Synthesis Of Quercetin Methylated Derivatives By Expression Of FOMT In Escherichia Coli

Quercetin is an important citrus flavonoid with many biological activities,such as anti-inflammatory,anti-cancer,and antioxidant.However,quercetin has poor water and fat solubility and low bioavailability,limiting its application in food and medicine.Studies have shown that quercetin methylation can effectively improve its biological activity,metabolic stability and bioavailability.In order to obtain a variety of quercetin methylated derivatives.In this study,11 flavonoid O-methyltransferases(FOMT)from different sources were screened from NCBI through synthetic biology and introduced into Escherichia coli(E.coli)for expression.The heterologous synthesis of four mono O-methylquercetin(tamarixetin,rhamnetin,isorhamnetin and 3-O-methylquercetin)was realized.By constructing the co-culture system of E.coli,di-O-methylquercetin((Ombuin)was synthesized for the first time.It lays a foundation for synthesising more rare methylated flavonoids or polymethoxyflavonoids by microorganisms in the future.The main research contents are as follows:(1)11 FOMT genes from different sources were screened from NCBI database.Their physical and chemical properties,protein secondary and tertiary structures,signal peptides,transmembrane domains,and protein conserved functional domains were predicted by bioinformatics analysis.The functional properties of proteins were analyzed and predicted by multiple sequence alignment and progressive tree analysis.It is expected to provide a theoretical reference for the study of FOMT at the molecular level and improve the heterologous catalytic activity of FOMT in the future.(2)according to the different methylation sites of FOMT,four strains of E.coli engineering bacteria containing F4’-OMT,four strains containing F7-OMT,two strains containing F3’-OMT,and one strain containing F3-OMT were constructed.Then,quercetin was used as a substrate for fermentation to produce four mono O-methylquercetin(tamarixetin,rhamnetin,isorhamnetin and 3-O-methylquercetin).Among the four strains expressing F4’-OMTs,the strain expressing Mp OMT4 had the highest catalytic activity for quercein reaching 31.17 mg/L tamarixetin.Among the four strains expressing F7-OMT,the strain expressing Os NOMT had the highest catalytic activity and synthesized 11.17 mg/L rhamnetin;The yields of isorhamnetin catalyzed by two strains expressing F3’-OMT(ROMT9 and SOMT9)were 8.72 mg/L and 8.90 mg/L,respectively;The yield of3-O-methylquercetin catalyzed by the strain expressing Sh MOMT3 was52.95 mg/L.(3)the co-culture system of E.coli was constructed.With quercetin as substrate,the biosynthesis of 4’,7-dioxymethylquercetin(Ombuin)was divided into two modules.Module 1 expressed Os NOMT and catalyzed the oxygen methylation of quercetin at 7-OH position to obtain the intermediate product 7-O-methylquercetin(rhamnetin);Module 2 expressed Mp OMT4 and catalyzed the oxygen methylation of 7-O-methylquercetin at 4’-OH position to obtain the final product ombuin.Changing different buffers,adjusting the addition sequence of module 1 and module 2,adjusting the proportion and biomass of bacteria in the two modules,and optimizing the culture system,a 1:2 ratio of the two strains,a dry weight of 24 g/L showed a highest catalytic performance.Ombuin production was boosted to 21.56mg/L in Tris-HCl(p H 7.4),demonstrating the potential of the two-step addition strategy to improve flavonoid production by biotransformation.