Project description:Comparison of T. reesei grown on lactose fed chemostat cultivations in different growth rates and cell densities The analysis is further described in paper Correlation of gene expression and protein production rate - a system wide study. Mikko Arvas, Tiina Pakula, Bart Smit, Jari Rautio, Heini Koivistoinen, Paula Jouhten, Erno Lindfors, Marilyn Wiebe, Merja Penttilä and Markku Saloheimo, Submitted. Abstract: Background:Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results: We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions: Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).

Project description:Comparison of T. reesei grown on lactose fed chemostat cultivations in different growth rates and cell densities The analysis is further described in paper Correlation of gene expression and protein production rate - a system wide study. Mikko Arvas, Tiina Pakula, Bart Smit, Jari Rautio, Heini Koivistoinen, Paula Jouhten, Erno Lindfors, Marilyn Wiebe, Merja Penttilä and Markku Saloheimo, Submitted. Abstract: Background:Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results: We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions: Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). A nine chip study using total RNA recovered from three separate cultures of T. reesei RutC-30 grown with growth rate 0.03, three separate cultures grown with growth rate 0.06 and three separate cultures grown with growth rate 0.03 in high cell density.

Project description:BackgroundThe model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown.Methodology/principal findingsHere, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2(G16V) allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway.Conclusions/significanceTogether, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.

Project description:BackgroundThe induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest.ResultsTrichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes' coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8.ConclusionsRecombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.

Dataset Information

N,N-dimethylformamide induces cellulase production in the filamentous fungus Trichoderma reesei.

Publications

<i>N</i>,<i>N</i>-dimethylformamide induces cellulase production in the filamentous fungus <i>Trichoderma reesei</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets