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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 CCL, VrROMTsyn and SbROMT3syn, and S-tagged STS recombinant proteins in E. coli cells. E. coli cells containing (A) CCL+STS, (B) CCL+STS-VrROMT, or (C) CCL+STS-SbROMT3 construct were grown and induced by the addition of 0.5 mM IPTG for 2 h and 4 h. Proteins were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Expression of Histag or S-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag or anti-S-tag antibodies. M, protein molecular marker in kDa; S, soluble fraction (15 μg); P, insoluble pellet fraction (1 μg). S-tagged STS, His-tagged CCL, and His-tagged ROMT recombinant proteins recognized by anti-S-tag and anti-His-tag antibody are shown with asterisks, arrows and arrowheads, respectively.
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f3-molce-38-4-318: Expression of His-tagged CCL, VrROMTsyn and SbROMT3syn, and S-tagged STS recombinant proteins in E. coli cells. E. coli cells containing (A) CCL+STS, (B) CCL+STS-VrROMT, or (C) CCL+STS-SbROMT3 construct were grown and induced by the addition of 0.5 mM IPTG for 2 h and 4 h. Proteins were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Expression of Histag or S-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag or anti-S-tag antibodies. M, protein molecular marker in kDa; S, soluble fraction (15 μg); P, insoluble pellet fraction (1 μg). S-tagged STS, His-tagged CCL, and His-tagged ROMT recombinant proteins recognized by anti-S-tag and anti-His-tag antibody are shown with asterisks, arrows and arrowheads, respectively.

Mentions: To improve the translational efficiency of the plant genes in a bacterial system, the RpSTS, VrROMT and SbROMT3 genes were synthesized following plant codon-optimization (Supplementary Fig. S3). The synthetic RpSTSsyn, VrROMTsyn and SbROMT3syn DNAs were introduced into the dual expression vector pETDuet-1 for the expression of the corresponding N-terminal His-tag or C-terminal S-tag fusion proteins (Fig. 1B). To examine the function of RpSTSsyn, VrROMTsyn and SbROMT3syn in E. coli, either pETDuet-STS or pETDuet-ROMT-STS was co-transformed into E. coli cells alongside pCOLADuet-CCL, and the bacteria were selected on LB media containing both kanamycin and ampicilin antibiotics. The most promising recombinant E. coli transformant was selected by SDS-PAGE and Western blot analyses, and used for further experiments (data not shown). Expression of recombinant CCL, STS and ROMT proteins was determined by SDS-PAGE and Western blot analysis with anti-His-tag or anti-S-tag antibody. As shown in Fig. 3, all the His-ScCCL, RpSTSsyn-S, His-VrROMTsyn and His-SbROMT3syn recombinant proteins corresponded to their theoretical molecular weights of 57.5, 45.3, 41.7 and 42.5 kDa, respectively, and were expressed at 2 and 4 h after IPTG induction in the soluble and insoluble fractions. Specific protein bands detected by His- or S-tag antibodies were detected, even in the presences of some degradation. All the recombinant CCL, STS and ROMT proteins appeared to be expressed in the both soluble and insoluble fractions of the E. coli cell lysate, but the major portions of the expressed proteins were observed in the insoluble fraction. The SbROMT3syn protein showed better expression than VrROMTsyn in the both soluble and insoluble fractions. Multiple protein bands hybridizing with His- or S-tag antibody were detected, which are likely to be the result of protein degradation. Producing soluble proteins at high levels in E. coli is still a major bottleneck for metabolic engineering. We observed that the codon-optimized RpSTSsyn improved the solubility in E. coli compared to the original RpSTS. However, we failed to obtain high-level expression of heterologous STS and ROMT recombinant proteins in the soluble fraction of E. coli, even with codon usage optimization and alterations affecting culture medium and temperature, IPTG induction, and the host strain.


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 CCL, VrROMTsyn and SbROMT3syn, and S-tagged STS recombinant proteins in E. coli cells. E. coli cells containing (A) CCL+STS, (B) CCL+STS-VrROMT, or (C) CCL+STS-SbROMT3 construct were grown and induced by the addition of 0.5 mM IPTG for 2 h and 4 h. Proteins were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Expression of Histag or S-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag or anti-S-tag antibodies. M, protein molecular marker in kDa; S, soluble fraction (15 μg); P, insoluble pellet fraction (1 μg). S-tagged STS, His-tagged CCL, and His-tagged ROMT recombinant proteins recognized by anti-S-tag and anti-His-tag antibody are shown with asterisks, arrows and arrowheads, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

f3-molce-38-4-318: Expression of His-tagged CCL, VrROMTsyn and SbROMT3syn, and S-tagged STS recombinant proteins in E. coli cells. E. coli cells containing (A) CCL+STS, (B) CCL+STS-VrROMT, or (C) CCL+STS-SbROMT3 construct were grown and induced by the addition of 0.5 mM IPTG for 2 h and 4 h. Proteins were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Expression of Histag or S-tag fusion proteins was confirmed by Western blot analysis with anti-His-tag or anti-S-tag antibodies. M, protein molecular marker in kDa; S, soluble fraction (15 μg); P, insoluble pellet fraction (1 μg). S-tagged STS, His-tagged CCL, and His-tagged ROMT recombinant proteins recognized by anti-S-tag and anti-His-tag antibody are shown with asterisks, arrows and arrowheads, respectively.
Mentions: To improve the translational efficiency of the plant genes in a bacterial system, the RpSTS, VrROMT and SbROMT3 genes were synthesized following plant codon-optimization (Supplementary Fig. S3). The synthetic RpSTSsyn, VrROMTsyn and SbROMT3syn DNAs were introduced into the dual expression vector pETDuet-1 for the expression of the corresponding N-terminal His-tag or C-terminal S-tag fusion proteins (Fig. 1B). To examine the function of RpSTSsyn, VrROMTsyn and SbROMT3syn in E. coli, either pETDuet-STS or pETDuet-ROMT-STS was co-transformed into E. coli cells alongside pCOLADuet-CCL, and the bacteria were selected on LB media containing both kanamycin and ampicilin antibiotics. The most promising recombinant E. coli transformant was selected by SDS-PAGE and Western blot analyses, and used for further experiments (data not shown). Expression of recombinant CCL, STS and ROMT proteins was determined by SDS-PAGE and Western blot analysis with anti-His-tag or anti-S-tag antibody. As shown in Fig. 3, all the His-ScCCL, RpSTSsyn-S, His-VrROMTsyn and His-SbROMT3syn recombinant proteins corresponded to their theoretical molecular weights of 57.5, 45.3, 41.7 and 42.5 kDa, respectively, and were expressed at 2 and 4 h after IPTG induction in the soluble and insoluble fractions. Specific protein bands detected by His- or S-tag antibodies were detected, even in the presences of some degradation. All the recombinant CCL, STS and ROMT proteins appeared to be expressed in the both soluble and insoluble fractions of the E. coli cell lysate, but the major portions of the expressed proteins were observed in the insoluble fraction. The SbROMT3syn protein showed better expression than VrROMTsyn in the both soluble and insoluble fractions. Multiple protein bands hybridizing with His- or S-tag antibody were detected, which are likely to be the result of protein degradation. Producing soluble proteins at high levels in E. coli is still a major bottleneck for metabolic engineering. We observed that the codon-optimized RpSTSsyn improved the solubility in E. coli compared to the original RpSTS. However, we failed to obtain high-level expression of heterologous STS and ROMT recombinant proteins in the soluble fraction of E. coli, even with codon usage optimization and alterations affecting culture medium and temperature, IPTG induction, and the host strain.

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.