Abstract
Resveratrol is an important antioxidant that confers several beneficial effects on human health. 4-coumarate coenzyme A ligase (4CL) and resveratrol synthase (RS) are key rate-limiting enzymes in the biosynthetic pathway of resveratrol. Using gene fusion technology, the fusion gene, 4CL::RS, was constructed by the 4CL gene from Arabidopsis thaliana and RS gene from Arachis hypogaea. DNAMAN analysis showed that the fusion gene encoded a 964-amino acid protein with an approximate weight of 104.7 kDa and a pI of 5.63. A prokaryotic expression vector containing Nco-I and EcoR-I restriction sites, pET-30a/4CL::RS, was identified by liquid culture bacterial PCR, enzyme digestion, and sequencing, and then used in the induction of expression. Subsequently, a biosynthetic pathway of resveratrol was constructed in Escherichia coli BL21(DE3) that harbored pET-30a/4CL::RS. The recombinant strains were induced to express the fusion protein at 28 °C for 8 h. After bacterial cells were disrupted by hypothermic ultrasonication, the 4CL::RS fusion protein was thoroughly separated from tags using Ni–NTA affinity chromatography, and then detected by SDS-PAGE analysis. When the recombinant strains expressed the fusion protein, the precursor, p-coumaric acid, was converted to resveratrol. In the present study, the final concentration of resveratrol derived from 1 mM p-coumaric acid was 80.524 mg/L, with a 35.28 % (mol/mol) conversion yield.
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This work was supported by a grant from the State Forestry Administration 948 Project (Program No. 2013-4-07), P. R. China.
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Erhao Zhang and Xuefeng Guo have contributed equally to this work.
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Zhang, E., Guo, X., Meng, Z. et al. Construction, expression, and characterization of Arabidopsis thaliana 4CL and Arachis hypogaea RS fusion gene 4CL::RS in Escherichia coli . World J Microbiol Biotechnol 31, 1379–1385 (2015). https://doi.org/10.1007/s11274-015-1889-z
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DOI: https://doi.org/10.1007/s11274-015-1889-z