(693f) Establishing a Novel and Efficient Biosynthetic Pathway for Anticoagulant Precursor 4-Hydroxycoumarin in Engineered Escherichia coli
4-hydroxycoumarin (4HC) serves as the building block for synthesis of various anticoagulants, such as warfarin, dicoumarol, phenprocoumon and acenocoumarol, which have great pharmaceutical significance in treatment of thromboembolic diseases. Currently, petroleum-derived chemical synthesis is the major approach for production of 4HC, which is expensive, unsustainable and harm to the environment. Notably, the natural 4HC biosynthetic route is not yet understood. In this work, we constructed an artificial pathway to achieve microbial production of 4HC from renewable carbon source for the first time. First, we proposed a novel pathway to produce 4HC from salicylate via salicoyl-CoA. Identifying enzymes suitable for catalyzing the sequential reactions enabled 2.3 mg/L of 4HC produced from salicylate. Second, a salicylate over-producer was constructed by screening the best candidates that capable of efficiently converting chorismate into salicylate. Third, the rate-limiting step in this pathway was eliminated by function-based enzyme bioprospecting. The replacement of an initially used plant biphenyl synthase with a microbial FabH-like quinolone synthase leads to high efficiency on the 4HC-forming reaction. Remarkably, this effort combined with pathway enzymes modular optimization yielded 108.9âmg/L 4HC from glucose. Then, this titer was further increased to around 500 mg/L in shake flasks by enhancing the availability of chorismate and malonyl-CoA. Furthermore, we stepwise identified 16 thioesterasesÂ candidates inÂ E. coliÂ and measured their enzymes activities for degradation of the pathway key intermediate salicoyl-CoA by bothÂ in vivoÂ andÂ in vitroÂ assays. Among them, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation. Remarkably, deletion ofÂ ydiIÂ in host strain further improved the 4HC titer to 935 mg/L, which is the highest titer reported to date. Finally, we exploredÂ in situÂ semi-synthesis of warfarin by feeding with the other precursor benzyldeneacetone and the catalyst (S,S)-1,2-diphenylethylenediamine, 43.7âmg/L of warfarin was generated via sonication. This work shows the potential for scaled-up microbial production of 4HC and opens up the possibility of biosynthesizing various coumarin molecules with pharmaceutical significance.