NDLI: Construction, expression, and characterization of Arabidopsis thaliana 4CL and Arachis hypogaea RS fusion gene 4CL::RS in Escherichia coli

Content Provider	Springer Nature Link
Author	Zhang, Erhao Guo, Xuefeng Meng, Zhifen Wang, Jin Sun, Jia Yao, Xi Xun, Hang
Copyright Year	2015
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.
Starting Page	1379
Ending Page	1385
Page Count	7
File Format	PDF
ISSN	09593993
Journal	World Journal of Microbiology and Biotechnology
Volume Number	31
Issue Number	9
e-ISSN	15730972
Language	English
Publisher	Springer Netherlands
Publisher Date	2015-06-20
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Resveratrol Fusion gene Linker Arabidopsis thaliana Arachis hypogaea Applied Microbiology Biotechnology Biochemistry Environmental Engineering/Biotechnology Microbiology
Content Type	Text
Resource Type	Article
Subject	Physiology Medicine Applied Microbiology and Biotechnology Biotechnology

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