Dataset Information

Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei.

ABSTRACT: Trichoderma reesei represents one of the most prolific producers of plant cell wall degrading enzymes. Recent research showed broad regulation by phosphorylation in T. reesei, including important transcription factors involved in cellulase regulation. To evaluate factors crucial for changes in these phosphorylation events, we studied non-essential protein phosphatases (PPs) of T. reesei. Viable deletion strains were tested for growth on different carbon sources, osmotic and oxidative stress response, asexual and sexual development, cellulase and protease production as well as secondary metabolism. Six PPs were found to be positive or negative regulators for cellulase production. A correlation of the effects of PPs on protease activities and cellulase activities was not detected. Hierarchical clustering of regulation patterns and phenotypes of deletion indicated functional specialization within PP classes and common as well as variable effects. Our results confirmed the central role of catalytic and regulatory subunits of PP2A which regulates several aspects of cell growth and metabolism. Moreover we show that the additional homologue of PPH5 in Trichoderma spp., PPH5-2 assumes distinct functions in metabolism, development and stress response, different from PPH5. The influence of PPs on both cellulase gene expression and secondary metabolite production support an interrelationship in the underlying regulation mechanisms.

SUBMITTER: Rodriguez-Iglesias A

PROVIDER: S-EPMC6662751 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei.

Rodriguez-Iglesias Aroa A Schmoll Monika M

Scientific reports 20190729 1

Trichoderma reesei represents one of the most prolific producers of plant cell wall degrading enzymes. Recent research showed broad regulation by phosphorylation in T. reesei, including important transcription factors involved in cellulase regulation. To evaluate factors crucial for changes in these phosphorylation events, we studied non-essential protein phosphatases (PPs) of T. reesei. Viable deletion strains were tested for growth on different carbon sources, osmotic and oxidative stress resp ...[more]

PMID: 31358805

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Project description:Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in ?gpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

Project description:Background:The filamentous ascomycete T. reesei is industrially used to produce cellulases and xylanases. Cost-effective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type xyr1 gene and locus. Results:Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose-there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol. Conclusion:During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1-Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.

Project description:Lignocellulose is an abundant waste resource and has been considered as a promising material for production of biofuels or other valuable bio-products. Currently, one of the major bottlenecks in the economic utilization of lignocellulosic materials is the cost-efficiency of converting lignocellulose into soluble sugars for fermentation. One way to address this problem is to seek superior lignocellulose degradation enzymes or further improve current production yields of lignocellulases. In the present study, the lignocellulose degradation capacity of a thermophilic fungus Chaetomium thermophilum was firstly evaluated and compared to that of the biotechnological workhorse Trichoderma reesei. The data demonstrated that compared to T. reesei, C. thermophilum displayed substantially higher cellulose-utilizing efficiency with relatively lower production of cellulases, indicating that better cellulases might exist in C. thermophilum. Comparison of the protein secretome between C. thermophilum and T. reesei showed that the secreted protein categories were quite different in these two species. In addition, to prove that cellulases in C. thermophilum had better enzymatic properties, the major cellulase cellobiohydrolase I (CBH1) from C. thermophilum and T. reesei were firstly characterized, respectively. The data showed that the specific activity of C. thermophilum CBH1 was about 4.5-fold higher than T. reesei CBH1 in a wide range of temperatures and pH. To explore whether increasing CBH1 activity in T. reesei could contribute to improving the overall cellulose-utilizing efficiency of T. reesei, T. reesei cbh1 gene was replaced with C. thermophilum cbh1 gene by integration of C. thermophilum cbh1 gene into T. reesei cbh1 gene locus. The data surprisingly showed that this gene replacement not only increased the cellobiohydrolase activities by around 4.1-fold, but also resulted in stronger induction of other cellulases genes, which caused the filter paper activities, Azo-CMC activities and ?-glucosidase activities increased by about 2.2, 1.9, and 2.3-fold, respectively. The study here not only provided new resources of superior cellulases genes and new strategy to improve the cellulase production in T. reesei, but also contribute to opening the path for fundamental research on C. thermophilum.

Project description:Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei. Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δare1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH4)2SO4. Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1, cbh2, egl1, and egl2, were dramatically decreased in Δare1. Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δare1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei, although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi.

Dataset Information

Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei.

Publications

Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei.

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