Defining Rational Photoelectron Routing for Targeted Intracellular Energy Transfer

ABSTRACT Microbial artificial photosynthesis offers a promising strategy for light‐driven biomanufacturing, yet its efficiency remains limited by the non‐selective conversion of photogenerated electrons into metabolically usable reducing power, causing energy dissipation and weak coupling between light capture and metabolic reactions. Here, we report a rational strategy using riboflavin (RF), a membrane‐permeable and biocompatible flavin photosensitizer, to selectively channel photonic energy into intracellular NADPH regeneration. Quantum chemical calculations and spectroscopic analyses reveal that light‐excited RF exhibits a specific binding affinity and favorable electron transfer trend toward NADP + . In vivo, RF activation markedly elevated intracellular NADPH levels and enhanced the synthesis of NADPH‐dependent metabolites through NADPH reductase‐associated pathways. Transcriptomic and inhibition analyses linked RF‐mediated NADPH regeneration to NADP + /NADPH redox enzymes rather than glucose‐6‐phosphate dehydrogenase‐mediated flux, while NADH‐related redox genes remained largely unaffected, demonstrating the selectivity of this reductive route. Cross‐species and multi‐product validations consistently reproduced these results, underscoring the generality of this mechanism across distinct NADPH‐dependent microbial chassis. This work establishes a mechanistically defined and broadly applicable framework for directing photogenerated electrons into specific cellular reducing equivalents, paving the way for efficient artificial photosynthetic and bioelectrochemical platforms.