Dataset Information

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

ABSTRACT: BACKGROUND: Trichoderma 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. RESULTS: We 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. CONCLUSIONS: aCGH 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.

SUBMITTER: Vitikainen M

PROVIDER: S-EPMC3091638 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Vitikainen Marika M Arvas Mikko M Pakula Tiina T Oja Merja M Penttilä Merja M Saloheimo Markku M

BMC genomics 20100719

<h4>Background</h4>Trichoderma 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 util ...[more]

PMID: 20642838

Similar Datasets

Project description:BACKGROUND:Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. RESULTS:Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. CONCLUSIONS:The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.

Project description:BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure