Screening And Characterization Of Peroxidase For Biosynthesis Of Ellagic Acid And Its Derivatives

Ellagic acid（EA）compounds are a class of natural products that are widely distributed in plants and are widely used in pharmaceuticals,nutraceuticals and cosmetics for their anti-inflammatory,anti-bacterial and anti-cancer activities,with an annual global market value of hundreds of millions of dollars.At present,the production of EA relies mainly on plant extraction methods,which have low yields and cannot meet market demand.In recent years,research on the production of natural products using synthetic biology methods has attracted much attention.Peroxidase（POD）has been applied to the biosynthesis of a variety of natural products due to its broad substrate spectrum and high catalytic efficiency.In this study,we constructed a pathway to catalyse the production of EA from methyl gallate（MG）using POD in Escherichia coli by screening for efficient POD.Firstly,based on the substrate-product structural similarity and the catalytic mechanism of the enzyme,six PODs were screened and the expression and solubility of the target protein in E.coli was successfully improved by fusing the maltose binding protein（MBP）.Subsequent in vitro enzyme activity assays were performed using MG as a substrate and revealed that the ascorbate peroxidase APX1 from Silybum marianum had the highest catalytic rate for MG with a k_catvalue of 0.618±0.07 s^-1.By optimizing the addition amount of H₂O₂in the in vitro reaction system,120.56 mg/L EA was generated by using APX1 to catalyze MG,with a yield of 52%.By optimizing the p H,755.79 mg/L EA was finally catalyzed by APX1 at a final concentration of 6 m M MG added at an optimized p H of 8.0 in the in vitro reaction,with a yield of 83%.Based on the in vitro synthesis of EA,we transferred APX1 into BL21（DE3）for fermentation experiments using MG as substrate and obtained 22.91mg/L EA with the addition of 100 mg/L MG,successfully achieving the first in vivo biosynthesis of EA in E.coli.Through the strategies of overexpressing hem A to improve the supply of cofactor heme,increasing the substrate addition and optimizing the fermentation conditions,the EA yield was finally increased to 213.55 mg/L with a yield of 57%,which laid the foundation for the de novo synthesis of EA in E.coli and provided a theoretical basis for the subsequent industrial scale-up.As another important nucleus of EA derivatives,3,3’-di-O-methylellagic acid has similar structure and function to EA.In this study,the horseradish peroxidase HRPO from Armoracia rusticana was found to have the best catalytic activity for the substrate by using the screened efficient POD for catalytic methyl-4-O-methylgallate.By optimizing the addition amount of H₂O₂and the p H in the in vitro reaction system,the synthesis of3,3’-di-O-methylellagic acid was finally achieved for the first time by the addition of 2 m M methyl-4-O-methylgallate at p H 6.0,catalyzed by HRPO to produce 24.10 mg/L 3,3’-di-O-methylellagic acid,which showed the great application potential of POD in the synthesis of EA compounds,and provided a new direction for the synthesis of other natural products with complex structures.