Project description:In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Experiment Overall Design: Cultivation of microorganisms in chemostats offers numerous advantages for studying the structure and regulation of metabolic networks (11). In chemostat cultures, individual culture parameters can be changed, while keeping other relevant physical and chemical culture parameters (composition of synthetic medium, pH, temperature, aeration, etc.) constant. An especially important parameter in this respect is the specific growth rate, which, in a chemostat, is equal to the dilution rate, which can be accurately controlled. This allows the experimenter to investigate the effects of environmental changes or genetic interventions at a fixed specific growth rate, even if these changes result in different specific growth rates in batch cultures. In a chemostat, growth can be limited by a single, selected nutrient. The very low residual concentrations of this growth-limiting nutrient in chemostat cultures alleviate effects of catabolite repression and inactivation. Furthermore, these low residual substrate concentrations prevent substrate toxicity, which, for example, occurs when S. cerevisiae is grown on ethanol or acetate as the carbon source in batch cultures . Experiment Overall Design: The central goal of the present study is to assess to what extent carbon source-dependent regulation of fluxes through central carbon metabolism in S. cerevisiae is regulated at the level of transcription. To this end, we compare the transcriptome of carbon-limited, aerobic chemostat cultures grown on four different carbon sources: glucose, maltose, ethanol, and acetate. Data from the transcriptome analysis are compared with flux distribution profiles calculated with a stoichiometric metabolic network model. Questions that will be addressed are as follows: (i) does glucose-limited aerobic cultivation lead to a complete alleviation of glucose-catabolite repression; (ii) how (in)complete is our understanding of the genes involved in the transcriptional response of S. cerevisiae to four of the most common carbon sources for this yeast; and (iii) to what extent do transcriptome analyses with microarrays provide a reliable indication of flux distribution in metabolic networks?

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:When the yeast Saccharomyces cerevisiae is subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from being purely respiratory to mixed respiratory and fermentative. This shift is characterized by ethanol production, a phenomenon known as the Crabtree effect due to its analogy with lactate overflow in cancer cells. It is well known that at high glycolytic fluxes there is glucose repression of respiratory pathways resulting in a decrease in the respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect (or overflow metabolism) is due to a limited respiratory capacity or caused by glucose-mediated repression of respiration. We addressed this issue by increasing respiration in S. cerevisiae by introducing a heterologous alternative oxidase, and observed reduced aerobic ethanol formation. In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, while NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, while alternative oxidase is directed to the mitochondria. The onset of aerobic ethanol formation is demonstrated to be a consequence of an imbalance in mitochondrial redox balancing. In addition to answering fundamental physiological questions, our findings are relevant for all biomass derived applications of S. cerevisiae. Experiment Overall Design: Heterologous gene expression in chemostats using Affymetrix Yeast Genome 2.0 arrays. Total RNA extraction and sample preparation, hybridization was done according to the manufacturer's protocol.

Project description:In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Keywords: medium composition

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Growth control of the eukaryote cell: a systems biology study in yeast

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Growth control of the eukaryote cell: a systems biology study in yeast.

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