Production of resveratrol in recombinant microorganisms

Abstract

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).

FIG. 1.

Biosynthetic pathway of resveratrol. The biosynthesis of resveratrol starts by the coupling of p-coumaric acid to CoA by the 4CL enzyme. Subsequently, coumaroyl-CoA is converted into resveratrol by sequential addition of three malonyl-CoA units with the release of carbon dioxide.

FIG. 2.

HPLC analysis of resveratrol produced by E. coli at 307 nm. The upper chromatogram (A) was derived from an extract of a culture of E. coli BL21 plus pAC-4CL-STS grown with p-coumaric acid for 20 h. Culture medium was separated by an HPLC setup as described in reference , and absorbance was recorded at 307 nm. The lower chromatogram (B) shows the same for control E. coli BL21 plus pACYC-DUET1. The peak at 17 min represents p-coumaric acid, and the peak at 28.9 min represents resveratrol. The absorption spectrum of the peak at 28.9 min is shown in the “PDA” inset, while the mass spectrum, recorded with a QTOF ULTIMA mass spectrometer (negative electrospray ionization mode) is shown in the “MS” inset.

FIG. 3.

Production of resveratrol in E. coli (A) and S. cerevisiae (B) over time. Each graph displays the concentration of resveratrol in mg/liter in the culture medium as measured after extraction, the concentration of p-coumaric acid (p-CA) in g/liter, and the density of the culture (no absolute values indicated). The values shown are averages of two measurements of individual cultures.

FIG. 4.

Cassette for integration of STS and 4CL into the yeast genome. LEU2_1-546, LEU2 gene from nucleotides 1 to 546 of the coding region; Pi, promoter of the ILV2 gene; ILV2-SMR, sulfometuron methyl resistance gene; Ti, terminator of the ILV2 gene; Tc, terminator of the CYC gene; STS, coding region of the grape STS gene; PGAL1, promoter of the GAL1 gene; PGAL10, promoter of the GAL10 gene; 4CL, coding region of the 4CL2 gene from tobacco; Ta, terminator of the ADH1 gene; LEU2_547-1060, LEU2 gene from nucleotides 547 to 1060 of the coding region; PacI, unique PacI restriction site.