De novo biosynthesis of 4,6-dihydroxycoumarin in Escherichia coli†

Coumarins and their derivatives possess crucial biochemical and pharmaceutical properties. However, the exploration of the coumarin biosynthesis pathways remains limited, restricting their microbial biosynthesis, especially for hydroxycoumarins. In this work, we designed and verified novel artificial pathways to produce a valuable compound 4,6-dihydroxycoumarin (4,6-DHC) in Escherichia coli. Based on the retrosynthesis analysis, multiple routes were designed and verified by extending the shikimate pathway, screening the potential enzymes, and characterizing the enzymes involved. Rare codon optimization and protein engineering strategies were applied to optimize the rate-limiting steps. De novo biosynthesis of 4,6-DHC was achieved using the cheap carbon source glycerol, and the titer can reach 18.3 ± 0.7 mg L⁻¹. Ultimately, inducible regulation of critical pathway genes with a tetracycline-inducible controller yielded a significant boost in 4,6-DHC production, achieving a titer of 56.7 ± 2.1 mg L⁻¹. This research successfully created a microbial platform for 4,6-dihydroxycoumarin production and demonstrated a generalizable strategy for synthesizing valuable compounds.