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Metabolic engineering for resveratrol derivative biosynthesis in Escherichia coli.

Jeong YJ, Woo SG, An CH, Jeong HJ, Hong YS, Kim YM, Ryu YB, Rho MC, Lee WS, Kim CY - Mol. Cells (2015)

Bottom Line: The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes.However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes.These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Eco-friendly Bio-Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 580-185, Korea.

ABSTRACT
We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

No MeSH data available.


HPLC analysis of resveratrol and pinostilbene produced by recombinant E. coli. E. coli cells harboring CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3 constructs were cultured for 48 h prior to extraction. Chromatogram STD represents the authentic standards of resveratrol (Res), pinostilbene (Pino), and pterostilbene (Ptero) with retention times of 9.423, 19.327, and 32.009 min, respectively. Chromatograms Control, CCL+STS, CCL+STS-VrROMT, and CCL+STS-SbROMT3 represent the samples produced from p-coumaric acid by recombinant E. coli. Insets indicate the chromatograms magnified to show small amounts of pterostilbene production.
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f6-molce-38-4-318: HPLC analysis of resveratrol and pinostilbene produced by recombinant E. coli. E. coli cells harboring CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3 constructs were cultured for 48 h prior to extraction. Chromatogram STD represents the authentic standards of resveratrol (Res), pinostilbene (Pino), and pterostilbene (Ptero) with retention times of 9.423, 19.327, and 32.009 min, respectively. Chromatograms Control, CCL+STS, CCL+STS-VrROMT, and CCL+STS-SbROMT3 represent the samples produced from p-coumaric acid by recombinant E. coli. Insets indicate the chromatograms magnified to show small amounts of pterostilbene production.

Mentions: To further investigate the production of resveratrol and its methylated derivatives in E. coli by expression of major enzymes involved in stilbene biosynthesis, we reconstructed recombinant E. coli producing CCL, STS and ROMT (Fig. 1B). For production of resveratrol and pterostilbene, E. coli cells containing either pCOLADuet-CCL and pETDuet-STS, or pCOLADuet-CCL and pETDuet-ROMT-STS, were cultured in modified M9 medium with 1 mM p-coumaric acid for 60 hrs after IPTG induction. As a negative control, E. coli cells containing empty vectors of pETDuet-1 and pCOLADuet-1 were cultivated with p-coumaric acid. Increasing concentrations of p-coumaric acid up to 1 mM enhanced the resveratrol production, but not at higher concentrations than 1 mM (data not shown). Based on this result, 1 mM concentration of p-coumaric acid was used as a substrate for all subsequent experiments. The concentrations of resveratrol and pinostilbene produced in recombinant E. coli were determined by comparing with standard curves generated from known concentrations of resveratrol and pinostilbene. Figure 5 shows the time-course of resveratrol and pinostilbene production in recombinant E. coli cells. When p-coumaric acid was used as the precursor, resveratrol biosynthesis in recombinant E. coli cells carrying the constructs (CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3) was first detected 6 h after addition of p-coumaric acid, and gradually increased up to 36 h. The highest levels of resveratrol produced in recombinant E. coli were 1.8 mg/L at 36 h in CCL+STS, 1.9 mg/L at 60 h in CCL+STS-VrROMT, and 1.2 mg/L at 24 h in CCL+STS-SbROMT3 samples. Resveratrol production in CCL+STS-SbROMT3 did not increase further after 24 h, but rather decreased. This is likely due to the consumption of resveratrol by conversion to pinostilbene by the action of SbROMT3syn. As shown in Fig. 5B, pinostilbene production in CCL+STS-SbROMT3 greatly increased from 24 h and the maximal level (2.4 mg/L) was reached at 48 h, after which production decreased. In addition, a very small amount of pterostilbene was detected at 48 h, only in CCL+STS-SbROMT3 (Fig. 6), indicating a low enzyme activity of recombinant SbROMT3syn in producing pterostilbene from resveratrol. As a result, the culture cells containing the pCOLADuet-CCL and pETDuet-SbROMT3-STS plasmids (CCL+STS-SbROMT3) were capable of producing both resveratrol and pinostilbene as the major products from p-coumaric acid along with the minor product of pterostilbene. This is consistent with our previous results showing that recombinant SbROMT3syn catalyzed the production of pinostilbene as the main compound from resveratrol with in vitro and in vivo methylation assays (Jeong et al., 2014). However, no pinostilbene and pterostilbene were produced in control, CCL+STS and CCL-STS-VrROMT. This indicates that VrROMTsyn has no or very poor enzyme activity toward resveratrol as a substrate.


Metabolic engineering for resveratrol derivative biosynthesis in Escherichia coli.

Jeong YJ, Woo SG, An CH, Jeong HJ, Hong YS, Kim YM, Ryu YB, Rho MC, Lee WS, Kim CY - Mol. Cells (2015)

HPLC analysis of resveratrol and pinostilbene produced by recombinant E. coli. E. coli cells harboring CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3 constructs were cultured for 48 h prior to extraction. Chromatogram STD represents the authentic standards of resveratrol (Res), pinostilbene (Pino), and pterostilbene (Ptero) with retention times of 9.423, 19.327, and 32.009 min, respectively. Chromatograms Control, CCL+STS, CCL+STS-VrROMT, and CCL+STS-SbROMT3 represent the samples produced from p-coumaric acid by recombinant E. coli. Insets indicate the chromatograms magnified to show small amounts of pterostilbene production.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400306&req=5

f6-molce-38-4-318: HPLC analysis of resveratrol and pinostilbene produced by recombinant E. coli. E. coli cells harboring CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3 constructs were cultured for 48 h prior to extraction. Chromatogram STD represents the authentic standards of resveratrol (Res), pinostilbene (Pino), and pterostilbene (Ptero) with retention times of 9.423, 19.327, and 32.009 min, respectively. Chromatograms Control, CCL+STS, CCL+STS-VrROMT, and CCL+STS-SbROMT3 represent the samples produced from p-coumaric acid by recombinant E. coli. Insets indicate the chromatograms magnified to show small amounts of pterostilbene production.
Mentions: To further investigate the production of resveratrol and its methylated derivatives in E. coli by expression of major enzymes involved in stilbene biosynthesis, we reconstructed recombinant E. coli producing CCL, STS and ROMT (Fig. 1B). For production of resveratrol and pterostilbene, E. coli cells containing either pCOLADuet-CCL and pETDuet-STS, or pCOLADuet-CCL and pETDuet-ROMT-STS, were cultured in modified M9 medium with 1 mM p-coumaric acid for 60 hrs after IPTG induction. As a negative control, E. coli cells containing empty vectors of pETDuet-1 and pCOLADuet-1 were cultivated with p-coumaric acid. Increasing concentrations of p-coumaric acid up to 1 mM enhanced the resveratrol production, but not at higher concentrations than 1 mM (data not shown). Based on this result, 1 mM concentration of p-coumaric acid was used as a substrate for all subsequent experiments. The concentrations of resveratrol and pinostilbene produced in recombinant E. coli were determined by comparing with standard curves generated from known concentrations of resveratrol and pinostilbene. Figure 5 shows the time-course of resveratrol and pinostilbene production in recombinant E. coli cells. When p-coumaric acid was used as the precursor, resveratrol biosynthesis in recombinant E. coli cells carrying the constructs (CCL+STS, CCL+STS-VrROMT, or CCL+STS-SbROMT3) was first detected 6 h after addition of p-coumaric acid, and gradually increased up to 36 h. The highest levels of resveratrol produced in recombinant E. coli were 1.8 mg/L at 36 h in CCL+STS, 1.9 mg/L at 60 h in CCL+STS-VrROMT, and 1.2 mg/L at 24 h in CCL+STS-SbROMT3 samples. Resveratrol production in CCL+STS-SbROMT3 did not increase further after 24 h, but rather decreased. This is likely due to the consumption of resveratrol by conversion to pinostilbene by the action of SbROMT3syn. As shown in Fig. 5B, pinostilbene production in CCL+STS-SbROMT3 greatly increased from 24 h and the maximal level (2.4 mg/L) was reached at 48 h, after which production decreased. In addition, a very small amount of pterostilbene was detected at 48 h, only in CCL+STS-SbROMT3 (Fig. 6), indicating a low enzyme activity of recombinant SbROMT3syn in producing pterostilbene from resveratrol. As a result, the culture cells containing the pCOLADuet-CCL and pETDuet-SbROMT3-STS plasmids (CCL+STS-SbROMT3) were capable of producing both resveratrol and pinostilbene as the major products from p-coumaric acid along with the minor product of pterostilbene. This is consistent with our previous results showing that recombinant SbROMT3syn catalyzed the production of pinostilbene as the main compound from resveratrol with in vitro and in vivo methylation assays (Jeong et al., 2014). However, no pinostilbene and pterostilbene were produced in control, CCL+STS and CCL-STS-VrROMT. This indicates that VrROMTsyn has no or very poor enzyme activity toward resveratrol as a substrate.

Bottom Line: The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes.However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes.These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Eco-friendly Bio-Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 580-185, Korea.

ABSTRACT
We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

No MeSH data available.