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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 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.

SUBMITTER: Hakkaart X

PROVIDER: S-EPMC7028085 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Hakkaart Xavier X Liu Yaya Y Hulst Mandy M El Masoudi Anissa A Peuscher Eveline E Pronk Jack J van Gulik Walter W Daran-Lapujade Pascale P

Biotechnology and bioengineering 20200122 3

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.P ...[more]

PMID: 31654410

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Project description:Saccharomyces cerevisiae metabolism produces ethanol and other compounds during the fermentation of grape must into wine. Thousands of genes change expression over the course of a wine fermentation, allowing S. cerevisiae to adapt to and dominate the fermentation environment. Investigations into these gene expression patterns previously revealed genes that underlie cellular adaptation to the grape must and wine environments, involving metabolic specialization and ethanol tolerance. However, the majority of studies detailing gene expression patterns have occurred in controlled environments that may not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, an analysis of the S. cerevisiae RC212 gene expression program is presented, drawing from 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. A core gene expression program was observed across all fermentations irrespective of vintage, similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-Saccharomyces microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into Saccharomyces cerevisiae biology critical to fermentation outcomes under industry-relevant conditions.IMPORTANCE This study characterized Saccharomyces cerevisiae RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using industry-relevant conditions. The reported gene expression patterns of RC212 are generally similar to those observed under laboratory fermentation conditions but also contain gene expression signatures related to yeast-environment interactions found in a production setting (e.g., the presence of non-Saccharomyces microorganisms). Key genes and pathways highlighted by this work remain undercharacterized, indicating the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.

Project description:BackgroundInterspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity.ResultsHere we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn't produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose.ConclusionsOur study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.

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

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Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO<sub>2</sub> levels.

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