One-pot enzymatic total synthesis of intermediates in the biosynthesis of kinamycin

Abstract:

[Objective] To reconstitute the partial biosynthetic pathway of the type II polyketide kinamycin in a cell-free system starting from CoA, acetyl-CoA and malonate.[Methods] The polyketide synthases (PKSs) and two supplemental proteins (MCAT and MatB) were purified and quantitated. The one-pot enzymatic synthesis of kinamycin intermediates was accomplished, and the products were analyzed by high performance liquid chromatography (HPLC). The system was used to explore the function and exact substrate of the stand-alone thioesterase, AlpS, in the biosynthetic pathway. The parameters including temperature, pH, and the concentrations of minimal PKSs and AlpS were optimized by the single-variable method. [Results] Eight PKSs and two supplemental proteins (MCAT and MatB) were expressed and purified. The intermediates and shunt products in kinamycin biosynthesis were produced in vitro, including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin. The yields of these products, especially prejadomycin and dehydrorabelomycin, were all increased when AlpS was added. The optimal conditions were 30℃, pH 7.0, 2.8 μmol/L minimal PKSs (AlpA, AlpB, and RavC, respectively), and 7.2 μmol/L AlpS, under which the yield of prejadomycin was increased to 302 mg/L. [Conclusion] A one-pot enzymatic synthesis system with high yields of kinamycin-related products including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin was successfully constructed. Furthermore, the chain-release function and substrate specificity of AlpS in the synthetic pathway were investigated.