So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h−1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals. IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

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 CO 2 . 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 CO 2 (50%) on slow-growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113-7D. Combined exposure to low pH and high CO 2 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 CO 2 on physiology, high CO 2 strongly affected genome-wide transcriptional responses to low pH. Interference of high CO 2 with low-pH signaling is consistent with low-pH and high-CO 2 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.

The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple nontargeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same setup yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

excess 12 13 14 15 16 17 18 19 20 21 a Current address: Royal Haskoning DHV, George Hintzenweg 85, 3009 AM Rotterdam, The Netherlands.prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which 46 growth is restricted by the limited supply of a non-energy substrate could therefore have a 47 wide range of industrial applications, but remains largely unexplored. In this work we 48 accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy 49 nutrient, namely the nitrogen or phosphorus source and carried out a quantitative 50 physiology study of the cells under these conditions. The possibility to achieve near-zero-51 growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer 52 interesting prospects to develop novel fermentation processes for high-yield production of 53 bio-based chemicals. 54 Growth and viability in ammonium-and phosphate-limited retentostat cultures130 Retentostat cultures were started by redirecting the effluent of steady-state ammonium-or 131 phosphate-limited chemostat cultures, grown at a dilution rate of 0.025 h -1 , through a 132 membrane filter unit placed inside the reactor (see Materials and Methods). Replicate 133 ammonium-limited retentostats were operated for 220 h with full biomass retention, after 134 which fouling caused the membrane filters to clog. Membrane fouling was not observed in 135 the phosphate-limited retentostats, which were operated with full biomass retention until, 136 after 400 h, the biomass concentration had reached a stable value. 137