Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast.

Jirasin Koonthongkaew, Yoichi Toyokawa, Masataka Ohashi, Christopher R L Large, Maitreya J Dunham, Hiroshi Takagi

Applied microbiology and biotechnology 2020 Sep

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In the yeast Saccharomyces cerevisiae, the mitochondrial branched-chain amino acid (BCAA) aminotransferase Bat1 plays an important role in the synthesis of BCAAs (valine, leucine, and isoleucine). Our upcoming study (Large et al. bioRχiv. 10.1101/2020.06.26.166157, Large et al. 2020) will show that the heterozygous tetraploid beer yeast strain, Wyeast 1056, which natively has a variant causing one amino acid substitution of Ala234Asp in Bat1 on one of the four chromosomes, produced higher levels of BCAA-derived fusel alcohols in the brewer's wort medium than a derived strain lacking this mutation. Here, we investigated the physiological role of the A234D variant Bat1 in S. cerevisiae. Both bat1∆ and bat1A234D cells exhibited the same phenotypes relative to the wild-type Bat1 strain-namely, a repressive growth rate in the logarithmic phase; decreases in intracellular valine and leucine content in the logarithmic and stationary growth phases, respectively; an increase in fusel alcohol content in culture medium; and a decrease in the carbon dioxide productivity. These results indicate that amino acid change from Ala to Asp at position 234 led to a functional impairment of Bat1, although homology modeling suggests that Asp234 in the variant Bat1 did not inhibit enzymatic activity directly. KEY POINTS: • Yeast cells expressing Bat1A234D exhibited a slower growth phenotype. • The Val and Leu levels were decreased in yeast cells expressing Bat1A234D. • The A234D substitution causes a loss-of-function in Bat1. • The A234D substitution in Bat1 increased fusel alcohol production in yeast cells.

Citation

Jirasin Koonthongkaew, Yoichi Toyokawa, Masataka Ohashi, Christopher R L Large, Maitreya J Dunham, Hiroshi Takagi. Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast. Applied microbiology and biotechnology. 2020 Sep;104(18):7915-7925