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

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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1118-9) contains supplementary material, which is available to authorized users.


Background
Phenylpropanoids, including flavonoids and stilbenes, are structurally diverse plant secondary metabolites that have significant potential as pharmaceuticals, nutraceuticals, and cosmetics owing to their antioxidation, cancer prevention, anticancer, antibacterial, and anti-inflammatory properties [1]. However, their availability from plants is limited owing to seasonal or regional variations, low abundance, and the difficulty in isolating single compounds from complex mixtures. Therefore, microbial production of these important metabolites by expressing plant biosynthetic genes in microbial hosts is an efficient alternative for large-scale and environment-friendly production [2]. The biosynthesis of the parent skeleton of flavonoids begins with the conversion of 4-coumaric acid, derived from phenylalanine or tyrosine, to 4-coumaroyl-CoA by 4-coumarate:CoA ligase (4CL). The subsequent condensation of 4-coumaroyl-CoA with three malonyl-CoA by chalcone synthase (CHS) produces naringenin chalcone, which is converted to simple flavanone naringenin by chalcone isomerase (CHI). The stilbene synthase (STS) acts on 4-coumaroyl-CoA and catalyzes sequential condensation, resulting in the production of resveratrol (one of the simplest stilbenes). The simple phenylpropanoid skeletons are further modified by various enzymes such as flavone synthase (FNS) and flavonol synthase as well as by diverse methylation and glycosylation ( Fig. 1) [2].
Here, we recharacterized the function of 7-OMT from Streptomyces avermitilis ATCC 31267 (SaOMT2). SaOMT2 expressed in E. coli reportedly exhibits 7-O-methylation activity toward naringenin as well as some isoflavones and flavones [18]. However, we found that SaOMT2 expressed in Streptomyces venezuelae was able to di-and tri-methylate resveratrol, naringenin, and apigenin. Furthermore, we developed a cocultivation system employing each of the engineered E. coli strains that produced pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant for achieving a high-yield production of multi-methylated stilbene, flavanone, and flavone. Optimization of cocultivation conditions resulted in the production of approximately 29 mg/L of 3,5,4′-trimethoxystilbene, which is the highest titer reported to date. To our knowledge, the production of a series of di-or tri-methylated naringenin and apigenin analogs based on a cocultivation system is the first report concerning microbial production of multi-methylated flavonoid compounds. This one-pot cocultivation system provides a facile tool for the high-yield production of such scarce and potentially valuable methylated flavonoids and stilbenes and will enable further development of these compounds.

In vitro characterization of S. avermitilis OMT expressed in E. coli and S. venezuelae
To examine the multi-methylation activity of SaOMT2 in vitro, we expressed the histidine-tagged recombinant SaOMT2 in both S. venezuelae and E. coli and subjected it to purification (Fig. 3a, c). When the purified SaOMT2 expressed in S. venezuelae was incubated overnight with apigenin in the presence of S-adenosyl-l-methionine (SAM) as a methyl donor, the products corresponding to genkwanin, 5-hydroxy-7,4′-dimethoxyflavone, and 5,7,4′-trimethoxyflavone were produced at conversion yields of approximately 26%, 10%, and 0.1%, respectively (Fig. 3b). In contrast, incubation of E. coli-expressed SaOMT2, whose codons were optimized for facilitating efficient expression in E. coli, with apigenin and SAM under the same conditions resulted in the production of UPLC peaks corresponding to genkwanin and 5-hydroxy-7,4′-dimethoxyflavone at 19% and 3% yields, respectively (Fig. 3d). These results confirmed that S. venezuelae-expressed SaOMT2 possesses mono-, di-, and tri-methylation activity although the conversion yield to tri-methylated 5,7,4′-trimethoxyflavone was low in vitro, whereas E. coli-expressed SaOMT2 exhibits only monoand di-methylation activities toward flavone. Increasing the enzyme amount and incubation period in the experiments using E. coli-expressed SaOMT2 did not produce 5,7,4′-trimethoxyflavone. Furthermore, S. venezuelaeexpressed SaOMT2 exhibited remarkably higher monoand di-methylation activities than E. coli-expressed SaOMT2. Interestingly, di-methylation activity of E. coli-expressed SaOMT2 has not been identified in the previous report in which the whole cell E. coli expressing SaOMT2 was used as a biocatalyst [18].

Development and optimization of the cocultivation system
The discovery of stronger and more diverse regiospecific methylation activity of S. venezuelae-expressed SaOMT2 in comparison with that of E. coli-expressed SaOMT2 motivated us to develop an E. coli-Streptomyces cocultivation system for the efficient microbial production of multi-methylated flavonoids and stilbenes because E. coli is a more favorable heterologous host for the production of simple phenylpropanoid backbone in terms of high titer, fast growth, and ease of genetic manipulation [2]. First, we engineered an E. coli strain producing a di-methylated stilbene, pterostilbene, rather than resveratrol using 4-coumaric acid as the substrate because the di-and tri-methylation activity of S. venezuelae-expressed SaOMT2 toward resveratrol is relatively weaker compared with that toward flavanone and flavone (Fig. 2); however, another OMT from V. vinifera (VvROMT; GenBank accession no. FM178870) is known to efficiently produce pterostilbene from resveratrol [22]. Naringenin-and apigenin-producing E. coli strains were also constructed. The genes for the synthesis of phenylpropanoid backbones were synthesized but were not codon-optimized because it had been reported that the plant genes could be successfully used for the production of desired compounds in E. coli [15,16,23]. The plasmid for pterostilbene synthesis, pPTS, was constructed using pET32a vector for expressing the synthetic 4CL gene from O. sativa (Os4CL; GenBank accession no. BAD27987) [23]; STS gene from V. vinifera (VvSTS; GenBank accession no. DQ459351) [24]; and OMT gene from V. vinifera (VvROMT) ( Table 1). The plasmid, pNRG, was constructed using the pCDFduet-1 vector for inducing the biosynthesis of naringenin by expressing the synthesized Os4CL gene, CHS gene from Populus euramericana (PeCHS; TIGR accession no. TC29789) [25], and CHI gene from Medicago truncatula  [16]. The ampicillin resistant gene was additionally inserted to the pCDFduet-1 vector as a selection marker during cocultivation with S. venezuelae, which is insensitive to ampicillin. The pAPG plasmid was designed for biosynthesizing apigenin by adding the synthetic FNS gene from Petroselinum crispum to pNRG (PcFNS; GenBank accession no. AY230247) [26]. These engineered plasmids were separately introduced into E. coli BL21(DE3) to generate BL21/PTS, BL21/NRG, and BL21/APG, respectively ( Table 1). The engineered E. coli strains, whose gene expression was induced by isopropyl β-d-1-thiogalactopyranoside (IPTG), were grown on LB medium for 3 h at 37 °C and subsequently transferred to R2YE medium [27] supplemented with 1.2 mM (196.8 mg/L) of 4-coumaric acid and further incubated at 30 °C for 18 h; the latter is considered to be a favorable growth medium and temperature for Streptomyces. UPLC-qTOF-HR-MS analysis of organic extracts obtained from the engineered E. coli strains revealed peaks that were consistent with those of authentic pterostilbene, naringenin, and apigenin standards ( Fig. 4; Additional file 1: Figure S4). Approximately, 79 mg/L, 134 mg/L, and 52 mg/L of pterostilbene, naringenin, and apigenin, respectively, were produced. These results validated that the engineered E. coli strains successfully produced and secreted the desired phenylpropanoid backbones when cultured under optimal growth conditions for Streptomyces, which allows further methylation by cocultivation with SaOMT2-expressing S. venezuelae.
Finally, to examine the possibility of direct production of multi-methylated flavonoids and stilbenes by E. coli strains, the BL21/NRG-SaOMT2 and BL21/ APG-SaOMT2 strains were constructed by additionally expressing the synthetic codon-optimized SaOMT2 in BL21/NRG and BL21/APG. The resulting BL21/NRG-SaOMT2 and BL21/APG-SaOMT2 strains produced only small amounts of the respective mono-methylated compounds, sakuranetin and genkwanin (Additional file 1: Figure S5). These results are consistent with those of a previous study in which SaOMT2-expressing E. coli strain showed only mono-methylation activity toward flavanone and flavone compounds [18], and thus, this study demonstrates the advantage of E. coli-Streptomyces cocultivation system for efficient multi-methylation of flavonoids and stilbenes.

Discussion
It has been known that the use of the whole cell E. coli expressing SaOMT2 as a biocatalyst can lead to monomethylation at the position 7 of flavanone, isoflavone, and flavone [18]. However, we found that the purified SaOMT2 expressed in E. coli is able to mediate in vitro di-methylation at the positions 7 and 4′ of flavone (apigenin) as well as mono-methylation at the position 7 of the same compound (Fig. 3). The expression of this S. avermitilis-derived OMT in the same Streptomyces species S. venezuelae showed broader regiospecificity than that shown by E. coli-expressed SaOMT2. We observed that the SaOMT2-expressing S. venezuelae (DHS2001/ SaOMT2) not only catalyzes 7,4′-and 5,7-di-methylation but also 5,7,4′-tri-methylation of flavanone (naringenin) and flavone (apigenin) by carrying out whole-cell bioconversion. Additionally, DHS2001/SaOMT2 mediates 3,5,4′-tri-methylation as well as 3,4′-di-methylation of stilbene (resveratrol) (Fig. 2). Moreover, the purified recombinant SaOMT2 obtained from S. venezuelae showed approximately 1.4-and 3.3-fold higher monoand di-methylation activities toward apigenin, respectively, than those shown by SaOMT2 expressed in E. coli (Fig. 3). At present, it is unclear why SaOMT2 s expressed in S. venezuelae and E. coli have different activities and regiospecificities. However, it is plausible that heterologous expression of SaOMT2 in the same species facilitates more productive folding of this enzyme compared with its expression in different species.
A total titer of 29 mg/L of 3,5,4′-trimethoxystilbene was produced by cocultivating BL21/PTS with DHS2001/ SaOMT2 (Fig. 6a). Previously, only 0.2 mg/L of this trimethylated stilbene compound was produced by the E. coli strain expressing resveratrol biosynthetic genes together with two OMTs from S. bicolor [17]. Moreover, the SaOMT2-associated cocultivation system proved to be useful for the production of well-known mono-methylated flavonoids such as sakuranetin and genkwanin. In previous reports, approximately 40 mg/L of sakuranetin and genkwanin were produced by the engineered E. coli using tyrosine as substrate [15,16]. In comparison to those previous reports, here, a higher level of sakuranetin (~ 79 mg/L) was produced along with a similar level of genkwanin (~ 42 mg/L) production from 4-coumaric acid (Fig. 6b, c).
Further optimization of culture conditions and metabolic engineering of both E. coli and S. venezuelae will improve the production levels of methylated phenylpropanoids. For instance, in the present study, the initial substrate, 4-coumaric acid, was fed at a concentration of 1.2 mM because this substrate concentration reportedly supports the production of phenylpropanoids in S. venezuelae without inhibiting the growth of the bacteria [19]. Additionally, the precultivation period for E. coli (3 h after induction) and S. venezuelae (40 h) was set in consideration of the different growth rates of these microbes before optimization of the mixing ratio. Therefore, further experimental optimization of the coculture conditions such as optimal substrate concentration and preculture period would improve the current titers. Metabolic engineering strategy such as the coexpression of SAM (a methyl donor) synthetase in the OMT-expressing S. venezuelae [31] as well as optimization of promoters and construct designs in the phenylpropanoid-producing E. coli [23] would also enhance the production levels. Finally, investigation of this multi-methylation activity of SaOMT2 toward more diverse phenylpropanoid substrates will expand the chemical diversity of this important class of natural products. Nevertheless, the cocultivation system involving phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae developed in this study proves to be an efficient biological tool for facilitating high-yield production of multi-methylated phenylpropanoid compounds.

Bacterial strains, plasmids, culture medium, and reagents
Escherichia coli DH5α was used for routine subcloning, whereas E. coli BL21(DE3) (Novagen, Madison, WI, USA) was used as a heterologous host for expressing the recombinant SaOMT2 and producing pterostilbene, naringenin, and apigenin. pGEM T-easy (Promega, Madison, WI, USA) vector was used for subcloning. The gene expression vectors for E. coli strain, namely pCDFduet-1 and pET32a, were purchased from Novagen (Madison, WI, USA). The pCDFduet-amp plasmid was prepared by inserting an ampicillin resistant marker for E. coli-Streptomyces coculture. E. coli strains were grown in LB medium [32] supplemented with suitable antibiotics. S. avermitilis ATCC 31267 was obtained from the American Type Culture Collection (Manassas, VA, USA). S. venezuelae DHS2001, in which the native pikromycin polyketide synthase gene was deleted [33], was used as a Streptomyces heterologous host. S. venezuelae mutants were grown in R2YE liquid medium [27] at 30 °C in a rotary shaker at 230 rpm. The high-copy number E. coli-Streptomyces shuttle vector pSE34 containing a strong constitutive ermE* promoter plus a thiostrepton resistance marker [21] was used as the expression vector in the Streptomyces heterologous host. For the expression of phenylpropanoid biosynthetic genes in an E. coli heterologous host, the Os4CL, VvSTS, PeCHS, MtCHI, PcFNS, VvROMT, and codon-optimized SaOMT2 genes were synthesized from Genotech (Daejon, Korea). All bacterial strains and plasmids used in this study are listed in Table 1.

Construction of SaOMT2-expressing S. venezuelae mutant
For achieving heterologous expression of the SaOMT2 gene in S. venezuelae, a pSE-SaOMT2 plasmid was constructed. The SaOMT2 gene was amplified from the S. avermitilis genomic DNA using PCR and by employing primers SaOMT2-1_F/SaOMT2-1_R (Additional file 1: Table S1). A PCR-amplified DNA fragment was cloned into pGEM T-easy vector, and the digested SaOMT2 fragment was ligated into the complementary sites of pSE34 to generate pSE-SaOMT2 (Table 1). The expression plasmid pSE-SaOMT2 was transformed into S. venezuelae DHS2001, yielding the corresponding recombinant strain DHS2001/SaOMT2 (Table 1). S. venezuelae DHS2001 protoplast formation and transformation procedures were performed as described previously [27].

Construction of the protein expression mutants for recombinant SaOMT2
The histidine-tagged recombinant SaOMT2 was expressed in both S. venezuelae and E. coli to characterize the methylation activity in vitro. For expression in S. venezuelae, PCR was performed using primers SaOMT2-1_F/SaOMT2-H1_R from S. avermitilis genomic DNA. The SaOMT2-H1_R primer sequence contained a sixhistidine tag to facilitate the purification of the resulting recombinant protein (Additional file 1: Table S1). The PCR fragment was cloned into pGEM T-easy vector and subsequently ligated into the complementary sites of pSE34 to yield pSE-SaOMT2-H. The pSE-SaOMT2-H plasmid containing the SaOMT2 gene fused with C-terminal histidine-tag was transformed into S. venezuelae DHS2001, yielding the corresponding recombinant strain DHS2001/SaOMT2-H (Table 1).
To construct the pET-SaOMT2-H plasmid for expressing recombinant SaOMT2 in E. coli, the synthetic codonoptimized SaOMT2 gene was cloned into pGEM T-easy vector and ligated into the XbaI/NotI sites of pET32a to generate pET-SaOMT2-H. The pET-SaOMT2-H plasmid containing the SaOMT2 gene fused with C-terminal histidine-tag was transformed into E. coli BL21(DE3), yielding the corresponding recombinant strain BL21/ SaOMT2-H ( Table 1).

Construction of phenylpropanoid-producing E. coli mutants
The pPTS plasmid containing Os4CL, VvSTS, and VvROMT genes was constructed for pterostilbene production. The restriction sites for the cloning of Os4CL, VvSTS, and VvROMT genes were introduced by PCR amplification using the primers Os4CL_F/Os4CL_R, VvSTS_F/VvSTS_R, and VvROMT_F/VvROMT_R, respectively (Additional file 1: Table S1). The PCR-amplified DNA fragments containing Os4CL, VvSTS, and VvROMT genes were cloned into pGEM T-easy vector, and the XbaI/NotI fragment carrying the entire combined DNA was transferred into the same sites of pET32a to generate pPTS. The expression plasmid, pPTS, was introduced into E. coli BL21(DE3), resulting in the formation of BL21/PTS ( Table 1).
The pNRG plasmid containing PeCHS, Os4CL, and MtCHI genes was constructed to produce naringenin. The DNA fragments, containing the appropriate restriction sites to facilitate cloning, were prepared by PCR amplification using the primers PeCHS_F/PeCHS_R, Os4CL-2_F/Os4CL-2_R, and MtCHI_F/MtCHI_R and were subsequently cloned into pGEM T-easy vector, respectively (Additional file 1: Table S1). The entire combined DNA fragments containing PeCHS, Os4CL, and MtCHI genes were ligated into pCDFduet-amp using NdeI/AvrII, yielding the corresponding recombinant strain pNRG. For the production of methylated naringenin, the codon-optimized SaOMT2 gene, in which the restriction sites were introduced into the synthetic DNA fragment by performing PCR using primers SaOMT2-2_F/SaOMT2-2_R, was cloned into pGEM T-easy vector (Additional file 1: Table S1); the digested SaOMT2 fragment was ligated into the complementary sites of pNRG to generate pNRG-SaOMT2 (Additional file 1: Table S1). The expression plasmids, pNRG and pNRG-SaOMT2, were introduced into E. coli BL21(DE3), resulting in the formation of BL21/NRG and BL21/NRG-SaOMT2 (Table 1).
For producing apigenin in E. coli heterologous host, the pAPG plasmid containing PcFNS gene was constructed. The restriction sites were introduced in the synthetic PcFNS gene by performing PCR amplification using primers PcFNS_F/PcFNS_R (Additional file 1: Table S1). The PCR product was digested with KpnI/AvrII and cloned into the complementary sites of pNRG to generate pAPG. Plasmid pAPG-SaOMT2 was constructed to produce the methylated apigenin. The AvrII/NotI-digested SaOMT2 fragment was ligated into the complementary sites of pAPG to generate pAPG-SaOMT2. The expression plasmids, pAPG and pAPG-SaOMT2, were introduced into E. coli BL21(DE3), thereby resulting in the formation of BL21/APG and BL21/APG-SaOMT2 (Table 1).

In vivo bioconversion of SaOMT2 expressed in S. venezuelae
For in vivo bioconversion using the S. venezuelae expressing SaOMT2, the DHS2001/SaOMT2 strain was cultivated at 30 °C for 36 h in R2YE medium. 0.5 mL of seed cultures was inoculated into 50 mL of R2YE medium. After 48 h, the cultures were supplemented with 100 mM resveratrol, naringenin, or apigenin, which were subsequently incubated for an additional 40 h period prior to extraction.

In vitro reaction using purified SaOMT2 expressed in E. coli and S. venezuelae
For the expression of recombinant SaOMT2 in Streptomyces host, 3 mL of 36 h-old cultivated DHS2001/ SaOMT2-H were inoculated into 300 mL of fresh R2YE medium. Cells were grown at 30 °C for 48 h. Meanwhile, the BL21/SaOMT2-H strain was cultivated overnight in 10 mL of LB medium at 37 °C; subsequently, 4 mL of seed cultures were inoculated into 400 mL of LB medium [32]. The cells were cultured until an OD 600 of 0.5-0.6, and the gene expression in the cell culture was induced with 1 mM IPTG. Simultaneously, the incubation temperature was shifted from 37 to 30 °C, and the cells were grown for an additional 6 h period.
The harvested cells were resuspended in Tris-HCl (pH 7.0) buffer solution and lysed by sonication using a VCX 500 from Sonics & Materials, Inc. (Newtown, CT, USA). The cell lysates were separated by centrifugation for 30 min at 4 °C, and then the supernatant was purified using Ni 2+ -NTA agarose chromatography columns. After washing the column with an NPI-40 buffer (300 mM NaCl, 40 mM imidazole, 50 mM sodium phosphate, pH 8.0), histidine-tagged recombinant proteins were eluted using NPI-500 buffer (300 mM NaCl, 500 mM imidazole, 50 mM sodium phosphate, pH 8.0). The purified proteins were analyzed by 12.5% SDS-PAGE and visualized using Coomassie brilliant blue. Typically, approximately 11 mg/L and 9 mg/L of purified SaOMT2 proteins were obtained from DHS2001/SaOMT2-H and BL21/ SaOMT2-H, respectively.
The enzyme reaction involving purified SaOMT2 was carried out using a mixture comprising 25 mM Tris-HCl (pH 7.0) supplemented with 1.23 mM of purified SaOMT2, 100 mM apigenin, 100 μM MgCl 2 , and 100 μM SAM; this mixture was allowed to react for 12 h at 37 °C. The reaction was stopped by adding two volumes of EA and centrifuged at 13,000 rpm for 10 min. After the organic phase was evaporated to dryness, the residue was dissolved in MeOH. A portion of this solution was analyzed by UPLC-qTOF-HR-MS.

Production of phenylpropanoid using E. coli mutants
The seed culture broth that was subjected to overnight incubation was subsequently inoculated into 30 mL LB medium at an OD 600 value of 0.2 and cultivated until an OD 600 of 0.7-0.8 was reached. At that point, the IPTG was added into the culture broth until the final concentration became 1 mM and continuously incubated at 30 °C for another 3 h. The cells were harvested at 4 °C by centrifugation (10 min at 6000 rpm) and resuspended with 15 mL of fresh R2YE medium. The culture was supplemented with 1.2 mM 4-coumaric acid plus 1 mM IPTG and further incubated for 18 h at 30 °C.

Production of O-methylated phenylpropanoids using E. coli-Streptomyces cocultivation
For methylated phenylpropanoid production using E. coli-Streptomyces cocultivation, the seed cultures of E. coli mutants (BL21/PTS, BL21/NRG, or BL21/APG), which were subjected to overnight incubation, were inoculated into 100 mL LB medium at an OD 600 value of 0.2. The culture broth was incubated at 37 °C until an OD 600 of 0.7-0.8 was reached. Subsequently, the biosynthetic genes were induced with 1 mM IPTG, and the cells were grown at 30 °C for another 3 h. Meanwhile, 1 mL of 36 h-old cultivated DHS2001/SaOMT2 was inoculated into 100 mL of R2YE medium and grown at 30 °C for 48 h. E. coli and Streptomyces cells were mixed volumetrically according to the indicated inoculation ratios. The cocultures were fed with 1.2 mM of 4-coumaric acid plus 1 mM IPTG and allowed to grow for different periods at 30 °C.