Analysis of acetohydroxyacid synthase variants from branched-chain amino acids-producing strains and their effects on the synthesis of branched-chain amino acids in Corynebacterium glutamicum

Abstract

Acetohydroxy acid synthase (AHAS) controls carbon flux through the branch point and determines the relative rates of the synthesis of isoleucine, valine and leucine, respectively. However, it is strongly regulated by its end products. In this study, we characterized AHAS variants from five branched-chain amino acids-producing strains. Amino acid substitution occurred in both catalytic subunit and regulatory subunit. Interestingly, AHAS variants reduced sensitivity to feedback inhibition by branched-chain amino acids (BCAAs). Although AHAS with amino acid substitution in regulatory subunit showed higher resistance, amino acid substitution in catalytic subunit could also endow AHAS with resistance to feedback inhibition. In addition, AHAS variants from V2 and L5 displayed about 1.4-fold higher specific activity compared to other AHAS variants. On the other hand, AHAS variant from V1 exhibited the highest resistance to BCAAs, 87% of original activity left even in the presence of 10mM BCAAs. Recombinant Corynebacteriumglutamicum strains were further constructed to investigate the effects of expressing AHAS variants on the synthesis of BCAAs and alanine (main by-product) in C. glutamicum. BCAAs production was increased with the increase of resistance to feedback inhibition, although valine showed a significant increase. For instance, C. g-1BN could accumulate 9.51g/l valine, 0.450g/l leucine and 0.180g/l isoleucine, and alanine was reduced to 0.477g/l. These AHAS variants are important for further improving performance of BCAAs-producing strain.