Abstract
BackgroundSaccharomyces 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.ResultsRetentostat 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0501-z) contains supplementary material, which is available to authorized users.
Highlights
Saccharomyces cerevisiae is an established microbial platform for production of native and non-native compounds
To predict the impact of these parameters on growth dynamics in aerobic retentostat cultures, a model based on the Pirt definition of resource allocation was used
maximum biomass yield on substrate (YXm/aSx) was estimated from published data on aerobic, glucose-limited chemostat cultures of the S. cerevisiae strain used in this study (0.5 g g−1 [28])
Summary
Saccharomyces cerevisiae is an established microbial platform for production of native and non-native compounds. Vos et al Microb Cell Fact (2016) 15:111 cerevisiae, a completely respiratory sugar metabolism requires aerobic conditions and sugar-limited cultivation at low to intermediate specific growth rates [5] In industry, these requirements are usually met by sugar-limited, aerobic fed-batch cultivation. Understanding and, breaking this correlation between growth and product formation by improving specific rates of product formation at low specific growth rates, is an important target for optimizing productivity and product yields in aerobic, sugar-limited fed-batch cultures. The Pirt equation describes how the fraction of the energy substrate that needs to be dissimilated to fulfil maintenance energy requirements increases as the specific growth rate in, for example, an aerobic, sugar-limited fed-batch process decreases. In slow-growing aerobic industrial fed-batch processes this increasing impact of maintenance requirements has a major negative impact on product yields and productivities [3]
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