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.

Heterologous production of resveratrol in bacterial hosts: current status and perspectives

“…A. hypogea STS has been reported to have a broad substrate specificity (Morita et al 2001;Abe et al 2004). In the same conditions Beekwilder et al (2006) and Zhang et al (2015) obtained a resveratrol production of 80.52 and 16 mg L −1 , respectively, using another E. coli strain, E. coli BL21. Lim et al (2011) achieved a resveratrol production of 2.39 g L −1 with an E. coli strain BW27784 harboring 4CL from A. thaliana and STS from V. vinifera, in the presence of 15 mM p-coumaric acid and cerulenin.…”

“…Protein engineering and mutagenesis of 4CL and STS have indeed been applied to improve resveratrol production capabilities in microorganisms. Zhang et al (2015) increased the resveratrol production in E. coli using gene fusion technology. The authors applied the fusion of the enzyme 4CL::STS (4CL from A. thaliana and STS from A. hypogaea) that enabled a resveratrol production of 80.5 mg L −1 from 1 mM p-coumaric acid.…”

Biotechnological Advances in Resveratrol Production and its Chemical Diversity

“…For the production of value-added bioactive resveratrol, pathway engineering is one of the pioneer methods in designing E. coli . Given an approximately 50% bioconversion rate, 105 mg/L of resveratrol was obtained from the engineered strain of E. coli harboring 4CL from A. thaliana and STS from A. hypogaea when 1 mM p -coumaric acid was used as a precursor [99], whereas about 80.5 mg/L resveratrol was obtained with the same amount of p -coumaric acid substrate in E. coli implanted with the fusion gene of 4CL and STS from A. thaliana , which clearly indicates a lower resveratrol production amount in the fusion genes from the same sources [100]. Similarly, Lim and colleagues made a different combination of 4CL and STS from two E. coli strains and obtained 1.3 g/L resveratrol by the strain E. coli BW27784.…”

“…For higher and more efficient resveratrol production, protein engineering and mutagenesis of 4CL and STS have been done and expressed in E. coli . With the thought that co-localization of the two enzymes’ active sites might improve the efficiency, the unnatural fusion of 4CL from A. thaliana and STS from A. hypogaea was incorporated in E. coli , which produced 80.5 mg/L resveratrol when fed 1 mM p -coumaric acid [100]. Previously with the similar strategies of introducing resveratrol biosynthesis pathway genes, such as RsTAL from R. shaeroides , At4CL from A. thaliana , and VvSTS of V. vinifera in S. cerevisiae , produced only 0.65 mg/L resveratrol starting from p -coumaric acid, but with the translational fusion of two proteins, At4CL and VvSTS, produced by substituting the stop codons of At4CL with three amino acids linker into the open reading frame of VvSTS, helped to increase the production to 5.25 mg/L resveratrol, which was almost 3500 times higher than in Becker and his team’s report in 2003.…”

Recent studies on resveratrol and its biological and pharmacological activity

“…It is derived from p -coumaric acid, which is an intermediate formed during lignin production. The 4-coumarate:coenzyme A (CoA) ligase converts p -coumaric acid to coumaroyl-CoA, along with three units of malonyl-CoA, which are then condensed to form resveratrol by resveratrol synthase or stilbene synthase (Deng et al, 2016[8]; Zhang et al, 2015[55]; Zheng et al, 2015[57]; Kim et al, 2013[21]).…”