Fig. 4: Improving the catalytic efficiency of the OMT-CGT module by semirational design and codon optimization.
From: Unravelling and reconstructing the biosynthetic pathway of bergenin
a Protein modeling of AjOMT2 and AjCGT1. The protein structures were modeled using CCoAOMT (5KVA) and SbCGTa (6LG0) as templates, respectively. GA and GA-Glc were docked into AjOMT2, while GA and 4-OMGA were docked into AjCGT1 using AutoDock Vina. The candidate sites, acceptors (GA, GA-Glc, and 4-OMGA), and donors (SAM and UDP-Glc) were shown in blue, yellow, and green, respectively. b, c The in vivo relative catalytic activity of AjOMT2 mutants of alanine scanning and site-saturated mutagenesis of the Y203 site. d The in vitro relative activity of AjCGT1 mutants of alanine scanning. e The in vivo relative activity of AjCGT1 mutants obtained by Hot-spot analysis. f Engineered strains of E. coli harboring different combinations (M1–M3) of AjOMT2, AjOMT2* and AjCGT1, AjCGT1*. g The relative catalytic activity of the engineered strains of M1–M3. h Engineered strains of E. coli harboring different combinations (Y1–Y5) of AjOMT2, AjOMT2opt, AjOMT2*opt and AjCGT1, AjCGT1opt, AjCGT1*opt. i The relative catalytic activity of the engineered strains of Y1–Y5; AjOMT2*, AjOMT2-Y203T; AjCGT1*, AjCGT1-A332F; AjOMT2opt, codon-optimized AjOMT2; AjOMT2*opt, codon-optimized AjOMT2-Y203T; AjCGT1opt, codon-optimized AjCGT1; AjCGT1*opt, codon-optimized AjCGT1-A332F; All data represent the means of three parallel experiments and error bars show standard deviation. Source data are provided as a Source data file.
