Mol Gen Genet (1992) 233:404-410 © Springer-Verlag 1992

Cloning and expression of a member of the Aspergillus niger gene family encoding a-galaetosidase Irma F. den Herder, Ana M. Mateo Rosell, Caren M. van Zuilen, Peter J. Punt, and Cees A.M.J.J. van den Hondel TNO Medical BiologicalLaboratory, PO Box 45, 2280 AA Rijswijk, The Netherlands Received December 13, 1991

Summary. An enzyme with a-galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of a-galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a )~ EMBL3 gene library of A. niger. The cloned gene (agIA) was shown to be functional by demonstrating that the 82 kDa a-galactosidase is absent from a strain with a disruption of the aglA gene, and is overproduced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa a-galactosidase A represents a minor extracellular a-galactosidase activity in A. niger. Key words: Aspergillus niger- Enzyme (a-galactosidase A) purification - aglA gene isolation - Gene disruption - Multicopy transformant

Introduction The filamentous fungi Aspergillus niger and A. oryzae are widely used in industry for the production of fermented foods, organic acids and enzymes (Barbesgaard 1977; Bennett 1985). Based on their capacity to secrete large amounts of protein (up to 30 g/1 culture medium), these organisms have also attracted much attention for the production and secretion of (heterologous) proteins, with the aid of recombinant DNA techniques. During the last decade such techniques have been rapidily developed for these organisms (van den Hondel et al. 1991). Although during that period a considerable amount of data has been obtained concerning fungal gene expression, only very little is known about the molecular mechanisms of

Correspondence to : C.A.M.J.J. van den Hondel

protein routing and secretion in filamentous fungi. Therefore, we have started a study on routing and secretion of proteins in A. niger. As a model protein for this study, we have chosen a-galactosidase, a-Galactosidases (a-D-galactoside galactohydrolases; E.C. 3.2.1.22)have been reported to occur widely in microorganisms, plants and animals (for review see Dey and Pridham 1972). The enzymes are able to hydrolyze a variety of simple a-galactosides as well as more complex polysaccharides. One of the attractions of using this enzyme as a model is the availability of simple, qualitative as well as quantitative, enzyme assays. As a first step in our study on protein secretion, an A. niger gene, encoding an extracellular a-galactosidase, was isolated and analysed. Materials and methods

Strains, plasmids and transformation procedures. A. niger N402 (Bos 1986) was used for the isolation of ~-galactosidase from culture fluid. A. niger AB4.1 (pyrG- ; van Hartingsveldt et al. 1987) was used as recipient in transformation experiments. Escheriehia coli JM109 was used for propagation of plasmids (Yanish-Perron et al. 1985). E. coli NM538 (supF, hsdR) and NM539 [supF hsdR (P2cox3)] and the replacement vector )~ EMBL3 were used for the construction and amplification of an A. niger gene bank (Frischauf et al. 1983). Plasmid pAO4--2 contains the A. oryzaepyrG gene (de Ruiter-Jacobs et al. 1989). Plasmid pAN4-1 is a derivative of pGW325 (Wernars 1986) and contains the A. nidulans amdS gene (Hynes et al. 1983). pAN52-7NotI is a derivative of pAN52-1NotI (van den Hondel et al. 1991). In this vector the upstream region of the gpdA gene is replaced by the upstream region of the glaA gene and a unique NcoI cloning site is present around the ATG. E. coli transformations were carried out as described by Hanahan (1983). Fungal transformations were carried out as described by Punt et al. (1987).

405

Growth conditions. A. niger was grown at 33 ° C and 300 rpm in a rotary shaker in minimal growth medium (Bennett and Lasure 1991) containing 1% galactomannan (Sigma, locust bean gum, Ceratonia siliqua) or 5 % maltodextrin (Sigma, dextrin from corn type I) as carbon source. One liter of medium was inoculated with 10 9 spores. Purification of a-galactosidase. After removal of the mycelium by filtration on Miracloth, the culture medium was acidified to pH 3 and 150 ml pre-swollen SPSephadex C-50 (Pharmacia) was added. After gentle stirring for 16 h at 4 ° C, the supernatant was removed and the beads were collected and packed into a column (diameter 2 cm, length 45 cm). Beads were washed with 100 ml 10 mM sodium formate buffer pH 3. Proteins were eluted with a salt gradient (0-0.5 M NaC1). The fraction with the highest specific a-galactosidase activity (eluting at 0.42 M NaC1) was further purified by reversed-phase chromatography with a C3 Ultrapore column. Elution was achieved in 40 rain with a 0-15% isopropanol gradient in 150 mM sodium acetate pH 4.5. Fractions with a-galactosidase activity (eluting at 10% isopropanol) were pooled, reloaded onto the column and eluted with an acetonitrile gradient in 0.1% trifluoroacetic acid, 5-18% acetonitrile in 10 min, 18-51% in 60 min, 51-70% in 20 min. The fractions with a-galactosidase activity (eluting at 15 % acetonitrile) were reloaded onto the column and eluted with the same solutions but using a different acetonitrile gradient; 5-21% in 50 min. The a-galactosidase peak, which was judged to be more than 95 % pure (Fig. 1), was lyophilized and subjected to automated Edman degradation. The N-terminal sequence was established a s S I E Q P S L L P T P P M G F N NWARFMCDLNET. Enzyme assay, a-Galactosidase activity was assayed with p-nitrophenyl-a-D-galactopyranoside (PNPaG, Sigma). The enzymatic assay was performed by mixing a 10 gl medium sample with 90 btl demineralized water and 100 gl 20 mM P N P a G in 0.1 M sodium acetate buffer (pH 4.5). After 5 min incubation at 37 ° C, the reaction was stopped by adding 2.8 ml 1 M N a z C O 3 and the color formation was measured at 405 nm. Activity is expressed as units per ml. One unit of a-galactosidase activity is defined as the amount of enzyme releasing 1 gmol of p-nitrophenol per min (Bahl and Agrawal 1972). The specific activity of the enzyme is defined as the number of units per mg of protein, u-Galactosidase activity was also assayed by staining isoelectrofocusing (IEF) gels with 5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside (X{zgal, Boehringer). This assay was performed by incubating the gel with an Xagal solution (2.25 mM Xagal in 0.1 M sodium acetate buffer pH 4.5) for 1-4 h at room temperature. Protein analysis. Isoelectrofocusing was carried out using the Pharmacia Phast System or Servalyte precotes IEF gels. SDS-PAGE was performed on the Pharmacia Phast System apparatus or with freshly polymerized gels. Medium samples, collected after 5 days of cultivation in

1% galactomannan or after 48 h in 5% maltodextrin, were concentrated 80-100 times in an Amicon cell with a PM10 membrane. Before electrophoresis, the samples were loaded onto PD10 Sephadex G25 columns for removal of non-protein material and to change the buffer to 20 mM sodium acetate pH 4.5.

DNA manipulations. The ~ EMBL gene library was constructed as described by van Hartingsveldt et al. (1987). DNA manipulations with restriction enzymes or T4 D N A ligase were carried out in accordance with the suppliers' instructions. For the isolation of the A. niger aglA gene from the )v EMBL3 gene library, a mixture of synthetic oligonucleotides: 5'ATG GG(T/C/A/G) TT(T/ C) AA(T/C) AA(T/C) TGG GC 3' (synthesized on a Biosearch Cyclone D N A synthesizer), was used as a probe. The oligonucleotide sequence was deduced from the N-terminal amino acid sequence of the purified protein, and encodes all the possible codons. The D N A of 12 out of 40 000 plaques tested hybridized with the probe. Restriction enzyme analysis of D N A isolated from the phage clones revealed that 7 of the 12 clones contained fragments with the same size as chromosomal fragments that hybridized with the oligonucleotide probe on a Southern blot. Hybridization experiments with the oligonucleotide mix were performed in 6 × SSC at 46 ° C. For cloning and analysis of the A. niger aglA gene and analysis of recombinant strains, standard recombinant D N A techniques were used (Sambrook et al. 1989). Doublestranded DNA sequencing of the agIA gene was performed according to Chen and Seeburg (1985). Construction and analysis of an A. niger aglA deletion mutant. To obtain an A. niger mutant which lacks the aglA gene, a vector was constructed as follows. A 4.4 kb EcoRV-EcoRI fragment of p A B I - I A (Fig. 2), containing the agIA gene, was replaced by a 2.8 kb SmaI-BglII fragment of pAO4~2 containing the pyrG gene of A. oryzae, resulting in pABl-lApyrG (Fig. 2). This plasmid was cut with SalI. The SalI fragment containing the pyrG gene flanked by aglA flanking sequences was gel-isolated and used for transformation of A. niger AB4.1 (pyrG-). Pyr + transformants were selected and purified. Subsequently, restriction enzyme-digested chromosomal D N A from a number of transformants was subjected to Southern analysis. An 0.95 kb EcoRI fragment, located downstream of the aglA gene, was used as a positive probe and an 0.5 kb BamHI fragment, located in the agIA gene, as a negative probe. Of 11 transformants tested, one, designated AA20, had the expected restriction enzyme profile and was used for further experiments. Construction and analysis of A. niger aglA multi copy strains. Two different strategies were used to obtain an A. niger aglA multicopy transformant. 1. The 6.8 kb SalI fragment from plasmid pABI-IA, containing the aglA gene, was inserted in the SalI site of pAN4-1 (containing the A. nidulans amdS gene), resulting in plasmid p A B I - I S (Fig. 2). A. niger AB4.1 AmdS + transformants were selected and purified. Restriction enzyme-digested D N A of these tranformants was sub-

406 jected to Southern analysis with the 0.5 kb BamHI agIA fragment mentioned above as probe. One of the transformants containing about ten copies of the aglA gene, was used for further experiments.

2. The aglA gene was placed under the control of the A. niger glaA promoter using plasmid pAN52-7NotI (van den Hondel et al. 1991). A 417 bp BspHI-AhaII fragment, encoding the putative 23 amino acid signal peptide and the N-terminal part of the a-galactosidase A protein, and a 2.2 kb AhaII-NeoI fragment, encoding the C-terminal part of the a-galactosidase A protein, were inserted into the unique NcoI site of pAN52-7NotI. Subsequently, a 5.0 kb NotI fragment containing the amdS gene of A. nidulans was cloned into the unique NotI site, resulting in plasmid pAB1-6S (Fig. 2). The A. niger AA20 strain was transformed using this plasmid. Ten AmdS + transformants were selected, purified and used for further experiments.

9/+ 67 43 30 20.1

Results

14.4

Purification and N-terminal amino acid sequencing of a-galactosidase : Isolation of the aglA gene An enzyme with a-galactosidase activity was purified from A. niger culture medium. The partially purified protein had an apparent molecular weight of 82 kDa, as deduced from its mobility in SDS-PAGE (Fig. 1). The isoelectric point was determined after IEF and was estimated to be 4.8. The N-terminal amino acid sequence, determined by automated Edman degradation, had

1 2 3 Fig. 1. SDS-polyacrylamidegel electrophoresisof purified a-galactosidase. Protein bands were visualized by Coomassie staining. Lane 1, molecularweightmarkers; lane 2, purifieda-galactosidase A; lane 3, Aspergillus niger culture fluid after cation-exchange chromatography

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GCGGATGAAAAGCGATCGCATTTTGCACTGTGGGCTTCTTTCTCGGCTCCACTTATTATC A D E K R S H F A L W A S F S A P L I I

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AGTGCTTATATACCTGCACTTTCG~GGATGAGATTGCCTTCTTGACG~CG~GCATTG S A Y I P A L S K D E I A F L T N E A L

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ATTGCGGTG~TCAGGATCCCCTAGCGCAGCAGGCCACGTTGGCGAGCCGCGATGATACA I A V N Q D P L A Q Q A T L A S R D D T

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CTGGATATATTGACCCGTAGTCTGGCAAACGGCGACAGGCTGCTGACGGTGCTT~T~G L D I L T R S L A N G D R L L T V L N K

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GGAAACAC~CTGT~CGAGGGACATTCCCGTAC~TGGTTGGGTCTCACAGAGACTGAC G N T T V T R D I P V Q W L G L T E T D

AC CGAGACTGC CGATACGATGG CTG CTAACGGTCTGCGGGACG CAGGCTACAATCG CATC T E T A D T M A A N G L R D A G Y N R I

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TGTACATACACGGCCGAGGATCTCTGGGATGGC~GACCCAG~GATCAGCGACCATATA C T Y T A E D L W D G K T Q K I S D H I

AATCTGGATGACTGCTGGATGGCTTATCAGCGATCCGACAATGGATCCCTACAGTGGAAC N L D D C W M A Y Q R S D N G S L Q W N

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AAAATTG~CTAGCCAGCCATGCGACAGCAGTCTTCCGGCTCAGTCTTCCGCAGGGTTGT K I E L A S H A T A V F R L S L P Q G C

ACGACTAAGTTTC C C CACGGCCTG C CTTG GCTAGC CAAATATGTCAAAG C CAAAGGGTTT T T K F P H G L P W L A K Y V K A K G F

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CATTTTGGGATTTATGAAGATTCTGG CAACATGACTTGTGGCGGATAT C CCGGATC CTAC H F G I Y E D S G N M T C G G Y P G S Y

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AAC CACGAGGAG CAGGACG C CAACACCTTTGCTTCCTGGGGGATTGACTAT CTCAAGCTC N H E E Q D A N T F A S W G I D Y L K L

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GTGACACCGTCAGGAGTCATTCGTCCAGTATCGCAGACCACAC~TGCTTGGCTGCTGAT V T P S G V I R P V S Q T T Q C L A A D

GAC GGTTG CAATGTCTACGCAACACAAGGTAGAACACTCGAG GAG GAATACAAGCAACG C D G C N V Y A T Q G R T L E E E Y K Q R

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GCCCCGGCATACTTCGCGGGCACAGACAACAACACAGACTGGTACACCGTGATGGACTGG A P A Y F A G T D N N T D W Y T V M D W

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Fig. 3A and B. Structure of the aglA gene (Genebank accession number A. niger aglA X63348). A Restriction enzyme map of the aglA gene. The shaded boxed area represents the putative protein encoding region. Abbreviations: A AhaII; B, BamHI; Bg, BglII; Bs, BspHI; E, EcoRV; H, HpaI; N, NcoI. B Nucleotide sequence and deduced amino acid sequence of aglA. Asterisks indicate stop codons. The CT-rich region preceding the coding region and the

consensus sequence for polyadenylation are underlined. The first amino acid of the mature polypeptide is indicated with an arrow and with 1, and the N-terminal sequence of a-galactosidase A as determined by Edman degradation is enclosed in a dashed box. Putative translation initiation codons are indicated with - 2 3 and - 3 1 , respectively. Putative N-glycosylation sites are indicated by #

40-50 % similarity to the N-terminal amino acid sequence of a-galactosidases from Saccharomyees carlsbergensis (Sumner-Smith et al. 1985), Cyamopsis tetragonoloba (Overbeeke et al. 1989) and Homo sapiens (Kornreich et al. 1989). This result provided additional evidence that the purified enzyme (aglA) was indeed an a-galactosidase. The A. niger gene aglA, coding for a-galactosidase A was isolated from a phage k genomic library by hybridization with a probe derived from the N-terminal sequence of the purified protein. From one of the positive clones obtained, a 6.8 kb SaII fragment was subcloned into pUC18, resulting in p A B I - I A (Fig. 2).

Nucleotide and deduced amino acid sequence of the aglA gene The location of the aglA gene in p A B I - I A was determined by digestion with restriction enzymes followed by Southern analysis with the mixed oligonucleotide as a probe (Fig. 3A). Both p A B I - I A and a 2.5 kb HpaI-BglII fragment from p A B I - I A subcloned into pUC18 were used for sequence analysis. The nucleotide sequence revealed an uninterrupted open reading frame (ORF) of 1635 bp, a size that is sufficient to encode a polypeptide of 545 amino acids. Northern analysis using an 0.5 kb BamHI fragment containing a part of the ORF identified a transcript of 1.8 kb (data not shown) which is consistent with the size of the ORF.

408 The N-terminal sequence of the purified protein corresponds precisely to the amino acid sequence deduced from codons 32-59 of the ORF, indicating that this O R F encodes the purified a-galactosidase. The predicted pI of the mature polypeptide is 4.85 which corresponds well with the pI of the purified protein. The predicted molecular weight of the mature protein (60 kDa) is clearly lower than that of the purified protein (82 kDa), suggesting that a-galactosidase A is a glycoprotein. Indeed, the deduced amino acid sequence contains seven consensus sites (AsnXxxThr/Ser) for N-glycosylation (Fig. 3B). Since a-galactosidase A is an extracellular protein, the presence of a signal peptide is expected. Two in-frame ATG codons are present upstream of the mature a-galactosidase A sequence which would result in a prepeptide sequence of either 23 or 31 amino acids (Fig. 3B). Codon usage of the O R F is remarkably unbiased (60 of the 61 possible codons are used). Comparison of the deduced amino acid sequences of the aglA gene with various a-galactosidases present in EMBL and GeneBank data bases revealed considerable amino acid sequence similarities in the region comprising the first 150 N-terminal amino acids of the mature protein and in the region between residues 250-320. The region from residues 150 to 250 hardly showed any similarity between the various a-galactosidases. The best overall similarity (37% identity) was found between A. niger and C. tetranogoloba (Overbeeke et al. 1989). Interestingly, the aglA gene codes for a protein that is about 100 amino acids larger than any of the other a-galactosidases. The 5' and 3' flanking regions of aglA contain sequence elements observed in many other fungal genes (Gurr et al. 1988; Turner et al. 1989; Punt et al. 1990). Upstream of the putative start codon (from nucleotides 28 to 92) a CT-rich region is observed (Fig. 3B). At nucleotide 1827, approximately 70 bp downstream of the two stop codons, a canonical polyadenylation signal (AATAAA) is observed (Fig. 3B).

Functional analysis of the aglA gene To test the functionality of the cloned aglA gene, the level of c~-galactosidase activity which was synthesized by an A. niger strain with a deletion of the aglA gene (AA20) was tested after cultivation in minimal medium supplemented with 1% galactomannan. The results showed that the a-galactosidase activity was not affected by deletion of the aglA gene. Furthermore, in a strain containing multiple copies of the aglA gene (AB 4.1/pAB 1-1S), c~-galactosidase activity was also not affected. An explanation for these unexpected results is that the a-galactosidase activity encoded by aglA represents a minor activity, whereas the major activity is encoded by another agl gene or genes. To assess the validity of this hypothesis, we first determined the activity of A. niger transformants containing multiple copies of the aglA gene (AA20/ pAB1-6S) which was placed under the control of expression signals of the glucoamylase promoter to in-

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Fig. 4a and b. Isoelecticfocusing(IEF) of culture medium samples (50 times concentrated), a Staining with 5-bromo-4-chloro-3indolyl-et-D-galactopyranoside. Lane 1, aglA deletion strain AA20; lane 2, wild-typestrain N402; lane 3, pAB 1-6S transformant number 37. b Coomassie staining. Lane 4, aglA deletion strain AA20; lane 5, wild-typestrain N402; lane 6, pABI-6S transformant number 37. The position of IEF markers (pI) is indicated. The location of the c~-galactosidase A band in the wild-type strain is indicated with a closedarrowhead crease the level of expression. These transformants showed a 2- to 3-fold increase in ct-galactosidase activity, compared to that of the parent strain, when cultured in minimal medium supplemented with 5 % maltodextrin. In order to prove that the increased activity is encoded by the aglA gene we have, subsequently, analysed by means of IEF the activity and the amount of ~t-galactosidase A synthesized by the wild-type, the deletion mutant (AA20) and the multicopy transformant strain (AA20/pAB 1-6S). After staining the gel with Xctgal, a protein with a-galactosidase activity and a pI of 4.8 was seen in medium of the multicopy transformant (Fig. 4 lane 3). No enzymatically active protein was seen at this position for the wild-type or deletion strain (compare lanes 1,2 and 3). Apparently, the amount of enzyme present in the wildtype strain is too low to be detected in this way. When the gel was stained with Coomassie a faint protein band with a pI of 4.8 (lane 5) could be seen in the wild-type strain. This protein was absent from the mutant strain but was present in greatly enhanced quantities in the multicopy strain (lanes 4 and 6, respectively). Subsequent 2D-gel analysis (IEF followed by SDS-PAGE) revealed that the apparent molecular weight of the protein is 82 kDa (data not shown). These data conclusively show that the a-galactosidase activity which was purified is encoded by the aglA gene. The Coomassie stained gel also showed the presence of proteins with pIs of 4.7, and a faint band at pI 5.0. These proteins, which were not detected in wild-type or deletion mutant strains, probably reflect differently processed forms of the product of the aglA gene or some degradation products of a-galactosidase A protein. Xagal staining of the gel also showed the presence of proteins with a-galactosidase activity with a pI of 4.2 in all three strains. The activity of this protein was neither affected by deletion nor by the presence of multiple copies of the agIA gene. Apparently, this protein represents a second a-galactosidase encoded by another agl gene. In the wild-type strain this protein represents a major a-galactosidase activity.

409

Discussion In this paper we describe the isolation and preliminary characterization of an extracellular a-galactosidase (agalactosidase A) from A. niger and present the nucleotide sequence of the gene, aglA, encoding the enzyme. The purified enzyme represents a minor species of the a-galactosidase family of A. niger, as can be deduced from a comparison of Xagal stained gels of a wild-type A. niger strain and a transformant harboring multiple copies of the aglA gene. Apparently, other ct-galactosidase activities were lost during the purification procedure, probably due to inactivation after H P L C isolation. The conclusion that the cloned agIA gene codes for the purified, enzymatically active a-galactosidase is based on the fact that the N-terminal amino acid sequence of the purified protein matches the nucleotide sequence near the 5' end of the aglA gene, and that the a-galactosidase activity is increased 2- to 3-fold in an A. niger transformant with multiple copies o f the aglA gene. The genomic aglA gene contains a single O R F of 1635 bp. The D N A sequence o f the mature a-galactosidase A is preceded by two in-frame A T G codons, leading to prepeptide sequences of either 23 or 31 amino acids. The 23 amin acid peptide fulfills all the requirements of a canonical signal sequence; i.e. charged N-terminus, hydrophobic core, cleavage downstream of small residues (Va1-3, Gly -1) (yon Heijne 1983; von Heijne and Abrahmsen 1989). Furthermore, a length of 23 amino acids corresponds to that of fungal signal peptides (17-23 amino acids), unlike the 31 amino acid prepeptide. Therefore it is more likely that the 23 amino acid prepeptide and not the 31 amino acid prepeptide represents the a-galactosidase A signal peptide. The purified a-galactosidase A protein has an apparent molecular weight of 82 kDa, which is considerably larger than that predicted from the D N A sequence. These data suggest that the aglA gene product undergoes post-translational modification, presumably glycosylation. Seven N-glycosylation consensus sites are present in the deduced ~-galactosidase A amino acid sequence. However, most glycosylation sites were found in hydrophobic regions of the protein, suggesting that not all sites will be available for the addition o f sugar side chains. Furthermore, at the C-terminal part of the polypeptide (from amino acids 420 to 480) a Ser- and Thr-rich region is present, providing possible sites for O-glycosylation. A similar O-glycosylated Ser/Thr-rich domain is present in the C-terminal end of the extracellular glucoamylase from A. niger (Svensson et al. 1983) and in the extracellular cellobiohydrolases C B H - I and C B H - I I from Trichoderrna reesei (Penttilfi et al. 1986). This Ser/Thr-rich hinge region separates the N-terminal catalytic domain from the Cterminal putative substrate binding domain in glucoamylase and the cellobiohydrolases. Probably, aglA has a similar structure. The C-terminal extension o f agIA, in which aglA is dissimilar to all other known a-galactosidases, could then be involved in binding insoluble substrates such as galactomannan. IEF analysis of culture medium from wild-type, deletion and multicopy strains, reveals the presence of more

than one protein band with a-galactosidase activity (Fig. 4). F r o m this result we assume that more than one gene encoding a-galactosidase is present in A. niger. The fact that A. niger produces more than one ~-galactosidase is not surprising, as a related species, A. tamarii, also produces several extracellular and intracellular ct-galactosidases (Civas et al. 1984a, b). Preliminary results, based on an analysis of other a-galactosidases activities, suggest the presence of at least three more a-galactosidases. We are presently analysing the complexity of the agl gene family. Subsequently, we will focus on the analysis of the routing signals for different types of a-galactosidase and study the mechanism of routing and localization of these proteins. The results o f this research will provide more information about the mechanism of protein secretion in A. niger.

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Communicated by W. Gajewski