Generation of mutant threonine dehydratase and its effects on isoleucine synthesis in Corynebacterium glutamicum

Isoleucine synthesis is strongly regulated by its end product (isoleucine) in Corynebacterium glutamicum, especially at threonine dehydratase (TD) node. Multiple alignments of TD sequences of C.glutamicum and other sources were performed. According to the structural analysis, three TD variants were constructed by site-directed mutagenesis. These TD variants improved the performance of the holoenzyme. The specific activity of V140M variant was 1.5-fold higher than that of the wild-type TD, whereas F383A variant showed complete resistance to feedback inhibition by isoleucine. V140M-F383A variant had all the advantages of V140M and F383A variants and displayed 1.5-fold specific activity and complete resistance to isoleucine. In C. glutamicum, overexpression of V140M, F383A, and V140M-F383A variants accumulated 0.55, 0.63, and 0.73 g/l isoleucine, and overexpression of wild-type TD produced 0.47 g/l isoleucine. Thus, these novel TD variants, particularly V140M-F383A, showed great potential in isoleucine synthesis.

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