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.

The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.

Stationary-phase (SP) batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability.