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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.


Expression of His-tagged plant STS and ScCCL recombinant proteins and time-course production of resveratrol in E. coli cells. (A) E. coli cells harboring both pCOLADuet-H::CCL and pET22b-STS::H (CCL+STS) constructs were grown in modified M9 media and induced by the addition of 0.5 mM IPTG at 25°C for 2 h and 4 h. Expression of His-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag antibody. M, protein molecular marker in kDa; S, soluble fraction (20 μg); P, insoluble pellet fraction (1 μg). Arrows and arrowheads indicate the expressed ScCCL and plant STS recombinant proteins, respectively. (B) E. coli cells harboring CCL+STS constructs were cultured and harvested at the indicated time points after the addition of 1 mM p-coumaric acid to the pre-induced cells. E. coli cells carrying both pCOLADuet-1 and pET-22b were used as a control. Samples were extracted with ethyl acetate and subjected to HPLC analysis. E. coli cells were pre-induced to express recombinant proteins by the addition of 0.1 mM IPTG. (C) HPLC analysis was performed using a C18 reverse-phase column. Chromatogram STD represents the authentic standards of resveratrol (Res) and p-coumaric acid (p-Cou) with retention times of 9.887 and 4.907 min, respectively.
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f2-molce-38-4-318: Expression of His-tagged plant STS and ScCCL recombinant proteins and time-course production of resveratrol in E. coli cells. (A) E. coli cells harboring both pCOLADuet-H::CCL and pET22b-STS::H (CCL+STS) constructs were grown in modified M9 media and induced by the addition of 0.5 mM IPTG at 25°C for 2 h and 4 h. Expression of His-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag antibody. M, protein molecular marker in kDa; S, soluble fraction (20 μg); P, insoluble pellet fraction (1 μg). Arrows and arrowheads indicate the expressed ScCCL and plant STS recombinant proteins, respectively. (B) E. coli cells harboring CCL+STS constructs were cultured and harvested at the indicated time points after the addition of 1 mM p-coumaric acid to the pre-induced cells. E. coli cells carrying both pCOLADuet-1 and pET-22b were used as a control. Samples were extracted with ethyl acetate and subjected to HPLC analysis. E. coli cells were pre-induced to express recombinant proteins by the addition of 0.1 mM IPTG. (C) HPLC analysis was performed using a C18 reverse-phase column. Chromatogram STD represents the authentic standards of resveratrol (Res) and p-coumaric acid (p-Cou) with retention times of 9.887 and 4.907 min, respectively.

Mentions: ScCCL gene product was shown to have distinct plant 4CL activity as a functional homologue (Choi et al., 2011; Kaneko et al., 2003). Thus, we used the ScCCL gene for the production of p-coumaroyl-CoA from p-coumaric acid. To examine whether plant STS proteins are capable of producing resveratrol from p-coumaric acid, the pET22b-STS::H constructs were co-expressed with the pCOLADuet-H::CCL plasmid in E. coli (Fig. 1B). The expression of His-tagged STS and CCL proteins was confirmed by Western blot analysis with anti-His-tag antibody (Fig. 2A). The recombinant His-ScCCL protein corresponding to its theoretical molecular weight (MW) of 57.5 kDa was expressed well at 2 and 4 h after IPTG induction in the soluble and insoluble fractions. However, the most portions of the expressed STS recombinant proteins with the theoretical MW of 45.3 kDa were observed in the insoluble fraction. Only AhSTS recombinant protein was detectable at low level in the soluble fraction but not in the RpSTS and VrSTS. We further tested whether co-expression of STS and CCL recombinant proteins in E. coli cells can produce resveratrol from p-coumaric acids. E. coli cells containing pET22b-STS and pCOLADuet-CCL were cultured in modified M9 medium with 1 mM p-coumaric acid for 60 h after IPTG induction. The culture broth and bacterial cells were collected at the indicated time points and the extracted samples were subjected to HPLC analysis (Figs. 2B and 2C). HPLC analysis indicates that a major peak corresponding to resveratrol was detected at the same retention time (9.904 min) as that of standard resveratrol (9.887 min) only in the pET22b-AhSTS and pCOLADuet-CCL culture sample. Out of three STS constructs, only AhSTS was able to produce significant amounts of resveratrol from p-coumaric acids when added to the E. coli cells. The resveratrol level began to increase at 6 h followed by dramatic increase at 12 h, continuing to accumulate up to 60 h after the addition of p-coumaric acid. The highest level (3.3 mg/L) of resveratrol was detected at 48 h in the pET22b-AhSTS culture cells. By contrast, the other pET22b-RpSTS and pET22b-VrSTS failed to convert effectively p-coumaric acid to resveratrol. This differences among plant STS is likely due to the solubility of the STS recombinant proteins in E. coli. As shown in Fig. 2A, the AhSTS recombinant protein showed better expression than RpSTS and VrSTS in the soluble fraction. This suggests that the solubility of recombinant proteins is one of important factors for the better production of resveratrol in E. coli. Thus, to improve the solubility of recombinant RpSTS protein in E. coli, the original RpSTS gene was synthesized following codon-optimization using bacterial-preferred codon usage. In this paper, the codon-optimized synthetic RpSTSsyn was used for further experiments.


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)

Expression of His-tagged plant STS and ScCCL recombinant proteins and time-course production of resveratrol in E. coli cells. (A) E. coli cells harboring both pCOLADuet-H::CCL and pET22b-STS::H (CCL+STS) constructs were grown in modified M9 media and induced by the addition of 0.5 mM IPTG at 25°C for 2 h and 4 h. Expression of His-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag antibody. M, protein molecular marker in kDa; S, soluble fraction (20 μg); P, insoluble pellet fraction (1 μg). Arrows and arrowheads indicate the expressed ScCCL and plant STS recombinant proteins, respectively. (B) E. coli cells harboring CCL+STS constructs were cultured and harvested at the indicated time points after the addition of 1 mM p-coumaric acid to the pre-induced cells. E. coli cells carrying both pCOLADuet-1 and pET-22b were used as a control. Samples were extracted with ethyl acetate and subjected to HPLC analysis. E. coli cells were pre-induced to express recombinant proteins by the addition of 0.1 mM IPTG. (C) HPLC analysis was performed using a C18 reverse-phase column. Chromatogram STD represents the authentic standards of resveratrol (Res) and p-coumaric acid (p-Cou) with retention times of 9.887 and 4.907 min, respectively.
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Related In: Results  -  Collection

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f2-molce-38-4-318: Expression of His-tagged plant STS and ScCCL recombinant proteins and time-course production of resveratrol in E. coli cells. (A) E. coli cells harboring both pCOLADuet-H::CCL and pET22b-STS::H (CCL+STS) constructs were grown in modified M9 media and induced by the addition of 0.5 mM IPTG at 25°C for 2 h and 4 h. Expression of His-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag antibody. M, protein molecular marker in kDa; S, soluble fraction (20 μg); P, insoluble pellet fraction (1 μg). Arrows and arrowheads indicate the expressed ScCCL and plant STS recombinant proteins, respectively. (B) E. coli cells harboring CCL+STS constructs were cultured and harvested at the indicated time points after the addition of 1 mM p-coumaric acid to the pre-induced cells. E. coli cells carrying both pCOLADuet-1 and pET-22b were used as a control. Samples were extracted with ethyl acetate and subjected to HPLC analysis. E. coli cells were pre-induced to express recombinant proteins by the addition of 0.1 mM IPTG. (C) HPLC analysis was performed using a C18 reverse-phase column. Chromatogram STD represents the authentic standards of resveratrol (Res) and p-coumaric acid (p-Cou) with retention times of 9.887 and 4.907 min, respectively.
Mentions: ScCCL gene product was shown to have distinct plant 4CL activity as a functional homologue (Choi et al., 2011; Kaneko et al., 2003). Thus, we used the ScCCL gene for the production of p-coumaroyl-CoA from p-coumaric acid. To examine whether plant STS proteins are capable of producing resveratrol from p-coumaric acid, the pET22b-STS::H constructs were co-expressed with the pCOLADuet-H::CCL plasmid in E. coli (Fig. 1B). The expression of His-tagged STS and CCL proteins was confirmed by Western blot analysis with anti-His-tag antibody (Fig. 2A). The recombinant His-ScCCL protein corresponding to its theoretical molecular weight (MW) of 57.5 kDa was expressed well at 2 and 4 h after IPTG induction in the soluble and insoluble fractions. However, the most portions of the expressed STS recombinant proteins with the theoretical MW of 45.3 kDa were observed in the insoluble fraction. Only AhSTS recombinant protein was detectable at low level in the soluble fraction but not in the RpSTS and VrSTS. We further tested whether co-expression of STS and CCL recombinant proteins in E. coli cells can produce resveratrol from p-coumaric acids. E. coli cells containing pET22b-STS and pCOLADuet-CCL were cultured in modified M9 medium with 1 mM p-coumaric acid for 60 h after IPTG induction. The culture broth and bacterial cells were collected at the indicated time points and the extracted samples were subjected to HPLC analysis (Figs. 2B and 2C). HPLC analysis indicates that a major peak corresponding to resveratrol was detected at the same retention time (9.904 min) as that of standard resveratrol (9.887 min) only in the pET22b-AhSTS and pCOLADuet-CCL culture sample. Out of three STS constructs, only AhSTS was able to produce significant amounts of resveratrol from p-coumaric acids when added to the E. coli cells. The resveratrol level began to increase at 6 h followed by dramatic increase at 12 h, continuing to accumulate up to 60 h after the addition of p-coumaric acid. The highest level (3.3 mg/L) of resveratrol was detected at 48 h in the pET22b-AhSTS culture cells. By contrast, the other pET22b-RpSTS and pET22b-VrSTS failed to convert effectively p-coumaric acid to resveratrol. This differences among plant STS is likely due to the solubility of the STS recombinant proteins in E. coli. As shown in Fig. 2A, the AhSTS recombinant protein showed better expression than RpSTS and VrSTS in the soluble fraction. This suggests that the solubility of recombinant proteins is one of important factors for the better production of resveratrol in E. coli. Thus, to improve the solubility of recombinant RpSTS protein in E. coli, the original RpSTS gene was synthesized following codon-optimization using bacterial-preferred codon usage. In this paper, the codon-optimized synthetic RpSTSsyn was used for further experiments.

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.