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Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

ABSTRACT: Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

SUBMITTER: Li WC

PROVIDER: S-EPMC7923544 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Li Wan-Chen WC Lee Chia-Yi CY Lan Wei-Hsuan WH Woo Tai-Ting TT Liu Hou-Cheng HC Yeh Hsin-Yi HY Chang Hao-Yen HY Chuang Yu-Chien YC Chen Chiung-Ya CY Chuang Chi-Ning CN Chen Chia-Ling CL Hsueh Yi-Ping YP Li Hung-Wen HW Chi Peter P Wang Ting-Fang TF

Proceedings of the National Academy of Sciences of the United States of America 20210201 8

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interho ...[more]

PMID: 33593897

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Project description:BACKGROUND:The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a widely used industrial host organism for protein production. In industrial cultivations, it can produce over 100 g/l of extracellular protein, mostly constituting of cellulases and hemicellulases. In order to improve protein production of T. reesei the transcriptional regulation of cellulases and secretory pathway factors have been extensively studied. However, the metabolism of T. reesei under protein production conditions has not received much attention. RESULTS:To understand the physiology and metabolism of T. reesei under protein production conditions we carried out a well-controlled bioreactor experiment with extensive analysis. We used minimal media to make the data amenable for modelling and three strain pairs to cover different protein production levels. With RNA-sequencing transcriptomics we detected the concentration of the carbon source as the most important determinant of the transcriptome. As the major transcriptional response concomitant to protein production we detected the induction of selected genes that were putatively regulated by xyr1 and were related to protein transport, amino acid metabolism and transcriptional regulation. We found novel metabolic responses such as production of glycerol and a cellotriose-like compound. We then used this cultivation data for flux balance analysis of T. reesei metabolism and demonstrate for the first time the use of genome wide stoichiometric metabolic modelling for T. reesei. We show that our model can predict protein production rate and provides novel insight into the metabolism of protein production. We also provide this unprecedented cultivation and transcriptomics data set for future modelling efforts. CONCLUSIONS:The use of stoichiometric modelling can open a novel path for the improvement of protein production in T. reesei. Based on this we propose sulphur assimilation as a major limiting factor of protein production. As an organism with exceptional protein production capabilities modelling of T. reesei can provide novel insight also to other less productive organisms.

Project description:BackgroundThe tropical ascomycete Trichoderma reesei (Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light.ResultsOur transcriptome analysis of strains lacking the photoreceptors BLR1 and BLR2 as well as ENV1 revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. We show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. In contrast to N. crassa, photoreceptor mediated regulation of carbon metabolism in T. reesei occurs primarily by BLR1 and BLR2 via their positive effect on induction of env1 transcription, rather than by a presumed negative effect of ENV1 on the function of the BLR complex. Nevertheless, genes consistently regulated by photoreceptors in N. crassa and T. reesei are significantly enriched in carbon metabolic functions. Hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved.Analysis of growth on different carbon sources revealed that the oxidoreductive D-galactose and pentose catabolism is influenced by light and ENV1. Transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by ENV1, BLR1 and/or BLR2. Additionally we detected an ENV1-regulated genomic cluster of 9 genes including the D-mannitol dehydrogenase gene lxr1, with two genes of this cluster showing consistent regulation in N. crassa.ConclusionsWe show that one major output pathway of light signalling in Trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. Targets of ENV1 and BLR1/BLR2 are for the most part distinct and indicate individual functions for ENV1 and the BLR complex besides their postulated regulatory interrelationship.

Project description:Background:In addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Among them, sorbicillinoids, a large group of hexaketide secondary metabolites derived from polyketides, are drawing a growing interest from researchers because they exhibit a variety of important biological functions, including anticancer, antioxidant, antiviral, and antimicrobial properties. The development of fungi strains with constitutive, hyperproduction of sorbicillinoids is thus desired for future industry application but is not well-studied. Moreover, although T. reesei has been demonstrated to produce sorbicillinoids with the corresponding gene cluster and biosynthesis pathway proposed, the underlying molecular mechanism governing sorbicillinoid biosynthesis remains unknown. Results:Recombinant T. reesei ZC121 was constructed from strain RUT-C30 by the insertion of the gene 12121-knockout cassette at the telomere of T. reesei chromosome IV in consideration of the off-target mutagenesis encountered during the unsuccessful deletion of gene 121121. Strain ZC121, when grown on cellulose, showed a sharp reduction of cellulase production, but yet a remarkable enhancement of sorbicillinoids production as compared to strain RUT-C30. The hyperproduction of sorbicillinoids is a constitutive process, independent of culture conditions such as carbon source, light, pH, and temperature. To the best of our knowledge, strain ZC121 displays record sorbicillinoid production levels when grown on both glucose and cellulose. Sorbicillinol and bisvertinolone are the two major sorbicillinoid compounds produced. ZC121 displayed a different morphology and markedly reduced sporulation compared to RUT-C30 but had a similar growth rate and biomass. Transcriptome analysis showed that most genes involved in cellulase production were downregulated significantly in ZC121 grown on cellulose, whereas remarkably all genes in the sorbicillinoid gene cluster were upregulated on both cellulose and glucose. Conclusion:A constitutive sorbicillinoid-hyperproduction strain T. reesei ZC121 was obtained by off-target mutagenesis, displaying an overwhelming shift from cellulase production to sorbicillinoid production on cellulose, leading to a record for sorbicillinoid production. For the first time, T. reesei degraded cellulose to produce platform chemical compounds other than protein in high yield. We propose that the off-target mutagenesis occurring at the telomere region might cause chromosome remodeling and subsequently alter the cell structure and the global gene expression pattern of strain ZC121, as shown by phenotype profiling and comparative transcriptome analysis of ZC121. Overall, T. reesei ZC121 holds great promise for the industrial production of sorbicillinoids and serves as a good model to explore the regulation mechanism of sorbicillinoids' biosynthesis.

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Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

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<i>Trichoderma reesei</i> Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

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