Isolation of Four Pepsin-Like Protease Genes from Aspergillus Niger and Analysis of the Effect of Disruptions on Heterologous Laccase Expression
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Available online at www.sciencedirect.com Fungal Genetics and Biology 45 (2008) 17–27 www.elsevier.com/locate/yfgbi 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 previ- ously 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. Tran- scriptome 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 tein production upon deletion of the protease genes (Archer et al., 1992; van den Hombergh et al., 1997b). Filamentous fungi represent excellent hosts for secre- Four extracellular proteases with acid pH optima (an tion of homologous and heterologous proteins (Conesa aspartic protease, PEPA, a glutamic protease, PEPB and et al., 2001). However, proteolytic degradation is a major two serine carboxypeptidases, PEPF and PEPG) have pre- problem during protein production even in a well-estab- viously been characterized (Berka et al., 1990; Inoue lished organism such as Aspergillus niger (Archer et al., et al., 1991; Krishnan and Vijayalakshimi, 1985; Mattern 1992). Proteolytic degradation affects mainly heterologous et al., 1992; van den Hombergh et al., 1994). A fifth pro- proteins evidenced by improvement of heterologous pro- tease gene, encoding an extracellular subtilisin-type serine protease, PEPD, has been cloned based on conserved * Corresponding author. Fax: +86 010 64807505. amino acid sequences within subtilisins (Jarai et al., E-mail address: [email protected] (A. Wang). 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. 2. Materials and methods niger; these are PEPE, a pepsin-type aspartyl endopro- tease that is the homologue of the vacuolar pep4 gene 2.1. Strains and growth conditions product in yeast (Jarai et al., 1994b), PEPC, a subtilisin- type serine endoprotease (Frederick et al., 1993) and A. niger GICC2773 (Valkonen et al., 2003) was used for CPY, a serine carboxypeptidase (Yaver et al., 1995). Fol- the disruption experiments of the putative aspartic protease lowing the whole-genome sequencing of A. niger (Pel genes. This strain contains the disruption mutant of the et al., 2007), approximately 200 predicted proteases have pepA gene and integrated heterologous gene (lcc1 of been identified, with similar numbers also seen following theTrametes versicolor laccase gene) expressed as fusion whole-genome sequencing of Aspergillus nidulans (Gala- to the glucoamylase. Escherichia coli DH5a served as host gan et al., 2005), Aspergillus oryzae (Machida et al., for routine DNA manipulations (Sambrook et al., 1989). 2005) and Aspergillus fumigatus (Nierman et al., 2005). Aspergillus niger GICC2773 strains were maintained on Pepsin-like enzymes such as aspartic proteases are solid sporulation medium, GMP (2% glucose, 2% maltose members of the A1 family of peptidases (Rawlings extract, 0.1% peptone, 1% agar) supplemented with 0.3% et al., 2004). This family comprises proteins with a casein when necessary. Disruption mutants were grown three-dimensional structure close to that of pepsin. Gen- on GMP supplemented with 200 lg hygromycin B per erally, these enzymes form two domains with different ml. Seed cultures of the A. niger strains in 30 ml of S3Y2 amino acid sequences, but basically similar folds. The medium (3% soluble starch, 2% yeast extracts, 0.5% catalytic site of the pepsin-like enzymes is formed at KH2PO4, 0.5% corn powder) were incubated at 30 °C with the junction of the two domains. It contains two aspartic 200 rpm agitation in a rotary G10 incubator (New Bruns- acid residues, Asp32 and Asp215 (human pepsin number- wick Scientific, NJ) for 14 h. For laccase production study, ing), one in each domain (Blundell et al., 1998). In accor- A. niger strains were grown in 30 ml of modified Promosoy dance with the accepted mechanism of the pepsin-like medium (Ward et al., 2004), where the promosoy was enzyme function (James, 2004), the Asp215 has to be substituted with tryptic soy broth (Difco, Detroit, MI). charged, whereas Asp32 has to be protonated. A remark- able property of the catalytic center is its adaptation for 2.2. Protease identification and sequence analysis the action in a wide range of pH, from 4.0 to 7.0. Aspar- tic proteinases have been identified from Botrystis cinerea The predicted pepsin-like proteases were identified as (ten Have et al., 2004) and A. oryzae (Machida et al., described previously (Sims et al., 2004a,b). Briefly, files 2005). containing all the predicted open reading frames (ORFs) Berka et al. showed that deletion of the pepA gene from species of filamentous fungi with recently completed increased bovine prochymosin production by more than whole-genome sequences (Table 2) were downloaded 66% (Berka et al., 1990). Van den Hombergh et al. dis- from their respective databases and filtered for sequences rupted three different acid-protease genes in A. niger, of 100 or more amino acids beginning with a methionine pepA, pepB and pepE, to determine the contribution of start codon. These were then searched for putative aspar- each protease to the overall protease spectrum using tic protease domains (PF00026) using the protein families bovine serum albumin as a broad substrate. They con- and HMM database (Pfam) release 11.0 (Bateman et al., cluded that the PEPA and PEPB proteases appeared to 2004). The results were compared to known protease constitute 84% and 6% of the extracellular acidic proteo- families in the peptidase database, MEROPS release 6.4 lytic activity, respectively, whereas the PEPE protease is (Rawlings et al., 2004). The coding sequences for pre- responsible for 68% of the intracellular acidic protease dicted proteases were manually compared using the Clu- activity (van den Hombergh et al., 1997a). Moralejo stalW program (Thompson et al., 1994) with default et al. also showed that a defect in the pepA gene reduced settings. Additional sequences of genes from other spe- degradation of overexpressed thaumatin in A. niger cies that have previously been cloned and characterized (Moralejo et al., 2000). were added to demonstrate that the ORFs represent Here, we report the isolation and characterization of putative orthologs of known genes. The Treeview pro- four ‘new’ genes (pepAa, pepAb, pepAc and pepAd) gram was used to draw and visualize hierarchical trees encoding pepsin-like aspartic proteases from A. niger based upon amino acid sequence alignments (Page, and demonstrate that these genes are conserved within 1996). The SignalP (Nielsen et al., 1997) and ProP the Aspergillus genus. The proteins possess different char- (Duckert et al., 2004) programs were used to identify acteristics and may have distinct localizations and func- prepro signal sequences and propeptide cleavage sites, tions. Transcription of the pepAa gene during respectively. The big-PI (http://mendel.imp.univie.ac.at/ recombinant protein production was observed with our sat/gpi/fungi_server.html) fungal predictor was also used A. nidulans cDNA microarray (Sims et al., 2005). There- to identify likely GPI modification sites (Eisenhaber fore, we examined the effect of disrupting these proteases et al., 2004). The disulfide bonds were predicted using on the secretion of the homologous and the heterologous DIpro 2.0 (http://contact.ics.uci.edu/bridge.html)(Cheng proteins in A. niger. et al., 2006). Y. Wang et al. / Fungal Genetics and Biology 45 (2008) 17–27 19 2.3. Microarray analysis sequence of the pepAx genes of A. niger. At the 50 end of each primer, a restriction enzyme recognition site of HpaI Transcriptome analysis was performed as described pre- was introduced. The PCR was carried out with Pfu DNA viously (Sims et al., 2005, 2004b,c). Briefly, a pyrG- single- polymerase and consisted of one cycle at 94 °C for 4 min copy recombinant chymosin-producing A. nidulans strain and 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C (Cullen et al., 1987) was compared to its parental strain for 2.5 min, and then a final extension at 72 °C for transformed with an empty vector.