Engineering and design of artificial enzymes for non-natural biocatalysis in whole cells

Artificial enzymes represent transformative biocatalysts that overcome the inherent limitations of natural enzymes in substrate scope and activity specificity, enabling challenging new-to-nature reactions. Despite remarkable advances in designing diverse artificial enzymes through synthetic cofactor or non-canonical amino acid incorporation, their widespread industrial application remains hindered by the high costs and operational complexity of in vitro systems. Whole-cell biocatalysis emerges as a promising alternative by capitalizing on the cellular environment, including cofactor regeneration, multi-enzyme cascades, and enhanced enzyme stability. This review provides a comprehensive introduction to recent progress in artificial enzyme design and their successful applications in whole-cell biocatalysis. We summarize innovative protein engineering tools for creating artificial enzymes, assembly strategies to enhance in vivo catalytic performance, and representative applications of artificial enzymes-containing whole-cell systems in non-natural transformations. We also discuss the challenges and prospects of whole-cell artificial enzyme catalysis in advancing sustainable and scalable biomanufacturing.