Dataset Information

Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator.

ABSTRACT:

Background

The filamentous fungus Trichoderma reesei (T. reesei) is a natural producer of cellulolytic and xylanolytic enzymes and is therefore industrially used. Many industries require high amounts of enzymes, in particular cellulases. Strain improvement strategies by random mutagenesis yielded the industrial ancestor strain Rut-C30. A key property of Rut-C30 is the partial release from carbon catabolite repression caused by a truncation of the repressor Cre1 (Cre1-96). In the T. reesei wild-type strain a full cre1 deletion leads to pleiotropic effects and strong growth impairment, while the truncated cre1-96 enhances cellulolytic activity without the effect of growth deficiencies. However, it is still unclear which function Cre1-96 has in Rut-C30.

Results

In this study, we deleted and constitutively expressed cre1-96 in Rut-C30. We found that the presence of Cre1-96 in Rut-C30 is crucial for its cellulolytic and xylanolytic performance under inducing conditions. In the case of the constitutively expressed Cre1-96, the cellulase activity could further be improved approximately twofold. The deletion of cre1-96 led to growth deficiencies and morphological abnormalities. An in silico domain prediction revealed that Cre1-96 has all necessary properties that a classic transactivator needs. Consequently, we investigated the cellular localization of Cre1-96 by fluorescence microscopy using an eYFP-tag. Cre1-96 is localized in the fungal nuclei under both, inducing and repressing conditions. Furthermore, chromatin immunoprecipitation revealed an enrichment of Cre1-96 in the upstream regulatory region of the main transactivator of cellulases and xylanases, Xyr1. Interestingly, transcript levels of cre1-96 show the same patterns as the ones of xyr1 under inducing conditions.

Conclusions

The findings suggest that the truncation turns Cre1 into an activating regulator, which primarily exerts its role by approaching the upstream regulatory region of xyr1. The conversion of repressor proteins to potential activators in other biotechnologically used filamentous fungi can be applied to increase their enzyme production capacities.

SUBMITTER: Rassinger A

PROVIDER: S-EPMC6100732 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Truncation of the transcriptional repressor protein Cre1 in <i>Trichoderma reesei</i> Rut-C30 turns it into an activator.

Rassinger Alice A Gacek-Matthews Agnieszka A Strauss Joseph J Mach Robert L RL Mach-Aigner Astrid R AR

Fungal biology and biotechnology 20180820

<h4>Background</h4>The filamentous fungus <i>Trichoderma reesei</i> (<i>T. reesei</i>) is a natural producer of cellulolytic and xylanolytic enzymes and is therefore industrially used. Many industries require high amounts of enzymes, in particular cellulases. Strain improvement strategies by random mutagenesis yielded the industrial ancestor strain Rut-C30. A key property of Rut-C30 is the partial release from carbon catabolite repression caused by a truncation of the repressor Cre1 (Cre1-96). I ...[more]

PMID: 30151221

Similar Datasets

Project description: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.