Harnessing photoenzymatic reactions for unnatural biosynthesis in microorganisms

Photoenzymatic catalysis enables new-to-nature transformations, but its scalability is limited by high enzyme loading, costly cofactors, and radical-induced instability. Here we report the integration of light-driven photoenzymatic reactions into the cellular metabolism of Escherichia coli, bridging flavin-based photobiocatalysis with biosynthesis. Using synthetic biology strategies, we engineered microbial cells to continuously produce olefin substrates and ene-reductase photoenzyme while regenerating cofactors directly from glucose. By externally supplying radical precursors or by introducing synthetic pathways for their in situ production, we enabled fermentation-based microbial photobiosynthesis, achieving high titers and demonstrating its feasibility for scale-up in bioreactor. This approach extends photobiocatalysis from in vitro applications to in vivo semi-biosynthesis and complete biosynthesis, revealing its full potential for integrating light-driven reactions into cellular me..., , # Harnessing photoenzymatic reactions for unnatural biosynthesis in microorganisms

Dataset DOI: [10.5061/dryad.ns1rn8q54](10.5061/dryad.ns1rn8q54)

## Description of the data and file structure

This dataset supports the research published in “Harnessing Photoenzymatic Reactions for Unnatural Biosynthesis in Microorganisms”. It includes all the necessary SnapGene files for the plasmids described in the article and the Supplementary Information. All files are provided in SnapGene (.dna) format and can be opened and edited using SnapGene or SnapGene Viewer.

OYE family enzymes (Old Yellow Enzymes / ene-reductases)

Auxiliary NADPH-regeneration systems

Promoter comparisons (T7, Trc, lac)

4-vinylphenol and styrene biosynthetic pathway construction

Photobiocatalysis pathways (using photoenzymes + NADPH regeneration)

Whole-cell screening of OYE mutants

**pACYC_receiver.dna**

* A p15A-origin backbone is used as a general template for constructing new plasmids.

**pML43_(pET30a(_)-vector...,