Maintenance-energy requirements and robustness of Saccharomyces cerevisiae at aerobic near-zero specific growth rates

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.

Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels

“…Physiological characterization of S. cerevisiae CEN.PK113‐7D in duplicate glucose‐limited, aerobic retentostat cultures, grown at pH 3 and 50% CO 2 (left column) and in quadruplicate cultures grown under reference conditions (pH 5, 0.04% CO 2 ; Vos et al, ). (a, b) Glucose concentration in influent during retentostat cultivation.…”

“…Time‐dependent regression analysis of substrate and product concentrations was previously shown to enable accurate estimates of specific growth rate, specific substrate‐consumption rate, first‐order death rate and m S in carbon‐ and energy‐limited yeast retentostat cultures (Vos et al, ). In contrast to growth under standard laboratory conditions, growth under industrially relevant conditions caused a strong decrease of the viable biomass concentration after the first 10 days of cultivation, which prevented use of regression analysis for data obtained beyond Day 10 (Figures c and g).…”

“…White bars: pH 5, gray bars: pH 3 (and 50% CO 2 for glucose‐limited cultures). Data for glucose‐limited cultures grown at pH 5 are from Vos et al (), data for ammonium‐limited cultivation at pH 5 are from Liu et al ()…”

“…For glucose‐limited chemostats, CFU counts were obtained by sorting 96 single events detected by a FACS Aria™ II SORP Cell Sorter (BD Biosciences, Franklin Lakes, NJ) on a YPD plate (in quintuplicate, see Supporting Information Appendix 1 for details). To measure viability based on membrane integrity, cells were stained with the fluorescent dye propidium iodide (PI; Vos et al, ). Staining of single‐cell esterase activity with 5‐CFDA‐AM was used to evaluate metabolic activity (Bisschops et al, ).…”

“…Despite its industrial relevance, quantitative understanding of maintenance‐related processes in S. cerevisiae and their sensitivity to industrially relevant process conditions is far from complete. Previous studies showed that, while growth‐rate independent (Boender, de Hulster, van Maris, Daran‐Lapujade, & Pronk, ; Vos et al, ), the maintenance‐energy requirement ( m S ; mmol glucose/g biomass/h) of S. cerevisiae can be affected by the cultivation conditions (Lahtvee, Kumar, Hallstrom, & Nielsen, ; Liu, el Bouhaddani, Pronk, & van Gulik, ; Vos et al, ). For example, growth at pH 2.5 substantially reduces the maximum specific growth rate in batch cultures (Carmelo et al, ; Della‐Bianca & Gombert, ; Della‐Bianca et al, ; Orij et al, ) and increases activity of the plasma‐membrane proton pumps, suggesting that low pH also affects m S (Carmelo et al, ; Eraso & Gancedo, ).…”