Integrating microbial cell factory with new-to-nature photobiocatalysis for de novo biosynthesis of D-homotryptophan.

Microbial cell factory is a powerful biological tool for synthesizing value-added molecules due to its unmatched sustainability and selectivity. However, the inherent catalytic specificity of natural enzymes limits product diversity. Although photobiocatalysis has expanded enzyme catalytic capabilities, challenges such as laborious enzyme purification, incompatibility with in vivo conditions, and complex unnatural substrate synthesis hinder photobiomanufacturing efforts. Herein, we combine a microbial cell factory with a new-to-nature photobiocatalytic transformation via a modular design that combines aerobic fermentation (Module I) and anaerobic photocatalysis (Module II) to achieve de novo biosynthesis of D-homotryptophan. In Module I, we engineer Escherichia coli equipped with a biosynthetic gene cluster to produce indole-3-acetic acid (IAA) via modifying metabolic flux and multi-copy genetic amplification strategies. In Module II, we develop an efficient synergistic photoredox/enzymatic synthesis of D-homotryptophan from L-serine and IAA by a pyridoxal phosphate (PLP)-dependent tryptophan synthase beta-subunit (TrpB) variant. This work synergistically merges emerging photobiocatalytic reactivity with natural biosynthesis, demonstrating a platform with potential for future biomanufacturing of non-natural products.