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

  • Applied genetics and molecular biotechnology
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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 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.

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Funding

This work was supported by a Grant-in-Aid for Research Activity start-up (19K21144) from the Japan Society for the Promotion of Science (JSPS) to YT and a grant from the Project of the NARO Bio-oriented Technology Research Advancement Institution (Research program on development of innovative technology) (30017B) to HT. CL was supported by NIH grant T32 HG00035. MD and CL were supported by the National Science Foundation under grant 1516330 and under Cooperative Agreement No. DBI-0939454. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The research of MD was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute.

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MD and HT conceived the study and designed the experiments. JK performed the experiments. JK, YT, MO, CL, and HT analyzed the data. JK, YT, MO, and HT wrote the manuscript. All authors reviewed and approved the final version of manuscript.

Corresponding author

Correspondence to Hiroshi Takagi.

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The authors declare that there is no conflict of interest.

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This article does not contain any studies with human participants performed by any of the authors.

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Koonthongkaew, J., Toyokawa, Y., Ohashi, M. et al. Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast. Appl Microbiol Biotechnol 104, 7915–7925 (2020). https://doi.org/10.1007/s00253-020-10800-y

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  • DOI: https://doi.org/10.1007/s00253-020-10800-y

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