Effects of BAT Genetic Modification on the Yield of Higher Alcohols from Saccharomyces cerevisiae
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Abstract:
Branched-chain amino acid transaminases are encoded by BAT genes in Saccharomyces cerevisiae, and BAT1 and BAT2 encode mitochondrial and cytosolic branched-chain amino acid transaminases, respectively. Owing to their different locations in cells, BAT1 and BAT2 have different physiological functions. BAT1 in the mitochondria has a tendency to carry out the catalytic synthesis of amino acids from α-keto acids, and BAT2 in the cytoplasm is responsible for converting amino acids into α-keto acids. This study attempted to reduce the synthesis of α-keto acids and increase the consumption of α-keto acids in S. cerevisiae by knocking out BAT2 and overexpressing BAT1, in order to decrease the level of higher alcohols in wine. Saccharomyces cerevisiae AY15 haploid α5 was used as the parental strain in this study, and fusion polymerase chain reaction (PCR) was adopted to construct the recombinant plasmid pUC-BABPB1K, and the recombinant gene cassette BA-PGK-BAT1-BB was obtained. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain B-8 overexpressing BAT1 and lacking BAT2. B-8, parental strain α5, and strain α5ΔBAT2 lacking BAT2 were used to conduct ethanol fermentation, and the fermentation performance and yield of higher alcohols were determined at the end of fermentation. The yields of isobutanol, isopentanol, and 2-methyl-1-butanol decreased by 25%, 15%, and 30%, respectively, compared with those of parental haploid α5, while the production of these higher alcohols were 0.7, 0.1, and 0.3 fold higher than those from the α5ΔBAT2 mutant, respectively.
