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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.


Related in: MedlinePlus

LC-MS analysis of resveratrol, pinostilbene and pterostilbene produced by recombinant E. coli cells. The molecular masses of resveratrol, pinostilbene and pterostilbene were analyzed with a linear ion trap quadrupole LC-MS at a positive mode of atmospheric pressure chemical ionization. Samples were taken from cell culture at 48 h after the addition of p-coumaric acid, and the extracted compounds were analyzed by LC-MS. The injection volume was 10 μl. Chromatograms (A), (B) and (C) represent the LC-MS spectra showing the peaks at m/z 229.2, 243.1 and 257.1 for reveratrol, pinostilbene and pterostilbene, respectively.
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f7-molce-38-4-318: LC-MS analysis of resveratrol, pinostilbene and pterostilbene produced by recombinant E. coli cells. The molecular masses of resveratrol, pinostilbene and pterostilbene were analyzed with a linear ion trap quadrupole LC-MS at a positive mode of atmospheric pressure chemical ionization. Samples were taken from cell culture at 48 h after the addition of p-coumaric acid, and the extracted compounds were analyzed by LC-MS. The injection volume was 10 μl. Chromatograms (A), (B) and (C) represent the LC-MS spectra showing the peaks at m/z 229.2, 243.1 and 257.1 for reveratrol, pinostilbene and pterostilbene, respectively.

Mentions: We further analyzed the 48 h culture cells by HPLC because of the high-level production of resveratrol, pinostilbene and pterostilbene at that time. When analyzing the culture cells, as shown in Fig. 6, a major peak corresponding to resveratrol was detected at the same retention times (9.694, 9.688 and 9.692 min in CCL+STS, CCL+STS-VrROMT and CCL+STS-SbROMT3 samples, respectively) as that of authentic resveratrol (9.423 min). Furthermore, another major peak corresponding to pinostilbene was observed at 19.825 min retention time, but only in CCL+ STS-SbROMT3. High-level production of resveratrol was detected in CCL+STS and CCL+STS-VrROMT, but the resveratrol level decreased after 24 h and the pinostilbene production instead increased in CCL+STS-SbROMT3 (Figs. 5 and 6). A very weak peak corresponding to pterostilbene, as shown in Fig. 6 (inset chromatograms), was also observed in CCL+STS-SbROMT3 only (retention time, 32.454 min). To confirm production of resveratrol, pinostilbene and pterostilbene in recombinant E. coli, subsequent analysis was carried out with LC-MS using 48 h cells. As shown in Fig. 7, the compounds in the peaks of the CCL+STS-SbROMT3 sample shown in Fig. 6 exhibited parent ion peaks [M+H]+ at m/z 229.2, 243.1 and 257.1. The corresponding mass spectra for these peaks revealed the compounds to be resveratrol, pinostilbene and pterostilbene, respectively. Collectively, the HPLC and LC-MS analyses indicate that recombinant E. coli cells co-expressing CCL, STS and ROMT proteins and fed with p-coumaric acid as the precursor are capable of producing stilbene compounds, such as resveratrol and pinostilbene as the major product, along with pterostilbene as a minor product.


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)

LC-MS analysis of resveratrol, pinostilbene and pterostilbene produced by recombinant E. coli cells. The molecular masses of resveratrol, pinostilbene and pterostilbene were analyzed with a linear ion trap quadrupole LC-MS at a positive mode of atmospheric pressure chemical ionization. Samples were taken from cell culture at 48 h after the addition of p-coumaric acid, and the extracted compounds were analyzed by LC-MS. The injection volume was 10 μl. Chromatograms (A), (B) and (C) represent the LC-MS spectra showing the peaks at m/z 229.2, 243.1 and 257.1 for reveratrol, pinostilbene and pterostilbene, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

f7-molce-38-4-318: LC-MS analysis of resveratrol, pinostilbene and pterostilbene produced by recombinant E. coli cells. The molecular masses of resveratrol, pinostilbene and pterostilbene were analyzed with a linear ion trap quadrupole LC-MS at a positive mode of atmospheric pressure chemical ionization. Samples were taken from cell culture at 48 h after the addition of p-coumaric acid, and the extracted compounds were analyzed by LC-MS. The injection volume was 10 μl. Chromatograms (A), (B) and (C) represent the LC-MS spectra showing the peaks at m/z 229.2, 243.1 and 257.1 for reveratrol, pinostilbene and pterostilbene, respectively.
Mentions: We further analyzed the 48 h culture cells by HPLC because of the high-level production of resveratrol, pinostilbene and pterostilbene at that time. When analyzing the culture cells, as shown in Fig. 6, a major peak corresponding to resveratrol was detected at the same retention times (9.694, 9.688 and 9.692 min in CCL+STS, CCL+STS-VrROMT and CCL+STS-SbROMT3 samples, respectively) as that of authentic resveratrol (9.423 min). Furthermore, another major peak corresponding to pinostilbene was observed at 19.825 min retention time, but only in CCL+ STS-SbROMT3. High-level production of resveratrol was detected in CCL+STS and CCL+STS-VrROMT, but the resveratrol level decreased after 24 h and the pinostilbene production instead increased in CCL+STS-SbROMT3 (Figs. 5 and 6). A very weak peak corresponding to pterostilbene, as shown in Fig. 6 (inset chromatograms), was also observed in CCL+STS-SbROMT3 only (retention time, 32.454 min). To confirm production of resveratrol, pinostilbene and pterostilbene in recombinant E. coli, subsequent analysis was carried out with LC-MS using 48 h cells. As shown in Fig. 7, the compounds in the peaks of the CCL+STS-SbROMT3 sample shown in Fig. 6 exhibited parent ion peaks [M+H]+ at m/z 229.2, 243.1 and 257.1. The corresponding mass spectra for these peaks revealed the compounds to be resveratrol, pinostilbene and pterostilbene, respectively. Collectively, the HPLC and LC-MS analyses indicate that recombinant E. coli cells co-expressing CCL, STS and ROMT proteins and fed with p-coumaric acid as the precursor are capable of producing stilbene compounds, such as resveratrol and pinostilbene as the major product, along with pterostilbene as a minor product.

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.


Related in: MedlinePlus