Correlation of gene expression and protein production rate - a system wide study

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.</p> <p>Results</p> <p>We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of <it>T. reesei</it>. 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.</p> <p>Conclusions</p> <p>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).</p

Differential Regulation of the Cellulase Transcription Factors XYR1, ACE2, and ACE1 in Trichoderma reesei Strains Producing High and Low Levels of Cellulase ▿ †

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2, and the corepressor gene ace1, during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d-galactose, but this induction was independent of d-galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C30 and the hyperproducer strain T. reesei CL847. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulase production

Genome wide analysis of protein production load in Trichoderma reesei

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.Peer reviewe