title: Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum.
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 30.7g/L L-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance L-isoleucine production in C. glutamicum.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Lianghong
authors: L
authors: Yin
authors: Xiaoqing
authors: X
authors: Hu
authors: Daqing
authors: D
authors: Xu
authors: Jianfei
authors: J
authors: Ning
authors: Jian
authors: J
authors: Chen
authors: Xiaoyuan
authors: X
authors: Wang
mesh_terms: D000095:Acetolactate Synthase; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007532:Isoleucine / Q000096:biosynthesis / Q000235:genetics; D020125:Mutation, Missense; D013913:Threonine Dehydratase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D007532:Isoleucine; D000095:Acetolactate Synthase; D013913:Threonine Dehydratase