Abstract:Objective This study modified the S-adenosylmethionine (SAM)-dependent methyltransferase to relieve the feedback inhibition of the product L-hypaphorine on this enzyme in Escherichia coli (E. coli), thereby enhancing L-hypaphorine production.Method E. coli BL21(DE3) was used as the chassis strain, and the expression of the SAM-dependent methyltransferase EgtDM252V, E282A (abbreviated as MsE, the encoding gene is egtD) was exogenously induced. The induction temperature and inducer concentration were optimized to enhance the soluble expression. It was observed that the enzyme activity suffered feedback inhibition when L-hypaphorine accumulated to a threshold mass concentration, which led to reduced catalytic efficiency. To address this problem, a protein engineering strategy was employed. Using Schrodinger (a molecular docking software) and PyMOL (a structural analysis software), the substrate-binding pocket and channels of MsE were simulated to screen potential mutation sites and perform alanine scanning.Result Glutamate-to-alanine mutation at position 143 (E143A) significantly attenuated product inhibition and enhanced the enzyme's catalytic capacity toward L-tryptophan. Under supplementation with 100 mg/L L-hypaphorine, the mutant MsEE143A exhibited markedly reduced inhibition. Consequently, the L-hypaphorine yield of the recombinant strain E. coli BL21(DE3)/pET-MsEE143A reached (10.85±0.26) mg/L, representing a 175% increase compared to that of the parental strain.Conclusion This study successfully engineered MsE through semi-rational design, not only resolving the feedback inhibition but also providing an effective strategy for enhancing L-hypaphorine biosynthesis via protein engineering.
