Investigation of resveratrol production by genetically engineered Saccharomyces cerevisiae strains
Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.