Dataset Information

Enhanced cellulase production by decreasing intercellular pH through H+-ATPase gene deletion in Trichoderma reesei RUT-C30.

ABSTRACT: Background:Cellulolytic enzymes produced by Trichoderma reesei are widely used for the industrial production of biofuels and chemicals from lignocellulose. We speculated that intracellular pH during the fermentation process can affect cellulase induction. Results:In this study, two H+-ATPase genes, tre76238 and tre78757, were first identified in T. reesei. Deletion of tre76238 and tre78757 in T. reesei RUT-C30 confirmed that tre76238 has a major function in maintaining intracellular pH, whereas tre78757 has a minor function. The tre76238 deletion strain ?76238 displayed a high level of cellulase production using cellulase-repressive glucose as a sole carbon source, along with intracellular acid accumulation and growth retardation. Our results indicated that intracellular acid accumulation in ?76238 stimulated a significant increase in the cytosolic Ca2+ levels. Ca2+ channels were shown to be necessary for cellulase production using glucose as the carbon source in ?76238. Delayed ?76238 growth could be reversed by optimizing the medium's nitrogen sources to produce ammonia for intracellular acid neutralization in the early phase. This may be useful for scale-up of cellulase production using glucose as the carbon source. Conclusions:This study provides a new perspective for significant alterations in the cellulase expression pattern of T. reesei ?76238, indicating a new mechanism for cellulase regulation under conditions of low intracellular pH.

SUBMITTER: Liu P

PROVIDER: S-EPMC6691542 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Enhanced cellulase production by decreasing intercellular pH through H+-ATPase gene deletion in Trichoderma reesei RUT-C30.

Liu Pei P Zhang Guoxiu G Chen Yumeng Y Zhao Jian J Wang Wei W Wei Dongzhi D

Biotechnology for biofuels 20190813

<h4>Background</h4>Cellulolytic enzymes produced by Trichoderma reesei are widely used for the industrial production of biofuels and chemicals from lignocellulose. We speculated that intracellular pH during the fermentation process can affect cellulase induction.<h4>Results</h4>In this study, two H+-ATPase genes, tre76238 and tre78757, were first identified in T. reesei. Deletion of tre76238 and tre78757 in T. reesei RUT-C30 confirmed t ...[more]

PMID: 31417630

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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:Background:The path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. Herein, we describe the rational engineering of the publicly available T. reesei RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products. Results:A CRISPR/Cas9 system was used to introduce six genetic modifications in RUT-C30. Implemented changes included the constitutive expression of a mutated allele of the cellulase master regulator XYR1, the expression of two heterologous enzymes, the β-glucosidase CEL3A from Talaromyces emersonii and the invertase SUC1 from Aspergillus niger, and the deletion of genes encoding the cellulase repressor ACE1 and the extracellular proteases SLP1 and PEP1. These alterations resulted in a remarkable increase of protein secretion rates by RUT-C30 and amended its well described β-glucosidase deficiency while enabling the utilization of sucrose and eliminating the requirement of inducing sugars for enzyme production. With a developed sugarcane molasses-based bioprocess, the engineered strain reached an extracellular protein titer of 80.6 g L-1 (0.24 g L-1 h-1), which is the highest experimentally supported titer so far reported for T. reesei. The produced enzyme cocktail displayed increased levels of cellulase and hemicellulase activities, with particularly large increments being observed for the specific activities of β-glucosidase (72-fold) and xylanase (42-fold). Notably, it also exhibited a saccharification efficiency similar to that of a commercially available cellulase preparation in the deconstruction of industrially pretreated sugarcane straw. Conclusion:This work demonstrates the rational steps for the development of a cellulase hyperproducing strain from a well-characterized genetic background available in the public domain, the RUT-C30, associated with an industrially relevant bioprocess, paving new perspectives for Trichoderma research on cellulase production.

Project description:BACKGROUND: Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. RESULTS: We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a ?pgi1 mutant. Both ?pgi1 and cre1+?pgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, ?pgi1 and cre1+?pgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. ?pgi1 mutants were also affected in the extracellular ?-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in ?pgi1 during growth on glucose. CONCLUSIONS: The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma ?pgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1.

Dataset Information

Enhanced cellulase production by decreasing intercellular pH through H+-ATPase gene deletion in Trichoderma reesei RUT-C30.

Publications

Enhanced cellulase production by decreasing intercellular pH through H<sup>+</sup>-ATPase gene deletion in <i>Trichoderma reesei</i> RUT-C30.

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