Construction Of Phloretin Biosynthetic Pathway In Escherichia Coli

Phloretin,one of the dihydrochalcones comprising the C6-C3-C6 skeleton,has various physicochemical and biological properties.In this study,we used p-dihydro-coumaric acid as precursor to construct a heterologous synthetic pathway of phloretin in E.coli BL21(DE3),and improved the efficiency of phloretin biosynthesis and reduced the derailment products by screening and mutation strategies.To construct the artificial synthetic pathway of phloretin,we respectively overexpressed the At4 CL from Arabidopsis thaliana,Aa4 CL from Aromatoleum aromaticum,Rj4 CL from Rhodococcus jostii RHA1 and Pa4 CL from Plagiochasma appendiculatum with HaCHS from Hypericum androsaemum in E.coli BL21(DE3)for fermentation,when p-dihydro-coumaric acid was added.The results showed that the strains harboring At4 CL and Pa4 CL could not synthesize phloretin,while strains harboring Aa4 CL and Rj4 CL synthesized phloretin with a large amount of derailment product 2H-BNY.The synthetic pathway built by the Aa4 CL and HaCHS synthesized 0.50 mg/L phloretin in E.coli.To screeCHSs,MdCHS3 and MdCHS5 from Malus x domestica,ErbCHS from Erigeron breviscapus,SorbCHS from Sorghum bicolor,PpCHS from Pyrus pyrifolia,AaCHS from Aquilaria sinensis and PhCHS from Petunia hybrid were overexpressed with Aa4 CL respectively to construct phloretin biosynthesis using HaCHS as the control.The results showed that,except for PpCHS which could not synthesize products,allCHSs could catalyze the substrate and produce phloretin and derailment products.Compared with HaCHS,SorbCHS increased phloretin and2H-BNY by 4-5 folds,and MdCHS3 slightly increased phloretin and 2H-BNY.We took homology modeling analysis for MdCHS3,and rationally designed 3surface sites,5 sites of substrate binding cavity,7 sites of CoA binding channel,V261 site outside the binding cavity and I254 site of circularization site.55 mutant variants were screened and 8 positive variants were obtained.The phloretin titers of variants L214 H and S63 R reached 1.02 mg/L and 1.19 mg/L,1.6 and 1.9 folds higher than the wild type,and 2H-BNY was decreased by more than 82%.Variant V261 A increased the phloretin production up to 1.84 mg/L,2.8 times higher than the wild type,however 2H-BNY was also increased by 193%.Compared with other flavonoids,the synthesis of phloretin in microorganisms falls far behind,and phloretin synthesis is currently only achieved in S.cerevisiae.In this study,gene screening andCHS mutation strategies were investigated,which paved the way for further improving the biosynthesis of phloretin in E.coli.