Abstract
BackgroundCellulolytic 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.ResultsTrichoderma 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%.ConclusionsThe 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.
Highlights
Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion
Phenotypic characteristics of T. reesei hyper‐cellulolytic mutant SS‐II We obtained the T. reesei hyper-cellulolytic mutant strain, SS-II, which was used for cellulase production, from Sunson Industry between 2002 and 2010. was derived from strain NG14 through NTG mutagenesis
To characterize the differences in growth rates between SS-II and RUT-C30, T. reesei strains were grown in MA medium with 2% (w/v) glucose, 2% (w/v) lactose, or 2% (w/v) Avicel as the sole carbon source
Summary
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. The production cost of biomass-degrading enzymes is still a significant challenge for commercial biofuel production [2]. Expression of cellulase and xylanase genes is subject to carbon catabolite repression (CCR) [6], regulated by carbon catabolite repressor 1 (CRE1) [7]. CCR facilitates preferential assimilation of metabolized carbon sources by inhibiting the expression of enzymes involved in the catabolism of other carbon sources. This is essential for the adaptation and survival of T. reesei [4, 6]
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