Biosynthesis Of 4-hydroxycoumarin Glycosides

4-Hydroxycoumarin has anticoagulant function and is used to treat thromboembolic disorders and is an important direct precursor of the anticoagulant drug warfarin.At the same time,it can also be used in anticancer,rodenticide,making synthetic spices,etc.,which has great applications.Traditional chemical synthesis methods suffer from drawbacks such as high cost,complicated process,need high temperature and pressure,and low yield.Therefore,biosynthesis of 4-hydroxycoumarin and its derived products are important.In this study,using synthetic biology technology and strategies,we optimized strains for biosynthesis of 4-hydroxycoumarin and achieved high yields of 4-hydroxycoumarin.By genomic modification of E.coli BW25113(integrating aro G^fbr,a key gene of the shikimate upstream pathway,and knocking out ydi I,a thioesterase gene that degrades intermediate products of the synthetic pathway),optimizaing the plasmid module for 4-hydroxycou-marin synthesis,and optimizaing the fermentation medium components,finally,we achieved the highest yield of 1157 mg/L for the current bio-synthesis of 4-hydroxycoumarin.Although high yield of 4-hydroxycoumarin has been preliminarily achieved,its higher cytotoxicity becomes a bottleneck to further improve the yield of 4-hydroxycoumarin.To solve the problem of high cytotoxicity of4-hydroxycoumarin,in this study,Os SGT1 was obtained by screening four glycosyltransferases,which successfully converted 4-hydroxycoumarin to4-hydroxycoumarin glycosides with low cytotoxicity.The glycosylation efficiency of the added experimental intracellular 4-hydroxycoumarin was0.65%.By purifying Os SGT1 in vitro for enzyme kinetic analysis,we know that the enzyme has a kcat/Km=0.45 m M^-1min^-1for 4-hydroxycoumarin.To enhance the intracellular glycosylation efficiency of Os SGT1,we used the way of adding chaperone,optimizating vector and medium component to make the addition experiment intracellular glycosylation efficiency close to 100%.The above optimal strains and media were used in extended fermenter cultures to prepare glycoside products.Addition of different concentrations of 4-hydroxycoumarin substrate（5 g/L,10 g/L,and20 g/L）,the glycosylation efficiency decreased with the substrate concentra-tion:96.7%,59.5%and 1.37%,respectively.The 4-hydroxycoumarin glycol-side product was prepared with fermenter cell broth,and the purity could reach 92%.Introduction of the Os SGT1 gene into the de novo 4-hydroxycoumarin pathway,followed by plasmid module optimization and promoter optimiza-tion,we achieved the first biosynthesis of 4-hydroxycoumarin glycosides with a yield of 1793 mg/L.Finally,in this study,hydrolysis of 4-hydroxy-coumarin glycosides was achieved by acid hydrolysis and enzymatic hydrolysis,respectively:we tested the effect of different temperatures,ethanol concentrations,and acid concentrations on the hydrolysis of4-hydroxycoumarin glycosides,which resulted in 32.0%hydrolysis;we screened of endogenous glycoside hydrolase in E.coli,with a hydrolysis rate of 18.6%.