Enhanced L-isoleucine Production by Corynebacterium glutamicum YILW through Overexpression of ilvBNC and cimA
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Abstract:
L-isoleucine is one of the eight essential amino acids for humans and animals, and it plays a vital role in numerous life functions. α-Ketobutyrate is the key precursor of L-isoleucine, and acetohydroxy acid synthase (AHAS; encoded by ilvBN) is the key enzyme in L-isoleucine synthesis. Therefore, L-isoleucine production can be theoretically increased either by increasing the supply of α-ketobutyrate or ilvBN expression. Citramalate synthase, encoded by cimA, catalyzes the generation of α-ketobutyrate from pyruvate and acetyl-CoA. α-Ketobutyrate is a direct precursor of L-isoleucine; thus, increasing cimA expression increases the metabolic flux of L-isoleucine synthesis. In the present study, the promoter of the ilvBNC operon in the L-isoleucine-producing strain Corynebacterium glutamicum YILW was replaced by the potent tac promoter to construct a recombinant strain, C. glutamicum YILWPtac, which overexpresses ilvBNC. Shake flask fermentation experiments showed that L-isoleucine production and glucose (Yp/s) conversion in the recombinant strain were increased by 14.8% and 18.6%, respectively. To further increase L-isoleucine production and Yp/s, a construct containing the cimA gene under the control of a strong promoter was introduced into C. glutamicum YILWPtac to construct C. glutamicum YILWPtacpXMJ19cimA. Compared to the original strain, in C. glutamicum YILWPtacpXMJ19cimA, L-isoleucine production and Yp/s were increased by 14.5% and 42.4%, respectively. Thus, the findings of this study provide a basis for constructing new and improved amino acid-producing C. glutamicum strains.
