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High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae.

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dc.contributor.author Takpho, Natthaporn en
dc.contributor.author Watanabe, Daisuke en
dc.contributor.author Takagi, Hiroshi en
dc.date.accessioned 2018-03-22T05:03:49Z en
dc.date.available 2018-03-22T05:03:49Z en
dc.date.issued 2018-03-19 en
dc.identifier.issn 1096-7176 en
dc.identifier.uri http://hdl.handle.net/10061/12216 en
dc.description.abstract Valine, which is one of the branched-chain amino acids (BCAAs) essential for humans, is widely used in animal feed, dietary supplements and pharmaceuticals. At the commercial level, valine is usually produced by bacterial fermentation from glucose. However, valine biosynthesis can also proceed in the yeast Saccharomyces cerevisiae, which is a useful microorganism in fermentation industry. In S. cerevisiae, valine biosynthesis is regulated by valine itself via the feedback inhibition of acetohydroxyacid synthase (AHAS), which consists of two subunits, the catalytic subunit Ilv2 and the regulatory subunit Ilv6. In this study, to improve the valine productivity of yeast cells, we constructed several variants of Ilv6 by introducing amino acid substitutions based on a protein sequence comparison with the AHAS regulatory subunit of E. coli. Among them, we found that the Asn86Ala, Gly89Asp and Asn104Ala variants resulted in approximately 4-fold higher intracellular valine contents compared with those in cells with the wild-type Ilv6. The computational analysis of Ilv6 predicted that Asn86, Gly89 and Asn104 are located in the vicinity of a valine-binding site, suggesting that amino acid substitutions at these positions induce conformational change of the valine-binding site. To test the effects of these variants on AHAS activity, both recombinant Ilv2 and Ilv6 were purified and reconstituted in vitro. The Ilv6 variants were much less sensitive to feedback inhibition by valine than the wild-type Ilv6. Only a portion of the amino acid changes identified in the E. coli AHAS regulatory subunit IlvH enhanced the valine synthesis, suggesting structural and/or functional differences between the S. cerevisiae and E. coli AHAS regulatory subunits. It should also be noted that these amino acid substitutions did not affect the intracellular pools of the other BCAAs, leucine and isoleucine. The approach described here could be a practical method for the development of industrial yeast strains with high-level production of valine or isobutanol. en
dc.language.iso en en
dc.publisher Elsevier en
dc.rights Copyright © 2018 Elsevier Inc. All rights reserved ja
dc.rights 出版社許諾条件により、本文は2019年3月19日以降に公開。 ja
dc.subject valine en
dc.subject yeast en
dc.subject acetohydroxyacid synthase en
dc.subject Ilv6 en
dc.subject feedback inhibition en
dc.subject branched-chain amino acids en
dc.title High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae. en
dc.type.nii Journal Article en
dc.contributor.transcription ワタナベ, ダイスケ ja
dc.contributor.transcription タカギ, ヒロシ ja
dc.contributor.alternative 渡邉, 大輔 ja
dc.contributor.alternative 高木, 博史 ja
dc.textversion author en
dc.identifier.jtitle Metabolic Engineering en
dc.identifier.volume 46 en
dc.identifier.issue March 2018 en
dc.identifier.spage 60 en
dc.identifier.epage 67 en
dc.relation.doi 10.1016/j.ymben.2018.02.011 en
dc.identifier.NAIST-ID 84365154 en
dc.identifier.NAIST-ID 74650151 en
dc.identifier.NAIST-ID 73290561 en
dc.relation.pmid 29477860 en

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