Abstract:Acetohydroxy acid synthase (AHAS, encoded by the gene ilvBN) is the first rate-limiting enzyme in L--leucine synthesis pathway. Corynebacterium glutamicum XL-3 was used as the chassis cell to increase its preference for pyruvate and improve the yield of L--leucine by analyzing and modifying AHAS. Initially, the amino acid sequence of AHAS was used for homologous modeling. Then potential mutation sites were identified through alanine scanning of the protein structure. The optimal mutant was selected by measuring the catalytic activity of the mutant and theL--leucine yield of the recombinant strain. The results showed that substituting Gln157 with Arg effectively enhanced the ability of AHAS to catalyze pyruvate, resulting in a final L--leucine production of (23.5±1.8) g/L in the recombinant strain, which was a 51% increase compared to the parent strain Corynebacterium glutamicum XL-3. In addition, the yield of by-products L--isoleucine decreased. Therefore, the rational modification of AHAS could promote the synthesis of L--leucine. The research results have important implications for the subsequent use of protein engineering to strengthen the microbial synthesis of branched-chain amino acids such as L--leucine.
