title: Construction, expression, and characterization of Arabidopsis thaliana 4CL and Arachis hypogaea RS fusion gene 4CL::RS in Escherichia coli.
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.
authors.affiliation: State Forestry Administration Key Open Laboratory, International Centre for Bamboo and Rattan, No. 8 Futong Dongdajie, Wangjing, Chaoyang District, Beijing, 100102, China.
authors.forename: Erhao
authors.initials: E
authors.lastname: Zhang
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Guo
authors.forename: Zhifen
authors.initials: Z
authors.lastname: Meng
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Jia
authors.initials: J
authors.lastname: Sun
authors.forename: Xi
authors.initials: X
authors.lastname: Yao
authors.forename: Hang
authors.initials: H
authors.lastname: Xun
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010367:Arachis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular / Q000379:methods; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011422:Propionates; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism
chemical_list: D003373:Coumaric Acids; D010940:Plant Proteins; D011422:Propionates; D011993:Recombinant Fusion Proteins; D013267:Stilbenes; D000217:Acyltransferases; C055200:resveratrol synthase; D003066:Coenzyme A Ligases; C495469:p-coumaric acid; D000077185:Resveratrol