Metabolic Engineering Of Escherichia Coli For The Efficient Synthesis Of Sakuranetin

Sakuranetin is a 7-O-methylated derivative of naringenin,which is present in many plants,such as Perilla frutescens,Oryza sativa,and cherry tree bark.It has many activities such as antioxidation,antibacterial,antiviral and antiallergic.At present,the researches on the biosynthesis of sakuranetin focus on exploring the catalytic activity of flavonoids methyltransferase（F7-OMT）acting on the 7-OH site of naringenin,and constructing the synthesis pathway of sakuranetin.There is no further analysis on the limiting factors for the synthesis of sakuranetin from naringenin.In this study,we used the FOMT that acts on the 7-OH of naringenin to synthetize sakuranetin in Escherichia coli by metabolic engineering.This study lays a foundation for the synthesis of other methylated flavonoids.The main strategies and results of this study are listed as follows:（1）Enhancement of methylation and stress tolerance to improve sakuranetin production.Firstly,we screened the methyltransferase with high catalytic ability,and optimized the substrate concentration.Subsequently,the expression of key enzymes（Met A,Cys E）and the production of cofactors（NADPH,ATP,PLP）were improved to promote the methylation reaction.In addition,the increase of the substate concentration inhibited the production of sakuranetin.To solve this problem,a stress tolerance gene rpo S was screened,which is an endogenous pressure response factor in E.coli.Finally,via the enhancement of methylation and stress tolerance,the titer of sakuranetin was improved to 496.2 mg·L^-1.（2）Improvement of the catalytic efficiency of Pf OMT3 to increase sakuranetin production.Alpha Fold was used to predict the structure of Pf OMT3,and the Discovery Studio software was used to perform enzyme-substrate molecular docking.Site-directed mutations were performed on the residues around the substrate pocket,and the optimal mutant Pf OMT3^M118Lwas obtained.The Km value of the mutant was lower than that of the wild type,indicating the higher substrate affinity of the mutant.The binding free energy of Pf OMT3 and Pf OMT3^M118Lto the ligand were calculated to be-16.08 kcal·mol^-1 and-21.20 kcal·mol^-1,respectively,indicating the improved structural stability of the mutant.The titer of sakuranetin was improved to 551.5 mg·L^-1.（3）Fermentation optimization to improve sakuranetin production.In shake flask scale,the substrate addition time,Mg²⁺concentration,glycerol contents and calcium carbonate concentration were optimized.The result showed that sakuranetin had the highest titer on the condition of 10μM Mg²⁺,8 g·L^-1 glycerol,8 g·L^-1 calcium carbonate,and the optimal substrate addition time was 3 h after the addition of 0.1m M IPTG.In 5 L fermenter,the effects of inducer addition time,substrate addition methods and inoculation quantity on the production of sakuranetin were compared.The result showed that the titer of sakuranetin reached 2742.3mg·L^-1 at 70 h,which is the highest titer ever reported.