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Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae - PubMed

Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae

Effendi Leonard et al. Appl Environ Microbiol. 2005 Dec.

Abstract

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4'-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.

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Figures

FIG. 1.
FIG. 1.

Flavone biosynthetic pathway. 4CL, 4-coumaroyl:CoA ligase; CHI, chalcone isomerase.

FIG. 2.
FIG. 2.

HPLC analysis of recombinant strain INV-4G+FSI and INV-4G+FS2+CPR1 fermentation. (A) Standard compounds separated by using HPLC profile 1. 1, cinnamic acid; 2, p-coumaric acid; 3, caffeic acid; 4, pinocembrin; 5, naringenin; 6, eriodictyol; 7, chrysin; 8, apigenin; 9, luteolin. (B) Apigenin and naringenin produced by the recombinant strains fed p-coumaric acid. (C) Luteolin and eriodictyol produced by the recombinant strains fed caffeic acid. (D) UV absorbance spectra of authentic compounds. (E) Standard compounds separated by using HPLC profile 2. (F) Chrysin, apigenin, pinocembrin, and naringenin produced by recombinant strain INV-4G+FSI fed cinnamic acid. The insets show the UV-visible spectra of flavonoid substances produced by the recombinant strains superimposed with the spectra of the authentic compounds.

FIG. 3.
FIG. 3.

Flavone and flavanone biosynthesis by two yeast recombinant strains. (A) Biosynthesis of apigenin from p-coumaric acid; (B) biosynthesis of naringenin from p-coumaric acid; (C) biosynthesis of luteolin from caffeic acid; (D) biosynthesis of eriodictyol from caffeic acid. ○, INV-4G+FSI; •, INV-4G+AFNS2+CPR1. All fermentations were carried out in SC-Leu−Trp− minimal medium with raffinose as the carbon source.

FIG. 4.
FIG. 4.

Flavanone production by yeast recombinant strain INV-4G. (A) Naringenin produced from p-coumaric acid in the presence of various concentrations of apigenin; (B) eriodictyol produced from caffeic acid in the presence of various concentrations of luteolin. ▪, 1 mg/liter of flavone; ▴, 3 mg/liter of flavone; ⧫, 10 mg/liter of flavone; •, control (no flavone).

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