Project description:Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites and cellular energy status are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires a net input of 2 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates, a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g., 27% at 0.03 h-1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in the chemostats. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g., ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways. The goal of the present study is to investigate the impact of specific growth rate on biomass-specific productivity, product yield, by-product formation and host strain physiology of an S. cerevisiae strain that was previously engineered for de novo production of resveratrol from glucose. To this end, (by)product formation, physiology and transcriptome were analysed in steady-state, glucose-limited chemostat cultures grown at different dilution rates.

Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness. The goal of the present study is to investigate the physiology of aerobic fully respiring S. cerevsiae at near-zero growth rates. Fundamental but industrially-relevant questions were addressed thanks to the design, implementation and study of aerobic retentostat cultivations enabling a rapid but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates.

Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness.

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: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).

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Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain

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