High-yield production of multiple O-methylated phenylpropanoids by the engineered Escherichia coli-Streptomyces cocultivation system

Abstract

Background: O-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date.

Results: A methyltransferase from Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids (including 3,5,4'-trimethoxystilbene, 5-hydroxy-7,4'-dimethoxyflavanone, 4'-hydroxy-5,7-dimethoxyflavanone, 5,7,4'-trimethoxyflavanone, 5-hydroxy-7,4'-dimethoxyflavone, and 5,7,4'-trimethoxyflavone) has been demonstrated for the first time.

Conclusions: This cocultivation system based on the phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.

Keywords: E. coli–Streptomyces cocultivation; O-methylated phenylpropanoids; O-methyltransferase.

Fig. 1

Schematic of the biosynthetic pathway from 4-coumaric acid to flavonoid and stilbene. 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; STS, stilbene synthase; CHI, chalcone isomerase; FNS, flavone synthase; OMT, O-methyltransferase

Fig. 2

UPLC–qTOF–HR-MS analyses of methylated stilbene, flavanone, and flavone produced by bioconversion using DHS2001/SaOMT2. a UPLC–qTOF–HR-MS chromatogram selected for m/z = 243.1016, 257.1172, and 271.1329 corresponding to desoxyrhapontigenin (P1), 5-hydroxy-3,4′-dimethoxystilbene (P2), and 3,5,4′-trimethoxystilbene (P3), respectively, of culture extracts obtained from DHS2001/SaOMT2 supplemented with resveratrol. b UPLC–qTOF–HR-MS chromatogram selected for m/z = 287.0914 corresponding to sakuranetin (P4), m/z = 301.1071 corresponding to 5-hydroxy-7,4′-dimethoxyflavanone (P5) and 4′-hydroxy-5,7-dimethoxyflavanone (P6), and m/z = 315.1227 corresponding to 5,7,4′-trimethoxyflavanone (P7) of culture extracts obtained from DHS2001/SaOMT2 supplemented with naringenin. c UPLC–qTOF–HR-MS chromatogram selected for m/z = 285.0757, 299.0914, and 313.1071 corresponding to genkwanin (P8), 5-hydroxy-7,4′-dimethoxyflavone (P9), and 5,7,4′-trimethoxyflavone (P10), respectively, of culture extracts obtained from DHS2001/SaOMT2 supplemented with apigenin

Fig. 3

Analysis of in vitro methylation reaction catalyzed by recombinant SaOMT2. a SDS-PAGE of purified SaOMT2 expressed in S. venezuelae (svSaOMT2). Lane M shows the molecular weight marker with the indicated masses. b UPLC–qTOF–HR-MS chromatogram selected for m/z = 285.0757, 299.0914, and 313.1071 corresponding to genkwanin (P2), 5-hydroxy-7,4′-dimethoxyflavone (P3), and 5,7,4′-trimethoxyflavone (P4), respectively, converted from apigenin (P1) by SaOMT2 expressed in S. venezuelae. c SDS-PAGE of purified SaOMT2 expressed in E. coli (ecSaOMT2). d UPLC– qTOF–HR-MS chromatogram selected for m/z = 285.0757 and 299.0914 corresponding to genkwanin (P2) and 5-hydroxy-7,4′-dimethoxyflavone (P3), respectively, converted from apigenin (P1) by SaOMT2 expressed in E. coli

Fig. 4

UPLC–qTOF–HR-MS analyses of pterostilbene, flavanone, and flavone produced by phenylpropanoid-producing E. coli mutants. a UPLC–qTOF–HR-MS chromatogram of pterostilbene (m/z = 257.1172) obtained from BL21/PTS supplemented with 4-coumaric acid. b UPLC–qTOF–HR-MS chromatogram of naringenin (m/z = 273.0757) obtained from BL21/NRG supplemented with 4-coumaric acid. c UPLC–qTOF–HR-MS chromatogram of apigenin (m/z = 271.0601) obtained from BL21/APG supplemented with 4-coumaric acid

Fig. 5

Production of methylated stilbene and flavonoid using different mixing ratios of E. coli and Streptomyces mutants. a The production of 3,5,4′-trimethoxystilbene by cocultivating BL21/PTS and DHS2001/SaOMT2. b The production of sakuranetin, 5-hydroxy-7,4′-dimethoxyflavanone, 4′-hydroxy-5,7-dimethoxyflavanone, and 5,7,4′-trimethoxyflavanone by cocultivating BL21/NRG and DHS2001/SaOMT2. c The production of genkwanin, 5-hydroxy-7,4′-dimethoxyflavone, and 5,7,4′-trimethoxyflavone by cocultivating BL21/APG and DHS2001/SaOMT2

Fig. 6

Production of methylated stilbene and flavonoid in the coculture system for a different cocultivation period. a The production of 3,5,4′-trimethoxystilbene by cocultivating BL21/PTS and DHS2001/SaOMT2. b The production of sakuranetin, 5-hydroxy-7,4′-dimethoxyflavanone, 4′-hydroxy-5,7-dimethoxyflavanone, and 5,7,4′-trimethoxyflavanone by cocultivating BL21/NRG and DHS2001/SaOMT2. c The production of genkwanin, 5-hydroxy-7,4′-dimethoxyflavone, and 5,7,4′-trimethoxyflavone by cocultivating BL21/APG and DHS2001/SaOMT2