Limits...
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.


In vitro assays for production of resveratrol from p-coumaric acid by purified recombinant CCL and STS. His-tagged CCL (A) and STS (B) recombinant proteins were expressed in E. coli and affinity-purified using Ni-NTA agarose. Protein samples (2 μg) of total lysates (T) after induction, unbound fraction (U) after Ni-NTA bead incubation, and purified His-CCL or His-STS eluted from agarose beads (E) were separated by 13% SDS-PAGE and stained with CBB. The partially purified enzymes (each 1.5 μM) were assayed for CCL and STS activity with 0.2 mM p-coumaric acid as a precursor in the presence of 0.3 mM malonyl-CoA and 0.3 mM CoA. The reaction mixtures were incubated at 30°C for 3 h prior to extraction. Concentrated 20 μl samples were analyzed by HPLC. Chromatogram STD represents the authentic standards of p-coumaric acid (p-Cou) and resveratrol (Res) with retention times of 4.75 and 9.51 min, respectively. Chromatograms Control and CCL+ STS represent the samples produced from p-coumaric acid by in vitro assays without and with recombinant proteins, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400306&req=5

f4-molce-38-4-318: In vitro assays for production of resveratrol from p-coumaric acid by purified recombinant CCL and STS. His-tagged CCL (A) and STS (B) recombinant proteins were expressed in E. coli and affinity-purified using Ni-NTA agarose. Protein samples (2 μg) of total lysates (T) after induction, unbound fraction (U) after Ni-NTA bead incubation, and purified His-CCL or His-STS eluted from agarose beads (E) were separated by 13% SDS-PAGE and stained with CBB. The partially purified enzymes (each 1.5 μM) were assayed for CCL and STS activity with 0.2 mM p-coumaric acid as a precursor in the presence of 0.3 mM malonyl-CoA and 0.3 mM CoA. The reaction mixtures were incubated at 30°C for 3 h prior to extraction. Concentrated 20 μl samples were analyzed by HPLC. Chromatogram STD represents the authentic standards of p-coumaric acid (p-Cou) and resveratrol (Res) with retention times of 4.75 and 9.51 min, respectively. Chromatograms Control and CCL+ STS represent the samples produced from p-coumaric acid by in vitro assays without and with recombinant proteins, respectively.

Mentions: To examine whether CCL and STS enzymes are capable of converting various cinnamate derivatives to resveratrol and its derivatives, the His-tagged CCL and STS recombinant proteins were purified using Ni-NTA agarose (Fig. 4A). Using the purified proteins, we conducted in vitro enzyme assays with p-coumaric acid, caffeic acid, ferulic acid, and cinnamic acid as phenolic substrates. As shown in Fig. 4B, the CCL and STS enzymes efficiently converted p-coumaric acid as a substrate to trans-resveratrol, when compared to the control without the enzymes. However, no reaction products corresponding to resveratrol and its derivatives were detected by HPLC when phenolic acids such as caffeic acid, ferulic acid, and cinnamic acid were used (data not shown). Here, we observed that the ScCCL enzyme catalyzed the conversion of cinnamic acid to the intermediate cinnamoyl-CoA, which was not further converted to resveratrol by the STS enzyme (data not shown). These results indicate that p-coumaric acid is a good substrate for CCL and STS in producing resveratrol derivatives in vitro, whereas the other compounds are very poor substrates for CCL and STS. Consistently, our results suggest that ScCCL efficiently converts p-coumaric acid to its coumaroyl-CoA ester, which in turn is synthesized to resveratrol by the action of RpSTSsyn. Kaneko et al. (2003) previously reported that the His-tagged ScCCL recombinant protein had distinct 4CL activity and possessed the ability to use p-coumarate as a substrate. ScCCL was also reported to have low activity toward caffeate and no activity toward ferulate. These results suggest that bacterial ScCCL in combination with RpSTS can be used to produce resveratrol and its derivatives from p-coumaric acid in E. coli.


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)

In vitro assays for production of resveratrol from p-coumaric acid by purified recombinant CCL and STS. His-tagged CCL (A) and STS (B) recombinant proteins were expressed in E. coli and affinity-purified using Ni-NTA agarose. Protein samples (2 μg) of total lysates (T) after induction, unbound fraction (U) after Ni-NTA bead incubation, and purified His-CCL or His-STS eluted from agarose beads (E) were separated by 13% SDS-PAGE and stained with CBB. The partially purified enzymes (each 1.5 μM) were assayed for CCL and STS activity with 0.2 mM p-coumaric acid as a precursor in the presence of 0.3 mM malonyl-CoA and 0.3 mM CoA. The reaction mixtures were incubated at 30°C for 3 h prior to extraction. Concentrated 20 μl samples were analyzed by HPLC. Chromatogram STD represents the authentic standards of p-coumaric acid (p-Cou) and resveratrol (Res) with retention times of 4.75 and 9.51 min, respectively. Chromatograms Control and CCL+ STS represent the samples produced from p-coumaric acid by in vitro assays without and with recombinant proteins, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

f4-molce-38-4-318: In vitro assays for production of resveratrol from p-coumaric acid by purified recombinant CCL and STS. His-tagged CCL (A) and STS (B) recombinant proteins were expressed in E. coli and affinity-purified using Ni-NTA agarose. Protein samples (2 μg) of total lysates (T) after induction, unbound fraction (U) after Ni-NTA bead incubation, and purified His-CCL or His-STS eluted from agarose beads (E) were separated by 13% SDS-PAGE and stained with CBB. The partially purified enzymes (each 1.5 μM) were assayed for CCL and STS activity with 0.2 mM p-coumaric acid as a precursor in the presence of 0.3 mM malonyl-CoA and 0.3 mM CoA. The reaction mixtures were incubated at 30°C for 3 h prior to extraction. Concentrated 20 μl samples were analyzed by HPLC. Chromatogram STD represents the authentic standards of p-coumaric acid (p-Cou) and resveratrol (Res) with retention times of 4.75 and 9.51 min, respectively. Chromatograms Control and CCL+ STS represent the samples produced from p-coumaric acid by in vitro assays without and with recombinant proteins, respectively.
Mentions: To examine whether CCL and STS enzymes are capable of converting various cinnamate derivatives to resveratrol and its derivatives, the His-tagged CCL and STS recombinant proteins were purified using Ni-NTA agarose (Fig. 4A). Using the purified proteins, we conducted in vitro enzyme assays with p-coumaric acid, caffeic acid, ferulic acid, and cinnamic acid as phenolic substrates. As shown in Fig. 4B, the CCL and STS enzymes efficiently converted p-coumaric acid as a substrate to trans-resveratrol, when compared to the control without the enzymes. However, no reaction products corresponding to resveratrol and its derivatives were detected by HPLC when phenolic acids such as caffeic acid, ferulic acid, and cinnamic acid were used (data not shown). Here, we observed that the ScCCL enzyme catalyzed the conversion of cinnamic acid to the intermediate cinnamoyl-CoA, which was not further converted to resveratrol by the STS enzyme (data not shown). These results indicate that p-coumaric acid is a good substrate for CCL and STS in producing resveratrol derivatives in vitro, whereas the other compounds are very poor substrates for CCL and STS. Consistently, our results suggest that ScCCL efficiently converts p-coumaric acid to its coumaroyl-CoA ester, which in turn is synthesized to resveratrol by the action of RpSTSsyn. Kaneko et al. (2003) previously reported that the His-tagged ScCCL recombinant protein had distinct 4CL activity and possessed the ability to use p-coumarate as a substrate. ScCCL was also reported to have low activity toward caffeate and no activity toward ferulate. These results suggest that bacterial ScCCL in combination with RpSTS can be used to produce resveratrol and its derivatives from p-coumaric acid in E. coli.

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.