd expression
on homologous and heterologous protein secretion
in A. niger was analyzed. The laccase activities were
increased by 21%, 42% and 30%, respectively, in the pepAa,
pepAb and pepAd gene disruption strains. The effect of the
pepAb gene disruption, despite the lack of a signal peptide
and disulfide bonds, suggesting the PEPAb is likely to be
another important protease leading to low production of
heterologous proteins, though the substrate specificity of
different proteases should be considered. The increased
production of laccase by disruption of the pepAd gene, is
relatively small compared with that obtained from pepAb
removal, which could support the hypothesis deduced from
the sequence characterization that PEPAd is likely to be a
GPI-anchored protease. The proteolytic effect of GPIanchored
or membrane-embedded protease may be limited,
and may only degrade heterologous proteins in the vicinity
of the cell wall as compared to extracellular proteases.
The effect of disruptions of these three proteases on
homologous proteins production was also studied. In comparison
with the parental strain, there was no significant
change in glucogenic enzymes production in the pepAd disruption
mutant which could represent a protective mechanism
to prevent native proteins from degradation by
nascent proteases (Calmels et al., 1991). However, in the
pepsins and the majority of other members of the family
show specificity for the cleavage of bonds in peptides of
at least six residues with hydrophobic amino acids in both
the Pl and Pl 0 positions. So the proteolytic activity of the
same protease to different heterologous proteins will vary
and the relative degradation ability compared with other
aspartic proteases needs to be examined with a number
of different substrates due to the substrate specificities for
enzymes.
To disrupt the pepAc gene in A. niger, a total of 286
transformants were generated in four experiments. All
transformants were screened by PCR, but none was identified
to contain the pepAc gene knockout. It is not known
why no disruptant was obtained, However, a number of
pepsinogen genes are reported to be located in the centromeric
region of human chromosome 11 (Taggart et al.,
1985), where homologous recombination in such area in
A. niger is so infrequent.
Table 3
Extracellular laccase and glucogenic enzyme activity secreted by protease-deficient and wild type strains
Strain
Mycelium dry
weight a,c (g/L)
Laccase
Activity a,b,c
(IU/L)
Activity relative to
mycelium dry weight
(IU/g)
Activity relative to
parental strain (%)
Glucogenic enzymes
Activity a,b,c
(U/L)
Activity relative to
mycelium dry
weight (U/g)
DpepAa#23 8.48 ± 0.36 479 ± 20.7 56.5 ± 1.22 121.0 7.96 ± 0.20 954.0 ± 70.2 118.7
DpepAb#17 8.37 ± 0.40 555 ± 31.0 66.3 ± 3.01 142.0 8.07 ± 0.28 119.2 ± 77.6 137.0
DpepAd#29 8.12 ± 0.30 491 ± 17.8 60.7 ± 1.76 130.0 6.98 ± 0.23 842.0 ± 66.9 105.2
GICC2773 10.22 ± 0.78 476 ± 10.7 46.7 ± 2.72 100 7.74 ± 0.13 800.6 ± 15.2 100
a Data are averages ± standard deviation of eight determinations (four replicates for two independent experiments).
b The supernatant was used as crude enzyme for the enzyme activity assay.
c The cultivation was carried out for 144 h. For all cultures the final pH was approximately 4.6.
Activity relative to
parental strain (%)

26 Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27
Residual proteolytic activity (other than that accredited
to identified proteases) remains a big challenge for the production
of heterologous protein such as laccase (Archer
et al., 1992; Conesa et al., 2001). The roles of these proteases
and their contribution to laccase degradation are
unclear at this time. Some of them might have intracellular
proteolytic activities required for adequate protein processing
and thus might be indispensable. On the other hand, we
do not know the substrate specificity for each of these proteases,
especially for the laccase molecule. Knowing the
specificities of the proteases will undoubtedly broaden
our methods to improve strains for heterologous protein
production by selectively disrupting one or more genes.
In summary, removal of proteolytic degradation due to
the presence of PEPAx is a useful method for increasing
laccase production in A. niger. This strategy, in association
with the removal of aspergillopepsin A (van den Hombergh
et al., 1997a) and other aspartic proteases by combining
multiple gene disruptions, by classical mutagenesis and
by physiological optimization of culture medium, represents
a good method to result in large increases in heterologous
protein production in A. niger. In future, it would be
interesting to look at the effects of disrupting multiple pep-
Ax genes or generating strains that silence all proteases that
are known to affect yields of recombinant proteins.
Acknowledgments
This work was supported by grants from Genencor, a
Danisco Division, Palo Alto, California, USA. Authors
would like to thank Michael Ward for his critical reading.
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