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 CO2. 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 CO2 (50%) on slow‐growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113‐7D. Combined exposure to low pH and high CO2 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 CO2 on physiology, high CO2 strongly affected genome‐wide transcriptional responses to low pH. Interference of high CO2 with low‐pH signaling is consistent with low‐pH and high‐CO2 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.
Highlights
Dicarboxylic acids are attractive platform molecules for production of a wide range of chemicals (Becker, Lange, Fabarius, & Wittmann, 2015).High‐yield microbial conversion of glucose to dicarboxylic acids can be achieved through the reductive branch of the TCA cycle and requires elevated concentrations of dissolved carbon dioxide (CO2) to promote carboxylation of pyruvate or phosphoenolpyruvate to oxaloacetate (Ahn, Jang, & Lee, 2016; Yin et al, 2015; Zelle, de Hulster, Kloezen, Pronk, & van Maris, 2010)
S. cerevisiae grows at high CO2, reduced biomass yields have been reported for respiring S. cerevisiae cultures grown at CO2 values of 50% and 79% (Aguilera et al, 2005; Eigenstetter & Takors, 2017; Richard et al, 2014)
Growth at pH 3 led to a significantly lower biomass yield than at pH 5, both at standard and at elevated CO2 Levels (7.4% and 9.7% decrease, respectively; 0.419 ± 0.009 gx/gs vs 0.388 ± 0.005 gx/gs; p < .001 for pH 5 vs pH 3 when sparged with compressed air and 0.411 ± 0.006 gx/gs vs 0.371 ± 0.004 gx/gs; p < .02 for pH 5 vs pH 3 at 50% CO2). These results showed that the higher ms in retentostat cultures grown at high CO2 and low pH resulted from the low pH rather from the high CO2
Summary
Dicarboxylic acids are attractive platform molecules for production of a wide range of chemicals (Becker, Lange, Fabarius, & Wittmann, 2015). S. cerevisiae can grow at pH values as low as pH 2.5, but only at significantly reduced specific growth rates (Carmelo, Bogaerts, & Sá‐Correia, 1996; Della‐Bianca & Gombert, 2013; Della‐Bianca, de Hulster, Pronk, van Maris, & Gombert, 2014; Eraso & Gancedo, 1987; Orij, Postmus, Beek, Brul, & Smits, 2009) Heterotrophic microorganisms dissimilate their carbon and energy substrate to supply ATP for biomass formation and for cellular maintenance (Pirt, 1965, 1982). Elevated CO2 and low pH are relevant industrial process conditions for dicarboxylic‐acid production and have both been reported to adversely affect yeast physiology, their effects on maintenance‐energy requirements and viability of slow growing S. cerevisiae cultures have not yet been quantitatively analyzed To address this knowledge gap, a nonproducing S. cerevisiae laboratory strain was grown at low and near‐zero specific growth rates using a combination of glucose‐limited chemostat and retentostat cultures, at a low pH (pH 3) and elevated CO2 concentrations (50% CO2). Transcriptome analysis was employed to elucidate regulatory responses to these conditions
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