Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties

BackgroundTrichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general.ResultsWe carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production.ConclusionsaCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

[1]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[2]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[3]  W. Nierman,et al.  Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis , 2008, PLoS pathogens.

[4]  K. H. Wolfe,et al.  Elusive Origins of the Extra Genes in Aspergillus oryzae , 2008, PloS one.

[5]  K. Isono,et al.  Genome sequencing and analysis of Aspergillus oryzae , 2005, Nature.

[6]  D. Hoyle,et al.  Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays , 2008, BMC Genomics.

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

[8]  E. S. Venkatraman,et al.  A faster circular binary segmentation algorithm for the analysis of array CGH data , 2007, Bioinform..

[9]  R. May.  The Arp2/3 complex: a central regulator of the actin cytoskeleton , 2001, Cellular and Molecular Life Sciences CMLS.

[10]  M. Rey,et al.  Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes , 1992, Molecular microbiology.

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

[12]  J. Martín,et al.  Molecular mechanisms of chromosomal rearrangement in fungi. , 1999, Critical reviews in microbiology.

[13]  David Edwards,et al.  Non-linear Normalization and Background Correction in One-channel CDNA Microarray Studies , 2003, Bioinform..

[14]  Martin Vingron,et al.  Variance stabilization applied to microarray data calibration and to the quantification of differential expression , 2002, ISMB.

[15]  M. Dante,et al.  Multifunctional yeast high-copy-number shuttle vectors. , 1992, Gene.

[16]  D. Eveleigh,et al.  Preparation of mutants of Trichoderma reesei with enhanced cellulase production , 1977, Applied and environmental microbiology.

[17]  Yasuji Koyama,et al.  Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation. , 2008, Fungal genetics and biology : FG & B.

[18]  D. Amberg,et al.  The secretory pathway mediates localization of the cell polarity regulator Aip3p/Bud6p. , 2000, Molecular biology of the cell.

[19]  B. Wold,et al.  Ligation-mediated PCR for genomic sequencing and footprinting. , 2001, Current protocols in molecular biology.

[20]  B. Emanuel,et al.  Chromosomal Translocations Mediated by Palindromic DNA , 2006, Cell cycle.

[21]  Ash A. Alizadeh,et al.  Genome-wide analysis of DNA copy-number changes using cDNA microarrays , 1999, Nature Genetics.

[22]  P. Broda,et al.  Rapid preparation of DNA from filamentous fungi , 1985 .

[23]  Jaideep P. Sundaram,et al.  Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus , 2008, PLoS genetics.

[24]  H. Döhner,et al.  Matrix‐based comparative genomic hybridization: Biochips to screen for genomic imbalances , 1997, Genes, chromosomes & cancer.

[25]  D. Pinkel,et al.  Comparative Genomic Hybridization for Molecular Cytogenetic Analysis of Solid Tumors , 2022 .

[26]  Gary E. Harman,et al.  Trichoderma and Gliocladium. Volume 2: Enzymes, biological control and commercial applications , 1998 .

[27]  H. van Tilbeurgh,et al.  The use of 4‐methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes , 1982 .

[28]  Wendy Schackwitz,et al.  Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing , 2009, Proceedings of the National Academy of Sciences.

[29]  Cathryn J. Rehmeyer,et al.  Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae , 2006, Nucleic acids research.

[30]  R. Contreras,et al.  Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei: a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30 , 2005, Applied and Environmental Microbiology.

[31]  Keiichiro Suzuki,et al.  Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora , 2006, Proceedings of the National Academy of Sciences.

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

[33]  D. Eveleigh,et al.  SELECTIVE SCREENING METHODS FOR THE ISOLATION OF HIGH YIELDING CELLULASE MUTANTS OF TRICHODERMA REESEI , 1979 .

[34]  B. Ylstra,et al.  High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides , 2004, Journal of Clinical Pathology.

[35]  Lukas Hartl,et al.  The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome , 2008, BMC Genomics.

[36]  Mark Gerstein,et al.  Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays , 2008, BMC Genomics.

[37]  Bradley P. Coe,et al.  A tiling resolution DNA microarray with complete coverage of the human genome , 2004, Nature Genetics.

[38]  Wolfgang Huber,et al.  Ringo – an R/Bioconductor package for analyzing ChIP-chip readouts , 2007, BMC Bioinformatics.

[39]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[40]  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.

[41]  D. Snakenborg,et al.  Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface , 2008, Nucleic acids research.

[42]  M. Penttilä,et al.  Enzyme production by recombinant Trichoderma reesei strains. , 1991, Journal of biotechnology.

[43]  M. Mandels,et al.  Enhanced cellulase production by a mutant of Trichoderma viride. , 1971, Applied microbiology.

[44]  Christopher M. Crew,et al.  A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors , 2006, Proceedings of the National Academy of Sciences.

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

[46]  Reese Et,et al.  History of the cellulase program at the U.S. army Natick Development Center. , 1976 .

[47]  S. Kauppinen,et al.  Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. , 1987, Gene.

[48]  Roland Brousseau,et al.  Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays. , 2004, Journal of microbiological methods.

[49]  M. Penttilä,et al.  Electrophoretic karyotyping of wild-type and mutant Trichoderma longibrachiatum (reesei) strains , 1992, Current Genetics.

[50]  E. Reese,et al.  History of the cellulase program at the U.S. army Natick Development Center. , 1976, Biotechnology and bioengineering symposium.

[51]  Erdogan Gulari,et al.  On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes , 2006, Nucleic acids research.