Unknown,Transcriptomics,Genomics,Proteomics

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Anaerobic carbon-limited retentostat cultivation of Lactobacillus plantarum WCFS1

ABSTRACT: Lactobacillus plantarum WCFS1 was grown under anaerobic carbon-limited conditions in a chemostat with complete biomass retention (retentostat). In this cultivation system, the biomass concentration progressively increases while the dilution rate is kept constant, resulting in decreased specific susbtrate availibility, and hence, a progressive decrease in the specific growth rate. During the progressive transition from growth to virtually no growth, the global changes occurring at the level of metabolism and gene expression were studied using a genome-scale metabolic model and DNA microarrays. Four different time-points are compared, corresponding to 4 different specific growth rates, and hence, 4 different ratios of energy used for maintenance and growth. The samples taken at the start of retentostat cultivation serves as a a reference sample, to which the three other samples (taken after 3 days, 17 days, and 31 days under retentostat conditions) are compared. No biological replicates: all samples were taken from the same retentostat fermentation.

ORGANISM(S): Lactobacillus plantarum

SUBMITTER: Jeroen Hugenholtz

PROVIDER: E-GEOD-18340 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The present study aims to explore the role of Rim15 in both physiology and genome wide expression in S. cerevisiae under severe caloric restriction. Non-growing but metabolically active cultures of S. cerevisiae are of major interest for application in industry and as model systems for aging in higher eukaryotes. Using retentostat cultivations, almost non-growing but metabolic active cultures can be obtained resulting from the severe caloric restriction, yet not starvation, yeast experiences. Rim15 plays an important role in several nutrient sensing pathways and is involved in activating stress response and glycogen accumulation upon nutrient shortage. To investigate the role of Rim15 in the extreme robustness and glycogen accumulation of anaerobic retentostat cultures, a rim15 deletion strain is compared with its parental strain under anaerobic calorie restriction on both physiology and transcriptome. Rim15 is described as essential for G0 entry and glycogen accumulation in yeast during diauxic shift and stationary phase. Anaerobic retentostat cultures display many stationary phase characteristics, including increased expression levels of Rim15 target genes, suggesting an important role for Rim15 under these conditions. Comparing a rim15 deletion strain and its parental strain revealed both on transcriptome level and physiology indeed a major role in the acquired robustness, glycogen accumulation, but also maintenance of viability and cell cycle arrest. The severe caloric restriction, but not starving, conditions applied together with a thorough physiological and transcriptome analysis of the cultures shows that Rim15 is essential in fine-tuning cell cycle progression with glucose availability under extreme growth-limiting conditions. To investigate the impact of rim15 deletion under severe calorie restricted conditions, transcriptome of a S. cerevisiae rim15 deletion mutant was monitored during anaerobic retentostat cultivation. Five time points were used, one in the starting point of the retentostat (T0 = chemostat) and four during the course of the retentostat (time points 2, 9, 16 and 20 days). Culture triplicates were performed for T0 in chemostat, while two independent cultures were run for retentostat.

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.

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Anaerobic carbon-limited retentostat cultivation of Lactobacillus plantarum WCFS1

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