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KMID : 1094720140190030456
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Biotechnology and Bioprocess Engineering
2014 Volume.19 No. 3 p.456 ~ p.467
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Generation of branched-chain amino acids resistant Corynebacterium glutamicum acetohydroxy acid synthase by site-directed mutagenesis
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Guo Yanfeng
Han Mei
Yan Weiliu
Xu Jianzhong
Zhang Weiguo
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Abstract
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In Corynebacterium glutamicum, acetohydroxy acid synthase (AHAS, encoded by ilvBN) is regulated by the end products in biosynthesis pathway, which catalyzes the first common reaction in the biosynthesis of branched-chain amino acids (BCAAs). In this study, conserved A42, A89 and K136 residues in AHAS regulatory subunit were chosen for site-directed mutagenesis, and the resulting mutations A42V, A89V and K136E exhibited higher resistance to inhibition by BCAAs than wild type AHAS. Furthermore, double-mutation was carried out on A42V, A89V and K136E mutations. Expectedly, A42V-A89V mutation exhibited nearly complete resistance to inhibition by all three BCAAs, which retained above 93% enzyme activity even at 10 mM. Strains were further studied to investigate the effects of over-expressing different mutant ilvBN on the biosynthesis of BCAAs. It was found that production of BCAAs was increased with the increase of resistance to BCAAs. However, the increase of isoleucine and leucine was slower than valine which showed a significant increase (up to 86.30 mM). Furthermore, strains harboring plasmids with different mutant ilvBN could significantly decrease production of alanine (main byproduct). This work gives additional understanding of roles of A42, A89 and K136 residues and makes the A42V, A89V, K136E and A42V-A89V mutations a good starting point for further development by protein engineering.
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KEYWORD
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acetohydroxy acid synthase, site-directed mutagenesis, branched-chain amino acids, resistance, residue
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