Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCA
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast Jirasin Koonthongkaew 1 & Yoichi Toyokawa 1 & Masataka Ohashi 2 & Christopher R. L. Large 3 & Maitreya J. Dunham 3 & Hiroshi Takagi 1 Received: 27 April 2020 / Revised: 17 July 2020 / Accepted: 27 July 2020 / Published online: 10 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 bat1Δ 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. Keywords Yeast . Saccharomyces cerevisiae . Mitochondrial branched-chain amino acid aminotransferase Bat1 . Branched-chain amino acids . Fusel alcohols . Beer brewing
Introduction Jirasin Koonthongkaew and Yoichi Toyokawa contributed equally to this work. * Hiroshi Takagi [email protected] 1
Division of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
2
Nara Prefecture Institute of Industrial Development, 129-1 Kashiwagi-cho, Nara, Nara 630-8031, Japan
3
Department of Genome Sciences, University of Washington, 3720 15th Ave NE, Seattle, WA 98195, USA
Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are essential and functional in humans (Mori et al. 1988; Hiroshige et al. 2001; Calder 2006; Howatson et al. 2012), leading to human diseases (Yamada et al. 2014; Wang et al. 2015; Manoli and Venditti 2016) or abnormal phenotypes in mice (She et al. 2007;
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