Analysis of α-isopropylmalate synthase variant and its effect on L-leucine synthesis in Corynebacterium glutamicum

Abstract

Purpose: L-Leucine is an essential amino acid for humans since it is only synthesized in fungi, bacteria, and plants. Its production has not been well documented as L-lysine and L-glutamic acid due to multivalent regulation and longer synthesis pathways. In Corynebacterium glutamicum, L-leucine synthesis is strongly regulated at α-isopropylmalate synthase (α-IMPS) node by the end product. Amino acid substitutions of the α-IMPS variant from strain ARTP-L04 were analyzed, and the α-IMPS variant was further characterized. To enhance the L-leucine production, the effect of α-IMPS variant was also evaluated.Methods: In the present study, construction of plasmid and recombination strain, protein expression and purification, enzymatic properties, fermentation, and amino acid analysis were performed.Results: Compared with leuA from wild-type strain ATCC 13032, 10 nucleotide exchanges were identified in the α-IMPS variant, leading to amino acid exchanges of Gly92Asp, Ile162Val, Gln494Arg, and Gly526Asp. Furthermore, the α-IMPS variant was expressed and characterized. It was found that the α-IPMS variant exhibited higher resistance to feedback inhibition by L-leucine. In the presence of 20 mM L-leucine, over 50% of α-IPMS variant activity was still observed. However, there was no significant difference in specific activity between the α-IPMS variant and wild-type α-IPMS. Therefore, the α-IPMS variant almost removed inhibition by L-leucine. Interestingly, co-expression of α-IPMS and AHAS both from strain ARTP-L04 could significantly enhance L-leucine production, accumulating 7.79 g/L L-leucine.Conclusions: Mutation that occurred in the α-IPMS variant played an important role in feedback resistance, almost removing feedback inhibition by L-leucine. In C. glutamicum, the expression of the α-IPMS variant could significantly enhance L-leucine production, especially when it was co-expressed with AHAS. Collectively, our findings provided valuable insights into further development in gene engineering.