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

Abstract

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.

Uncoupling growth and succinic acid production in an industrial Saccharomyces cerevisiae strain

“…However, long-term cultivation of S. cerevisiae at the near-zero growth rate in retentostat cultures has been reported to result in accumulation of nonviable, nonproducing cells (Boender et al, 2009;Vos et al, 2016). A previous study with the nonproducing laboratory reference strain CEN.PK113-7D (Hakkaart et al, 2020) showed that, at pH of 3, the specific death rates (k d ) in glucose-and ammonium-limited retentostat cultures (0.0039 ± 0.0005 and 0.0030 ± 0.0004 h −1 , respectively) were approximately eightfold higher than in corresponding cultures grown at pH of 5. However, cultivation of the reference strain at near-zero growth rate as such did not result in an increased k d , because the death rates in glucoseand ammonium-limited retentostat cultures performed at pH of 3 were respectively two and tenfold lower than in corresponding F I G U R E 6 Intracellular levels of the TCA cycle metabolites (a-f) and mass action ratio's of fumarase and aconitase (g,h) during aerobic, ammonium-limited retentostat cultivation of SUC632.…”

“…Data represent the averages and standard errors of measurements from duplicate cultures. αKG, α-ketoglutarate; Cit, citrate; Fum, fumarate; IsoCit, isocitrate; Mal, Malate; Suc, succinic acid; TCA, tricarboxylic acid [Color figure can be viewed at wileyonlinelibrary.com] chemostat cultures grown at a specific growth rate of 0.025 h −1 at pH of 3 (Hakkaart et al, 2020).…”

“…However, chemostat cultivation in laboratory bioreactors is impractical at specific growth rates below 0.025 h −1 , due to the long time periods needed to reach a steady‐state. As an alternative to chemostat cultivation, retentostat cultures have proven to be excellent tools to study the physiology of S. cerevisiae and other microorganisms at low to near‐zero growth rates under various nutrient limitations (Boender et al, 2009; Ercan et al, 2015; Hakkaart et al, 2020; Liu et al, 2019; Vos et al, 2016).…”

“…The strain was grown under industrially relevant conditions, that is, at an elevated CO 2 level to increase the driving force towards SA biosynthesis via the reductive pathway and a culture pH of 3.0 to facilitate downstream processing (Hakkaart et al, 2020).…”

Engineering Saccharomyces cerevisiae for Succinic Acid Production From Glycerol and Carbon Dioxide

“…The biomass concentrations were determined by filtering duplicate, exact volumes of culture broth (5 mL), over pre-dried Supor 47 membrane filters with a 0.45 μm pore size, washed and then dried in a microwave oven for 20 min at 360 W and weighed again (Pall Laboratory, Port Washington, NY, United States) as described by Postma et al (1989) . Extracellular metabolites of the cultures were obtained by centrifuging culture samples (10 min at 3500 g) of which the supernatant was analyzed by HPLC as described previously by Hakkaart et al (2020) .…”