Microbial Synthesis of Daphnetin by Engineering 2-Oxoglutarate-Dependent Dioxygenases.

Daphnetin is a clinically established coumarin, but its native biosynthetic pathway remains elusive. In this study, a 2-oxoglutarate-dependent dioxygenase (2OGD), AtS8H, was identified that hydroxylates umbelliferone (UMB) to daphnetin , enabling the design of an artificial biosynthetic pathway. However, the pathway efficiency was constrained by the poor solubility of AtS8H and the low catalytic activity of the upstream 2OGD enzyme IbC2'H. To enhance AtS8H solubility, we developed an integrated strategy combining ProteinMPNN-guided sequence redesign with surface charge engineering. The obtained quintuple mutant S8H 2-6 exhibited significantly improved solubility and a 7.2-fold increase in catalytic efficiency. For IbC2'H, we developed a fluorescence-based high-throughput screening method, and a quadruple mutant C6 was obtained by directed evolution, which displayed a 2.7-fold higher kcat and a 3.2 °C improvement in thermal stability. Implementing both engineered enzymes into an optimized Escherichia coli strain enabled the de novo production of daphnetin at a titer of 46 mg/L. This work reports, to the best of our knowledge, the first microbial de novo production of daphnetin from a simple carbon source and demonstrates an integrated enzyme engineering approach that synergistically refines biosynthetic pathways for efficient microbial production.