Production of (2)-sakuranetin from (2)-naringenin in by strengthening methylation process and cell resistance.

(2)-Sakuranetin is a 7--methylflavonoid that has anticancer, antiviral, and antimicrobial activities. Methylation process is involved in biosynthesizing (2)-sakuranetin from (2)-naringenin, in which -adenosylmethionine (SAM) serves as the methyl donor. In this study, after methyl donor and substrate inhibition were identified as limiting factors for (2)-sakuranetin biosynthesis, an efficient (2)-sakuranetin-producing strain was constructed by enhancing methyl donor supply and cell tolerance to (2)-naringenin. Firstly, PfOMT3 from was selected as the optimal flavonoid 7-O-methyltransferase (F7-OMT) for the conversion of (2)-naringenin to (2)-sakuranetin. Then, the methylation process was upregulated by regulating pyridoxal 5'-phosphate (PLP) content, key enzymes in methionine synthesis pathway, and the availability of ATP. Furthermore, genes that can enhance cell resistance to (2)-naringenin were identified from molecular chaperones and sRNAs. Finally, by optimizing the fermentation process, 681.44 mg/L of (2)-sakuranetin was obtained in 250-mL shake flasks. The titer of (2)-sakuranetin reached 2642.38 mg/L in a 5-L bioreactor, which is the highest titer ever reported. This work demonstrates the importance of cofactor PLP in methylation process, and provides insights to biosynthesize other -methylated flavonoids efficiently in . .