Characterization of the resveratrol O-methyltransferase via a bioconversion experiment in recombinant E. coli
Previous reports have noted that the two resveratrol O-methyltransferase genes (sbOMT1 and sbOMT3) from Sorghum bicolor are capable of using resveratrol as a substrate that yields methylated analogs of resveratrol [12, 13]. It was claimed that the sbOMT3 O-methyltransferase catalyzes the A-ring specific 3,5-bis-O-methylation of resveratrol, which in turn yields pterostilbene (3,5-dimethoxy-4’-hydroxystilbene) in the co-expression system of sbOMT3 with a stilbene synthase from peanuts (AhSTS3) [13]. In addition, sbOMT1, which had previously been identified as a potential eugenol O-methyltransferase, predominantly catalyzes the resveratrol B-ring (4’-O-methylation), which yields 3,5-dihydroxy-4’-methoxystilbene [12, 13].
In this study, the functions of the codon-optimized resveratrol O-methyltransferase genes (sbOMT1 and sbOMT3) were re-evaluated using a bioconversion experiment with resveratrol in recombinant E. coli. The codon-optimized synthetic sbOMT1 and sbOMT3 genes were cloned in the expression vector pET-22b(+) on the NdeI/HindIII sites (pET22-sbCOM1 and pET22-sbCOM3, respectively; Figure 2). Each construct was transformed in the E. coli C41 (DE3) cells, and the recombinant E. coli was selected based on the expression analysis using SDS-PAGE (Additional file 1: Figure S1). In order to investigate whether the recombinant O-methyltransferases can catalyze the production of methylated resveratrol derivatives, resveratrol was added to the cultured recombinant E. coli harboring pET22-sbCOM1 and pET22-sbCOM3. The culture broth and bacterial cells were collected after 36 hours and were then subjected to HPLC and LC/MS analyses (Figure 3). Under the bioconversion conditions employed in this study most of the feeding resveratrol disappeared and each methylated form was detected as a main peak in the HPLC (Figure 3A). However, when the bioconversion rate was calculated based on a quantitative comparison with feeding substrates and the products, the recombinant E. coli harboring pET22-sbCOM1 and pET22-sbCOM3 showed roughly 42% and 12% conversion ratios, respectively (data not shown). These bioconversion ratios are consistent with results recently reported by Jeong et al.[16]. It is presumed that a significant amount of additive resveratrol was decomposed in the E. coli culture medium.
The recombinant E. coli cells that harbored the pET22-sbCOM1 plasmid produced a 12.2 min retention time peak, which is a slightly later retention time than the pinostilbene (3,4’-dihydroxy-5-methoxystilbene) as seen in Figure 3A(d). The major peak in Figure 3A(d) exhibited parent mass ion peaks at m/z 243.05 [M + H]+, which corresponded to one methylation of resveratrol (an addition of 14 Da; Figure 3B(a)). It was expected that this methylated compound could have a methoxy group located in the 4’ position in the B-ring (Figure 1). In the pET22-sbCOM3 clone, a major peak was present at the same retention time (11.6 min) as that in the HPLC analysis and at the same mass ion peaks at m/z 243.08 [M + H]+ with authentic pinostilbene, which is one methylation of resveratrol (Figure 3B(b)). However, this recombinant produced a very low level minor peak of 15.9 min in the HPLC, which was the same retention time as the pterostilbene (3,5-dimethoxy-4’-hydroxystilbene; Figure 3A(e)). This minor peak was also accepted based on the mass spectra as m/z 257.13 [M + H]+, which corresponded to two methylations of resveratrol (an addition of 28 Da; Figure 3B(c)). However, the peak intensity was not sufficient to allow a detailed structural characterization. These results exhibit a controversial conclusion that contrasts with the results reported previously by Rimando et al., who demonstrated that the sbOMT3 produced pterostilbene as a major product [13]. However, our results agreed with Jeong et al.’s reported results with the in vitro and bioconversion activity of the sbOMT3 [16].
Construction of artificial biosynthetic pathways for the production of methylated resveratrol analogs in E. coli
We have previously produced resveratrol in E. coli harboring an artificial biosynthetic gene cluster in which the TAL from Saccharothrix espanaensis, CCL from Streptomyces coelicolor, and STS from the peanut plant Arachis hypogaea were contained [18]. In order to produce the methylated resveratrol analogs in E. coli using a simple sugar medium, a series of plasmids containing the artificial biosynthetic pathway were constructed (Figure 2). The artificial resveratrol and methylated resveratrol biosynthetic plasmids were constructed using the previously described cloning methods [18], and each plasmid contained genes with their own T7 promoter, ribosome-binding site (RBS), and terminator sequence as in the parental vectors. In this study, a new resveratrol-producing construct of pET-opTLS, which contained codon-optimized tal and sts genes, and cloned ccl gene from S. coelicolor, was constructed using previously reported cloning method of the parental vector pET-TLkS [18]. The E. coli cells with the pET-opTLS clone exhibited a higher production yield of resveratrol (5.2 mg/L) in the culture system with a modified M9 medium compared with the original pET-TLkS clone (1.4 mg/L) [18]. The cause of this improvement remains unknown, but it is possible that these protein expression ratios may have better optimized the resveratrol production than the original combination.
In order to construct an expression vector that contains the additional O-methyltransferase gene(s) that are under the control of the T7 promoter, the DNA fragment containing the promoter, O-methyltransferase coding region, and terminator using pET22-sbCOM1 and pET22-sbCOM3 plasmids as templates were amplified. Then, the amplified fragments were inserted into pET-opTLS, which resulted in pET-opTLO1S and pET-opTLO3S, respectively. Similarly, in order to construct the plasmid containing the two O-methyltransferases biosynthetic pathways, the O-methyltransferase fragment containing the sbOMT3 coding region was inserted into the pET-opTLO1S, which resulted in pET-opTLO13S. This is useful assembly method for the reconstruction of multi-enzyme biosynthetic pathways such as stilbenes and flavones biosynthesis.
The recombination cells that harbor the artificial biosynthetic gene cluster were cultured in a modified M9 medium. A comparison of the fermentation products of the E. coli cells that harbored pET-opTLS, pET-opTLO1S, pET-opTLO3S, and pET-opTLO13S revealed that new peaks were detected in the engineered strain (Figure 4). The retention times for peaks 1 and 2, which were produced in the recombinant pET-opTLO3S, were identical to those of the original resveratrol and pinostilbene, respectively (Figure 4(e)). These peaks were further analyzed using LC/MS/MS in the positive ion mode. Using the LC/MS analysis, peak 1 (resveratrol) and peak 2 (pinostilbene) were identified as m/z 229.02 [M + H]+ and m/z 243.08 [M + H]+, respectively, when comparing the obtained fragmentation pattern with that of the original standards. Furthermore, peak 3, which was produced in the recombinant pET-opTLO1S and pET-opTLO13S, was identified as m/z 243.05 [M + H]+ using the LC/MS. However, the MS/MS analysis of peak 3 exhibited different fragments in the ion pattern with the pinostilbene, and the peaks of m/z 134.88 [M + H]+ that represent the two hydroxyl group located in the A-ring was also identified (Figure 5(a)). The fragment ion at m/z 134.88 [M + H]+, which contained the phenolic A-ring of a compound with a triple bond, has already been reported as a distinctive mass fragment of resveratrol [26]. The A-ring cleavage of the resveratrol produced a mass peak of a triple bond (A-ring) at m/z 134.86 [M + H]+ due to the hydroxy group at the C-3 and C-5 positions (Figure 5(c)). However, due to the presence of a methoxy group in the A-ring in pinostilbene, the triple bond mass peak appeared at m/z 148.89 [M + H]+. In addition, peak 5 exhibited a very similar retention time and the same mass peak (m/z 257.13 [M + H]+) as pterostilbene (Additional file 1: Figure S2); however, the major daughter ion peaks of m/z 148.89 [M + H]+ represented one methoxy group located in the A-ring (Figure 5(b)). Interestingly, the mass fragmentation pattern of pterostilbene, which was found in a detectable amount in the bioconversion experiment with the recombinant pET22-sbCOM3 as mentioned earlier, was not detected in the recombinant pET-opTLO3S and pET-opTLO13S. These results support that peak 5 was not pterostilbene (3,5-dimethoxy-4’-hydroxystilbene), but rather that it was 3,4’-dimethoxy-5-hydroxystilbene, which has methoxy groups located in both the A-ring and B-ring.
Production and purification of methylated resveratrol analogs in E. colivia artificial biosynthetic pathways
The 3,4’-dimethoxy-5-hydroxystilbene synthesis levels in the E. coli cells that harbored pET-opTLO13S were not sufficient for structure elucidation. For a higher production of the bis-methylated resveratrol, the recombinant E. coli cells that harbored pET-opTLO13S were investigated using metabolite pattern analyses according to the culture times for 85 hours (Figure 6). This recombinant strain produced a relatively large amount of 3,5-dihydroxy-4’-methoxystilbene from the early stages of the culture. However, at the late stage of the culture, the resveratrol was consumed and the production of methylated resveratrol analogs slightly increased. The production level of 3,4’-dimethoxy-5-hydroxystilbene (peak 5) increased following the culturing and a new peak (peak 6) was also detected that exhibited a more delayed retention time (Figure 6A). The new peak was identified via LC/MS (m/z 271.14 [M + H]+) and it corresponded to the three methylations of resveratrol (an addition of 42 Da; Additional file 1: Figure S3). This result represents the methoxy moiety located at the C-3 and C-5 positions in the A-ring and the C-4’ position in the B-ring (Figure 1).
In order to obtain NMR-accessible amounts from the present culture conditions, it was scaled up to 4 liters of E. coli fermentation that harbored pET-opTLO13S. Three methylated resveratrol analogs were purified from the large culture broth. The mass spectra of the purified methylated resveratrol analogs (3,5-dihydroxy-4’-methoxystilbene, 3,4’-dimethoxy-5-hydroxystilbene, and 3,5,4’-trimethoxystilbene) are characterized with mass peaks at m/z 243, 257, and 271 [M + H]+, respectively. The 1H NMR spectra of the purified compounds were similar to those of the one, bi, and tri-methyl analogs that were isolated, respectively (Additional file 2: Table S1). The structures of the purified resveratrol analogs were identified through spectral data interpretation and comparison with the values reported in the literature [27]. The coexistence of sbOMT1 and sbOMT3 produced a relatively large amount of 3,5-dihydroxy-4’-methoxystilbene from the early stage of the culture; however, the pinostilbene was not significantly detected (Figure 6). These results indicate that sbOMT1 has superior methylation activity against resveratrol compared with that of sbOMT3. However, at the late stage of the culture, the production yield of 3,4’-dimethoxy-5-hydroxystilbene increased; it finally produced tri-methyl resveratrol (3,5,4’-trimethoxystilbene) (Figure 6B). Therefore, 3,4’-dimethoxy-5-hydroxystilbene is a major bis-methylated intermediate in the biosynthesis of 3,5,4’-trimethoxystilbene in this E. coli system. These results indicate that sbOMT3 has methylation activity in the A ring hydroxyl group against resveratrol as well as 3,5-dihydroxy-4’-methoxystilbene. Furthermore, the presence of 3,5,4’-trimethoxystilbene without the detection of pterostilbene indicates that the C-5 methylation is expected to be the final modification step in the coexistence of the sbOMT1 and sbOMT3 enzymes.
Although the production yield of these compounds is too low for commercial purposes, the results presented here were obtained using the wild type E. coil. This is the first report of an artificial biosynthetic pathway to obtain methylated resveratrol compounds used a single vector system joined with resveratrol biosynthetic genes and two resveratrol O-methyltransferase genes. The recombinant E. coli produces methylated resveratrol analogs beginning with simple fermentation rather than with bioconversion of intermediates. Recently, large-scale production of plant metabolites via metabolic engineered microbes has provided a promising alternative to chemical synthesis and extraction from raw plant sources [28, 29]. Further application with a metabolically optimized host strain, i.e. a tyrosine and/or malonyl CoA high producer [23, 24], may be very useful for obtaining industrial scale production yields.