Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO2. To quantify and understand how these process parameters affect yeast physiology, this study investigates individual and combined impacts of low pH (3.0) and high CO2 (50%) on slow-growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113-7D. Combined exposure to low pH and high CO2 led to increased maintenance-energy requirements and death rates in aerobic, glucose-limited cultures. Further experiments showed that these effects were predominantly caused by low pH. Growth under ammonium-limited, energy-excess conditions did not aggravate or ameliorate these adverse impacts. Despite the absence of a synergistic effect of low pH and high CO2 on physiology, high CO2 strongly affected genome-wide transcriptional responses to low pH. Interference of high CO2 with low-pH signaling is consistent with low-pH and high-CO2 signals being relayed via common (MAPK) signaling pathways, notably the cell wall integrity, high-osmolarity glycerol, and calcineurin pathways. This study highlights the need to further increase robustness of cell factories to low pH for carboxylic-acid production, even in organisms that are already applied at industrial scale.@en

Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state. In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/orchemostat cultivation.@en

Background: Saccharomyces cerevisiae is an established microbial platform for production of native and non-native compounds. When product pathways compete with growth for precursors and energy, uncoupling of growth and product formation could increase product yields and decrease formation of biomass as a by-product. Studying non-growing, metabolically active yeast cultures is a first step towards developing S. cerevisiae as a robust, non-growing cell factory. Microbial physiology at near-zero growth rates can be studied in retentostats, which are continuous-cultivation systems with full biomass retention. Hitherto, retentostat studies on S. cerevisiae have focused on anaerobic conditions, which bear limited relevance for aerobic industrial processes. The present study uses aerobic, glucose-limited retentostats to explore the physiology of non-dividing, respiring S. cerevisiae cultures, with a focus on industrially relevant features. Results: Retentostat feeding regimes for smooth transition from exponential growth in glucose-limited chemostat cultures to near-zero growth rates were obtained by model-aided experimental design. During 20 days of retentostats cultivation, the specific growth rate gradually decreased from 0.025 h-1 to below 0.001 h-1, while culture viability remained above 80 %. The maintenance requirement for ATP (mATP) was estimated at 0.63 ± 0.04 mmol ATP (g biomass)-1 h-1, which is ca. 35 % lower than previously estimated for anaerobic retentostats. Concomitant with decreasing growth rate in aerobic retentostats, transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes resembled transcriptional regulation patterns observed for anaerobic retentostats. The heat-shock tolerance in aerobic retentostats far exceeded previously reported levels in stationary-phase batch cultures. While in situ metabolic fluxes in retentostats were intentionally low due to extreme caloric restriction, off-line measurements revealed that cultures retained a high metabolic capacity. Conclusions: This study provides the most accurate estimation yet of the maintenance-energy coefficient in aerobic cultures of S. cerevisiae, which is a key parameter for modelling of industrial aerobic, glucose-limited fed-batch processes. The observed extreme heat-shock tolerance and high metabolic capacity at near-zero growth rates demonstrate the intrinsic potential of S. cerevisiae as a robust, non-dividing microbial cell factory for energy-intensive products.@en

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.@en

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.@en

In industrial biotechnology, micro-organisms are used for making a wide range of products, including neutraceuticals, transport fuels, pharmaceuticals and plastics. The raw materials for these microbial processes are often sugars derived from natural resources. Industrial biotechnology offers an advantage with respect to the petrochemical synthesis of these products with respect to its sustainability in CO2 emissions. Yeasts are important industrial workhorses in industrial biotechnology, as they are used for making many of the abovementioned products. Baker’s yeast – Saccharomyces cerevisiae – is used for the large-scale production of, for example, bioethanol, farnesene, human insulin and succinic acid. To achieve cost-efficient production processes, the high-yield conversion of substrate (e.g. glucose) to the product of interest is essential. A possible approach to achieve this goal is to uncouple microbial product formation from growth. This approach is challenging since, during evolution, microbial metabolism has been optimized for the use of substrate for growth and maintenance of cellular integrity and viability. For dissimilatory products, whose synthesis by micro-organisms results in the net production of ATP, such as ethanol for S. cerevisiae, uncoupling of growth and product formation has previously been investigated under anaerobic conditions. For non-dissimilatory products such as succinic acid, farnesene and proteins, a net input of ATP is required and their production is in direct competition with the use of ATP and energy substrate for growth and maintenance. High-yield production of non-dissimilatory products therefore requires a high energy efficiency in dissimilation. During fully respiratory growth of S. cerevisiae, the ATP yield from glucose dissimilation is eight-fold higher than during fermentative growth. Aerobic, respiratory dissimilation of glucose is therefore highly favorable for non-dissimilatory product formation. Systematic characterization of yeasts can be performed under strictly controlled conditions in bioreactors. When bioreactors are operated as steady-state chemostat cultures, the specific growth rate is determined by the dilution rate, which is set by the experimenter. Chemostat cultivation therefore permits comparisons between strains and/or conditions independent of the specific growth rate. Due to technical limitations in the rate of medium supply, chemostat cultivation in bench-top laboratory bioreactors is practically not feasible at extremely low dilution rates (< 0.015 h-1). The retentostat, a modification of the chemostat in which 100 % biomass retention in the reactor is achieved by placing a filter in the outflow line, offers a suitable alternative for the investigation of such extremely low growth rates. @en