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

Abstract

(2S)-Sakuranetin is a 7-O-methylflavonoid that has anticancer, antiviral, and antimicrobial activities. Methylation process is involved in biosynthesizing (2S)-sakuranetin from (2S)-naringenin, in which S-adenosylmethionine (SAM) serves as the methyl donor. In this study, after methyl donor and substrate inhibition were identified as limiting factors for (2S)-sakuranetin biosynthesis, an efficient (2S)-sakuranetin-producing strain was constructed by enhancing methyl donor supply and cell tolerance to (2S)-naringenin. Firstly, PfOMT3 from Perilla frutescens was selected as the optimal flavonoid 7-O-methyltransferase (F7-OMT) for the conversion of (2S)-naringenin to (2S)-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 (2S)-naringenin were identified from molecular chaperones and sRNAs. Finally, by optimizing the fermentation process, 681.44 mg/L of (2S)-sakuranetin was obtained in 250-mL shake flasks. The titer of (2S)-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 O-methylated flavonoids efficiently in E. coli.

Keywords: (2S)-Sakuranetin; Cell tolerance; Flavonoid 7-O-methyltransferases; Metabolic engineering; Methylation.

Graphical abstract

Fig. 1

Biosynthesis pathway of (2S)-sakuranetin from (2S)-naringenin. The biosynthesis pathway of (2S)-sakuranetin from (2S)-naringenin involves a methylation reaction, where SAM (S-adenosylmethionine) is used as the methyl donor. SAM biosynthesis involves methionine metabolism and the regeneration of cofactors ATP, NADPH, and PLP. Met represents l-Methionine; Hcys represents l-Homocysteine; SRH represents S-Ribosyl-l-homocysteine; SAH reprsents S-Adenosyl-L-homocysteine.

Fig. 2

Effects of different sources of F7-OMTs on the titer of (2S)-sakuranetin. (A) The (2S)-sakuranetin production of F7-OMTs under different concentrations of (2S)-naringenin. (B) Summary of the production of (2S)-sakuranetin with strain 7-FOMT-3 at different (2S)-naringenin concentrations. (C) LC-MS analysis of the fermentation simple. (D) LC-MS analysis of the (2S)-sakuranetin standard simple. SAK represents (2S)-sakuranetin; NAR represents (2S)-naringenin.

Fig. 3

Effects of methionine, ATP and NADPH on (2S)-sakuranetin production. (A) (2S)-Sakuranetin titer at different methionine concentrations. (B) Effects of metA, cysE, ydaO and metK expression on (2S)-sakuranetin production. (C) Effect of POS5 expression on (2S)-sakuranetin production. SAK represents (2S)-sakuranetin; NAR represents (2S)-naringenin; Met represents methionine. ****P < 0.0001.

Fig. 4

Effects of PLP on (2S)-sakuranetin production. (A) Effects of different concentrations of PLP on (2S)-sakuranetin accumulation. (B) Effects of SNZ3, RFC4 and RPS18B expression on PLP and (2S)-sakuranetin titer. ****P < 0.0001. SNZ3 encoding pyridoxal-5′-phosphate synthase; RFC4 is a DNA binding protein; RPS18B is the component of the ribosomal subunit, which is homologous to E. coli ribosomal protein S13.

Fig. 5

Selecting the optimal genes combination for enhancing methylation process. Effect of combinatorial expression of genes that can upregulate methylation reaction on (2S)-sakuranetin production. The (2S)-naringenin was added in batches.

Fig. 6

Improving substrate tolerance of strain to improve (2S)-sakuranetin production. (A) Effects of overexpressing stress resistance genes on (2S)-sakuranetin production and cell growth when 400 mg/L (2S)-naringenin was added. (B) Effects of overexpressing stress resistance genes on (2S)-sakuranetin production and cell growth when 500 mg/L (2S)-naringenin was added. (C) Effects of overexpressing stress resistance genes on (2S)-sakuranetin production and cell growth when 600 mg/L (2S)-naringenin was added. (D) Spot assay of strain tolerance ability to (2S)-naringenin. (E) The growth status and production of (2S)-sakuranetin when rpsQ^His31Pro and secB were co-expressed with rpoS. ****P < 0.0001.

Fig. 7

Fermentation process optimization to improve (2S)-sakuranetin production. (A) The accumulation of (2S)-sakuranetin when rpoS and secB were co-expressed with methylation-strengthening genes. (B) Effects of different (2S)-naringenin addition time on the production of (2S)-sakuranetin. The final concentration of (2S)-naringenin was 700 mg/L. (C) Effects of different Mg²⁺ levels on the production of (2S)-sakuranetin. (2S)-Naringenin was first added after IPTG was added for 3 h, and different concentrations of Mg²⁺ were added at the same time. The final concentration of (2S)-naringenin was 700 mg/L. (D) Strain NS34 was fermented in a 5-L bioreactor, and (2S)-naringenin was added in batches to a final concentration of 4 g/L.