Construction And Mechanism Analysis Of 4-Hydroxycoumarin High-Producing Strain

4-hydroxycoumarin is a precursor to the synthesis of the anticoagulant drug warfarin.At present,4-hydroxycoumarin is mainly produced by chemical synthesis,which has problems such as poor safety and high energy consumption.The biosynthesis of 4-hydroxycoumarin from glucose and other raw materials has the advantages of green sustainability,but its efficient biosynthesis is limited due to the insufficient supply of precursors and the high cytotoxicity of the product,its efficient biosynthesis is limited.In this paper,using Escherichia coli as the chassis strain,a strain for efficient synthesis of 4-hydroxycoumarin was constructed by enhancing the shikimic acid pathway,enriching malonyl-Co A,and adapting to the laboratory evolution of the strain,and the specific work content is as follows:In order to enhance the flux of shikimic acid pathway and increase the supply of PEP and E4P,the genes pps A,tkt A,aro G^fbrand aro L were overexpressed by CRISPR/Cas9 gene editing technology.And the yield of 4-hydroxycoumarin reached 596.71 mg/L.Furthermore,the endogenous thiolipase gene ydi I of Escherichia coli was knocked out,and the degradation of salicyl-Co A was inhibited,resulting in a yield of 809.84 mg/L.To increase malonyl-Co A content,gene knockout or promoter substitution was used to inhibit fatty acid synthesis.By replacing the promoter of the key lethal gene cluster fab D/H/G with the stable promoter P_flgc,the content of malonyl-Co A was increased by 27.27%,and the production of 4-hydroxycoumarin was 913.25 mg/L.The key non-lethal gene fab F was knocked out,and the content of malonyl-Co A increased by 40.91%,and the yield of 4-hydroxycoumarin reached 1117.96 mg/L.To solve the cytotoxicity problem of 4-hydroxycoumarin,the Adaptive laboratory evolution（ALE）of Escherichia coli was performed to obtain the 7g/L 4-hydroxycoumarin tolerant strain JRX00.Whole genome sequencing was performed on JRX00 and parental strains to obtain 14 differential mutant genes.By overexpressing and knocking out differential genes in parental strains,two single-target effective genes,yja A and kat E,were screened out.By knocking out kat E,the yield of engineered strain 4-hydroxycoumarin increased to 1249.54 mg/L,which was the highest yield of biosynthetic 4-hydroxycoumarin at present.In this study,an engineering strain of Escherichia coli with efficient synthesis of 4-hydroxycoumarin was constructed,which laid the foundation for its green and sustainable production.The target genes of 4-hydroxycoumarin tolerance were obtained,which provided direction and theoretical support for the analysis of its toxicity mechanism.