Construction of a high-performance universal catalyst
To obtain a high-performance universal catalyst, W1 and W2 were combined into variant W3 (A variant that combines W1 and W2 mutation sites). However, contrary to expectations, the catalytic efficiency of W3 toward L-Leu, L-Met, L-Val, L-Phe, L-Arg, and L-Glu decreased by 79.7%, 60.2%, 50.4%, 40.3%, 15.6%, and 5.8%, respectively (Figure 4A). To address this drawback, a one-pot two-enzyme cascade strategy was applied, whereby W1 and W2 were co-expressed in E. coli (Figure 6). The newly constructed S1 strain benefited from the advantages provided by both W1 and W2, and exhibited better catalytic performance than Pmi LAAD. Specifically, conversion of L-Val, L-Arg, and L-Glu was increased by 20.8%, 131.3%, and 487.4%, respectively, compared to the wild-type enzyme; whereas conversion of L-Phe and L-Met was only 15.3% and 10.7% higher (Figure 6H). The catalytic activity of W2, which has a preference for L-Phe and L-Met, constituted the limiting factor and required enhancement to improve overall efficiency of the whole-cell catalyst. To this end, the level of W2 was optimized by expressing two to four copies of W2 behind ribosome binding site (RBS) connectors. Among the newly generated strains S2, S3, and S4, respectively (Figure 6D–6G), S3 exhibited the best transformation performance, with >90% conversion toward all of the six selected substrates.
The substrate scope of S3 was analyzed for 13 different amino acids (100 g/L). Conversion of L-Met L-Val, L-Phe, L-Arg, L-Asp, and L-Glu was improved by 20.4%, 26.3%, 36.6%, 62.2%, 63.3%, and 81.5% compared with the wild type; whereas conversion of L-Trp, L-Tyr, L-Ala, L-Ser, and L-Thr was increased by 23.0%, 21.5%, 20.7%, 5.9%, and 5.6%, respectively (Figure 7). Conversion of L-Leu and L-Ile remained >99%.