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%.