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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.


(A) Biosynthetic pathway of stilbene compound production from phenylalanine and (B) construction of recombinant plasmids carrying the genes (ScCCL, STS, the synthetic RpSTSsyn, VrROMTsyn, and SbROMT3syn) involved in stilbene biosynthesis. The sequential actions of PAL or TAL, C4H, 4CL (CCL), STS, and ROMT result in the conversion of phenylalanine to stilbenes, resveratrol, and its methylated derivatives. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; TAL, tyrosine ammonia-lyase; 4CL, 4-coumarate:coenzyme A ligase; CCL, cinnamate/4-coumarate:coenzyme A ligase; STS, stilbene synthase; ROMT, resveratrol O-methyltransferase; T7 Pro, T7 RNA polymerase promoter; H, His-tag; S, S-tag.
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f1-molce-38-4-318: (A) Biosynthetic pathway of stilbene compound production from phenylalanine and (B) construction of recombinant plasmids carrying the genes (ScCCL, STS, the synthetic RpSTSsyn, VrROMTsyn, and SbROMT3syn) involved in stilbene biosynthesis. The sequential actions of PAL or TAL, C4H, 4CL (CCL), STS, and ROMT result in the conversion of phenylalanine to stilbenes, resveratrol, and its methylated derivatives. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; TAL, tyrosine ammonia-lyase; 4CL, 4-coumarate:coenzyme A ligase; CCL, cinnamate/4-coumarate:coenzyme A ligase; STS, stilbene synthase; ROMT, resveratrol O-methyltransferase; T7 Pro, T7 RNA polymerase promoter; H, His-tag; S, S-tag.

Mentions: Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic phytoalexin produced by a few plant species, such as grapes, peanuts, and berries, under biotic or abiotic stress conditions such as pathogen infection and UV irradiation. Resveratrol has diverse properties beneficial to health, including anti-inflammatory effects, anti-tumor activity, and anti-aging effects (Baur et al., 2006; Frémont, 2000; Ulrich et al., 2005). However, the beneficial effects of resveratrol are limited due to its instability when exposed to light and oxygen or in environments with harsh pH conditions. These stimuli may cause trans-to-cis isomerization or oxidation that leads to a reduction in the bioavailability and bioactivity of the compound (Walle et al., 2004). For this reason, it is important to develop resveratrol derivatives with enhanced stability. Furthermore, structure activity studies revealed that the substitution of hydroxyl groups of resveratrol with methoxy groups substantially potentiate its cytotoxic activity (Lee et al., 2003). The substitution of hydroxy with methoxy groups may give methylated resveratrol derivatives an increased lipophilicity compared to resveratrol, resulting in better bioavailability (Remsberg et al., 2008). For these reasons, the metabolic engineering of resveratrol and its methylated derivatives in plants and microbes is of great interest for the development of more stable and potent chemoprotective agents. There are various naturally occurring methylated derivatives, including pinostilbene (3,4′-dihydroxy-5-methoxy-trans-stilbene), pterostilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene), 3,4′,5-trimethoxystilbene, and desoxyrhapotigenin (3,5-dihydroxy-4′-methoxy-trans-stilbene) (Wang et al., 2010), but their biological activities remain largely unknown. One effective approach for stabilization of resveratrol is to produce methylated resveratrol derivatives via enzymatic O-methyltransaferases (OMTs). Plant OMTs constitute a large family of enzymes that methylate the hydroxyl groups of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids. However, a few plant OMT genes have been isolated and characterized as possible resveratrol OMTs (ROMT) (Rimando et al., 2012; Schmidlin et al, 2008). We previously reported that SbROMT3syn catalyzed the methylation of resveratrol, yielding pinostilbene as the major product alongside pterostilbene as a minor product (Jeong et al., 2014). More studies are required to determine the enzymatic functions of various plant OMTs using biochemical approaches. Rhubarb (Rheum) species have been used as medicinal plants in Asian traditional medicine to treat constipation, inflammation and cancer (Foust, 1992; Lee et al., 2012). They are rich in polyketides, including stilbenes, phenylbutanoids, anthraquinones, and naphthalenes (Kashiwada et al., 1988). Six stilbene derivatives such as desoxyrhapontigenin, rhapontigenin, trans-resveratrol, piceatannol, piceatannol-3′-O-β-d-glucopyrano-side, and isorhapontin were isolated from rhubarb rhizomes. These stilbene derivatives showed a good anti-inflammatory activity (Lee et al., 2012). However, little is known about stilbene biosynthesis in rhubarb plants. Furthermore, there are still no reports on metabolic engineering of stilbene biosynthesis in microbes using stilbene biosynthetic genes from rhubarb plants. In plants, resveratrol is synthesized by STS action as a branch from the phenylpropanoid pathway (Sparvoli et al., 1994). As an intermediate, p-coumaric acid is converted to p-coumaroyl-CoA by 4-coumrate:coenzyme A ligase (4CL). STS catalyzes the condensation of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA as substrates. Methylated resveratrol derivatives of pinostilbene and pterostilbene are produced by ROMT from resveratrol (Fig. 1). Extensive metabolic engineering studies have been carried out to increase the production of resveratrol in plants and microbes (Becker et al., 2003; Beekwilder et al., 2006; Delaunois et al., 2009; Horinouchi, 2009; Katsuyama et al., 2007; Melchior and Kindl, 1990; Ververidis et al., 2007; Watts et al., 2006; Zhang et al., 2006). However, little is known about the production of methylated resveratrol derivatives in recombinant microorganisms.


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)

(A) Biosynthetic pathway of stilbene compound production from phenylalanine and (B) construction of recombinant plasmids carrying the genes (ScCCL, STS, the synthetic RpSTSsyn, VrROMTsyn, and SbROMT3syn) involved in stilbene biosynthesis. The sequential actions of PAL or TAL, C4H, 4CL (CCL), STS, and ROMT result in the conversion of phenylalanine to stilbenes, resveratrol, and its methylated derivatives. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; TAL, tyrosine ammonia-lyase; 4CL, 4-coumarate:coenzyme A ligase; CCL, cinnamate/4-coumarate:coenzyme A ligase; STS, stilbene synthase; ROMT, resveratrol O-methyltransferase; T7 Pro, T7 RNA polymerase promoter; H, His-tag; S, S-tag.
© Copyright Policy
Related In: Results  -  Collection

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

f1-molce-38-4-318: (A) Biosynthetic pathway of stilbene compound production from phenylalanine and (B) construction of recombinant plasmids carrying the genes (ScCCL, STS, the synthetic RpSTSsyn, VrROMTsyn, and SbROMT3syn) involved in stilbene biosynthesis. The sequential actions of PAL or TAL, C4H, 4CL (CCL), STS, and ROMT result in the conversion of phenylalanine to stilbenes, resveratrol, and its methylated derivatives. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; TAL, tyrosine ammonia-lyase; 4CL, 4-coumarate:coenzyme A ligase; CCL, cinnamate/4-coumarate:coenzyme A ligase; STS, stilbene synthase; ROMT, resveratrol O-methyltransferase; T7 Pro, T7 RNA polymerase promoter; H, His-tag; S, S-tag.
Mentions: Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic phytoalexin produced by a few plant species, such as grapes, peanuts, and berries, under biotic or abiotic stress conditions such as pathogen infection and UV irradiation. Resveratrol has diverse properties beneficial to health, including anti-inflammatory effects, anti-tumor activity, and anti-aging effects (Baur et al., 2006; Frémont, 2000; Ulrich et al., 2005). However, the beneficial effects of resveratrol are limited due to its instability when exposed to light and oxygen or in environments with harsh pH conditions. These stimuli may cause trans-to-cis isomerization or oxidation that leads to a reduction in the bioavailability and bioactivity of the compound (Walle et al., 2004). For this reason, it is important to develop resveratrol derivatives with enhanced stability. Furthermore, structure activity studies revealed that the substitution of hydroxyl groups of resveratrol with methoxy groups substantially potentiate its cytotoxic activity (Lee et al., 2003). The substitution of hydroxy with methoxy groups may give methylated resveratrol derivatives an increased lipophilicity compared to resveratrol, resulting in better bioavailability (Remsberg et al., 2008). For these reasons, the metabolic engineering of resveratrol and its methylated derivatives in plants and microbes is of great interest for the development of more stable and potent chemoprotective agents. There are various naturally occurring methylated derivatives, including pinostilbene (3,4′-dihydroxy-5-methoxy-trans-stilbene), pterostilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene), 3,4′,5-trimethoxystilbene, and desoxyrhapotigenin (3,5-dihydroxy-4′-methoxy-trans-stilbene) (Wang et al., 2010), but their biological activities remain largely unknown. One effective approach for stabilization of resveratrol is to produce methylated resveratrol derivatives via enzymatic O-methyltransaferases (OMTs). Plant OMTs constitute a large family of enzymes that methylate the hydroxyl groups of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids. However, a few plant OMT genes have been isolated and characterized as possible resveratrol OMTs (ROMT) (Rimando et al., 2012; Schmidlin et al, 2008). We previously reported that SbROMT3syn catalyzed the methylation of resveratrol, yielding pinostilbene as the major product alongside pterostilbene as a minor product (Jeong et al., 2014). More studies are required to determine the enzymatic functions of various plant OMTs using biochemical approaches. Rhubarb (Rheum) species have been used as medicinal plants in Asian traditional medicine to treat constipation, inflammation and cancer (Foust, 1992; Lee et al., 2012). They are rich in polyketides, including stilbenes, phenylbutanoids, anthraquinones, and naphthalenes (Kashiwada et al., 1988). Six stilbene derivatives such as desoxyrhapontigenin, rhapontigenin, trans-resveratrol, piceatannol, piceatannol-3′-O-β-d-glucopyrano-side, and isorhapontin were isolated from rhubarb rhizomes. These stilbene derivatives showed a good anti-inflammatory activity (Lee et al., 2012). However, little is known about stilbene biosynthesis in rhubarb plants. Furthermore, there are still no reports on metabolic engineering of stilbene biosynthesis in microbes using stilbene biosynthetic genes from rhubarb plants. In plants, resveratrol is synthesized by STS action as a branch from the phenylpropanoid pathway (Sparvoli et al., 1994). As an intermediate, p-coumaric acid is converted to p-coumaroyl-CoA by 4-coumrate:coenzyme A ligase (4CL). STS catalyzes the condensation of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA as substrates. Methylated resveratrol derivatives of pinostilbene and pterostilbene are produced by ROMT from resveratrol (Fig. 1). Extensive metabolic engineering studies have been carried out to increase the production of resveratrol in plants and microbes (Becker et al., 2003; Beekwilder et al., 2006; Delaunois et al., 2009; Horinouchi, 2009; Katsuyama et al., 2007; Melchior and Kindl, 1990; Ververidis et al., 2007; Watts et al., 2006; Zhang et al., 2006). However, little is known about the production of methylated resveratrol derivatives in recombinant microorganisms.

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.