Photoenzymatic Enantioselective [2+2] Cycloaddition of Alkynes Enabled by Whole-Cell Directed Evolution

Abstract Enantioselective excited-state photocycloadditions represent an attractive yet challenging strategy for accessing optically active strained molecules. Here, we report a whole-cell directed evolution platform for engineering artificial triplet photoenzymes that catalyze the enantioselective [2 + 2] photocycloaddition of alkynes. By employing calcium chloride-mediated membrane permeabilization, we overcome substrate accessibility limitations in whole-cell systems and enable high-throughput evolution without the need for cell lysates or purified enzymes. Through ten rounds of directed evolution, a primitive ancestor (APC1.0; 46% yield, 6% ee) was transformed into a highly efficient variant (APC10.0; 92% yield, 94% ee under whole-cell conditions; 99% yield, 97% ee as the purified enzyme). The enhanced activity of APC10.0 enabled a total turnover number of 1800. This work establishes a generalizable strategy for the evolution of artificial photoenzymes and expands the scope of biocatalysis toward stereoselective excited-state transformations.