Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

High temperature and UV-C treatments affect stilbenoid accumulation and related gene expression levels in Gnetum parvifolium

Background: Gnetum parvifolium stems and roots have been used for a long time in traditional Chinese medicines. Stilbenes are bioactive compounds present in G. parvifolium plants, and they possess antioxidative and anticancer properties. However, little is known about the responses of G. parvifolium stilbene biosynthetic pathways to stress conditions. Therefore, we investigated stilbene biosynthesis, including the expression of relevant genes, in G. parvifolium exposed to high-temperature and ultraviolet-C treatments. Results: High temperatures did not influence the accumulation of total stilbenes in stems but decreased stilbene concentrations in roots at 3 h, with a subsequent restoration to control levels. In contrast, ultraviolet irradiation induced the accumulation of total stilbenes in stems but not in roots. We also observed that high temperatures inhibited the production of resveratrol and piceatannol in G. parvifolium stems and roots, whereas ultraviolet treatments initially inhibited their accumulation (up to 6 h) but induced their production at later time points. Analyses of specific genes (i.e., PAL, C4H, 4CL, STS, and CYP) revealed that their expression levels generally increased in stress-treated stems and roots, although there was some variability in the expression profiles during treatments. Conclusions: Our results indicated that high temperatures and ultraviolet irradiation differentially affect the biosynthesis of specific stilbenes in G. parvifolium stems and roots. Therefore, cultivating G. parvifolium seedlings under optimal stress conditions may increase the biosynthesis of specific stilbene compounds.

Stimulation of trans-resveratrol biosynthesis in Vitis vinifera cv. Kyoho cell suspension cultures by 2, 3-dihydroxypropyl jasmonate elicitation

Background: Plant cell suspension culture of Vitis vinifera is a promising technology for investigating different factors that are able to induce and/or modify stilbenes biosynthesis. Jasmonates have been reported to play an important role in a signal transduction pathway that regulates defence responses as well as the production of secondary metabolites. In this study, 2, 3-dihydroxypropyl jasmonate (DHPJA) was used to investigate its effect on stimulating trans-resveratrol (t-R) accumulation and the plant defence responses in Vitis vinifera cv. Kyoho cell suspension cultures for the first time. Results: It demonstrated that DHPJA had superior effects on stilbenoids accumulation over methyl jasmonate (MeJA). The optimal condition was 150 uM DHPJA added on day 15 of cultivation period, with the highest level of t-R accumulation which was increased 1.8-fold and 1.3-fold compared with the control and 150 uM MeJA respectively. DHPJA induced stronger plant defence responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL) than MeJA. H2O2 generation induced by DHPJA played a significant role in enhancing t-R accumulation. Adding a specific inhibitor of H2O2 signalling pathway inhibited DHPJA-induced t-R accumulation, but had no effects on DHPJA-induced other metabolites accumulation, which resulted in regulations of product diversity. Conclusions: This study demonstrated that DHPJA was an efficient elicitor to enhance t-R accumulation by activating stronger oxidative burst, and H2O2 signalling pathway could regulate product diversity in DHPJA-induced V. vinifera cv. Kyoho cell suspension cultures.