Abstract
During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex–mediated degradation. Next, we investigated the effects of GID protein–coding genes (GID1–GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle–related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation.
Key Points
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An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2.
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The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production.
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The protein expression profiles in the GID disruptants differed from one another.
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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Hiroaki Negoro and Fumio Matsuda. The first draft of the manuscript was written by Hiroaki Negoro and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Negoro, H., Matsumura, K., Matsuda, F. et al. Effects of mutations of GID protein–coding genes on malate production and enzyme expression profiles in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 104, 4971–4983 (2020). https://doi.org/10.1007/s00253-020-10573-4
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DOI: https://doi.org/10.1007/s00253-020-10573-4