Yuhui Sheng, Yi Fu, Zhi Zhou
Significant progress has been made in the design of artificial enzymes using noncanonical amino acids; however, the field still faces considerable challenges. In this study, we present a novel approach for the design of an artificial enzyme capable of catalyzing a tandem Friedel–Crafts alkylation–enantioselective protonation reaction by incorporating in situ-biosynthesized noncanonical amino acids (ncAAs). By using a system of ncAA biosynthesis and genetic incorporation in Escherichia coli, the designer enzyme with a biosynthesized unnatural S -(3-aminophenyl)- l -cysteine (mAPhC) was constructed and promoted the tandem Friedel–Crafts alkylation–enantioselective protonation reaction with excellent enantioselectivity and reactivity after directed evolution. The developed system demonstrates the power of in situ ncAA biosynthesis in creating highly specialized biocatalysts with tailored functions. This work provides valuable insights into the potential of ncAAs to expand the catalytic versatility of artificial enzymes for new-to-nature reactions.