Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

BackgroundThe soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei.ResultsWe have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes.ConclusionsTranscriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

[1]  Merja Penttilä,et al.  The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei. , 2005, Microbiology.

[2]  M. Penttilä,et al.  Isolation of the ace1 Gene Encoding a Cys2-His2 Transcription Factor Involved in Regulation of Activity of the Cellulase Promoter cbh1of Trichoderma reesei * , 2000, The Journal of Biological Chemistry.

[3]  Matthias G. Steiger,et al.  Transcriptional Regulation of xyr1, Encoding the Main Regulator of the Xylanolytic and Cellulolytic Enzyme System in Hypocrea jecorina , 2008, Applied and Environmental Microbiology.

[4]  M. Rep,et al.  Mutation of CRE1 in Fusarium oxysporum reverts the pathogenicity defects of the FRP1 deletion mutant , 2009, Molecular microbiology.

[5]  Bernard Henrissat,et al.  Corrigendum: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina) , 2008, Nature Biotechnology.

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  Monika Schmoll,et al.  Biology and biotechnology of Trichoderma , 2010, Applied Microbiology and Biotechnology.

[8]  J. Bono,et al.  Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi , 2001, Molecular microbiology.

[9]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[10]  W. Timberlake,et al.  Primary structure of the trpC gene from Aspergillus nidulans , 2004, Molecular and General Genetics MGG.

[11]  H. Nakazawa,et al.  Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli , 2008, Applied Microbiology and Biotechnology.

[12]  P. Pouwels,et al.  Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene. , 1991, Journal of biotechnology.

[13]  M. Penttilä,et al.  ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression , 2003, Applied and Environmental Microbiology.

[14]  C. Kubicek,et al.  Expression of Biomass-Degrading Enzymes Is a Major Event during Conidium Development in Trichoderma reesei , 2011, Eukaryotic Cell.

[15]  M. Penttilä,et al.  The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form , 1996, Molecular and General Genetics MGG.

[16]  R. Mach,et al.  Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina , 2006, Eukaryotic Cell.

[17]  M. Bailey,et al.  Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy , 2003, Applied Microbiology and Biotechnology.

[18]  J. P. Craig,et al.  Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi , 2012, Proceedings of the National Academy of Sciences.

[19]  D. Ramón,et al.  CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans. , 2008, Fungal genetics and biology : FG & B.

[20]  G. Braus,et al.  Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction. , 2012, Fungal genetics and biology : FG & B.

[21]  S. Baker,et al.  Functional Analyses of Trichoderma reesei LAE1 Reveal Conserved and Contrasting Roles of This Regulator , 2013, G3: Genes | Genomes | Genetics.

[22]  Monika Schmoll,et al.  Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina , 2009, Biotechnology for biofuels.

[23]  M Penttilä,et al.  A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. , 1987, Gene.

[24]  Axel A. Brakhage,et al.  Regulation of fungal secondary metabolism , 2012, Nature Reviews Microbiology.

[25]  S. Zeilinger,et al.  The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element , 2001, Molecular Genetics and Genomics.

[26]  F. Henrique-Silva,et al.  Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei. , 1995, Biochemistry.

[27]  J. Kelly,et al.  The Aspergillus nidulans creC gene involved in carbon catabolite repression encodes a WD40 repeat protein , 2000, Molecular and General Genetics MGG.

[28]  T. Pakula,et al.  The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina). , 2012, Microbiology.

[29]  M. Moo-young,et al.  Interlaboratory testing of methods for assay of xylanase activity , 2002 .

[30]  S. Kuhara,et al.  A new Zn(II)(2)Cys(6)-type transcription factor BglR regulates β-glucosidase expression in Trichoderma reesei. , 2012, Fungal genetics and biology : FG & B.

[31]  Mikko Arvas,et al.  Correlation of gene expression and protein production rate - a system wide study , 2011, BMC Genomics.

[32]  Robert L. Mach,et al.  Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) , 2008, Applied Microbiology and Biotechnology.

[33]  Merja Penttilä,et al.  Transcriptional regulation of plant cell wall degradation by filamentous fungi. , 2005, FEMS microbiology reviews.

[34]  S. Baker,et al.  The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei , 2012, Molecular microbiology.

[35]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[36]  T. Houfek,et al.  Transcriptional Regulation of Biomass-degrading Enzymes in the Filamentous Fungus Trichoderma reesei* , 2003, Journal of Biological Chemistry.

[37]  M. Penttilä,et al.  Molecular biology of Trichoderma and biotechnological applications , 2004 .

[38]  J. Kelly,et al.  Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cataboiite repression in Aspergillus nidulans , 1993, Molecular microbiology.

[39]  Lokesh Kumar,et al.  Mfuzz: A software package for soft clustering of microarray data , 2007, Bioinformation.

[40]  Mikko Arvas,et al.  Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates , 2012, Microbial Cell Factories.

[41]  M. Penttilä,et al.  ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei * , 2001, The Journal of Biological Chemistry.

[42]  M. Penttilä,et al.  The Effects of Drugs Inhibiting Protein Secretion in the Filamentous Fungus Trichoderma reesei , 2003, Journal of Biological Chemistry.

[43]  S. Sze,et al.  Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei. , 2011, Journal of proteome research.

[44]  D. Ussery,et al.  Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina , 2007, BMC Genomics.

[45]  Gordon K. Smyth,et al.  Use of within-array replicate spots for assessing differential expression in microarray experiments , 2005, Bioinform..

[46]  Robert D. Finn,et al.  InterPro in 2011: new developments in the family and domain prediction database , 2011, Nucleic acids research.

[47]  C. Kubicek,et al.  Systems Analysis of Lactose Metabolism in Trichoderma reesei Identifies a Lactose Permease That Is Essential for Cellulase Induction , 2013, PloS one.

[48]  Monika Schmoll,et al.  Trichoderma in the light of day – Physiology and development , 2010, Fungal genetics and biology : FG & B.