Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

Abstract:

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter P_gpmA* from six candidate promoters for expressing ilvBN. P_gpmA* showcased the expression strength 23.3-fold that of the native promoter P_ilvBN. Moreover, three synthetic RBS libraries based on the promoter P_gpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that “R₍₉₎N₍₆₎” was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of P_ilvBN. Finally, the promoter P_gpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBN^S155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.