Article ID Journal Published Year Pages File Type
31569 Metabolic Engineering 2012 9 Pages PDF
Abstract

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7 g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.

► Mutant TD and AHAS from JHI3-156 were characterized. ► Mutant TD was 100% resistant to l-isoleucine inhibition. ► The activity of mutant TD was enhanced in the presence of l-isoleucine. ► Overexpression of mutant TD or AHAS increased l-isoleucine production. ► l-isoleucine could reach 30.7 g/L when mutant TD and AHAS co-expressed.

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Physical Sciences and Engineering Chemical Engineering Bioengineering
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