The production of resveratrol by wine yeast

[摘要] ENGLISH ABSTRACT:Grapevine is constantly under attack from a wide variety of pathogens including viruses,bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array ofdefense mechanisms to combat invading organisms. A key element of this diseaseresistance is the production of phytoalexins, of which resveratrol is the primary component.The synthesis of resveratrol, together with other structural and biochemical defensemechanisms equips the plant to combat a number of pathogens resulting in the productionof healthy grapes for the vinification of top quality wine. As part of the active diseaseresponse resveratrol is synthesised de novo in the berry skin at the site of infection, onrecognition of the pathogen. Here it is able to limit the damage caused by the pathogen aswell as preventing it from spreading. This gives the plant the opportunity to initiate itssystemic acquired resistance thereby protecting the rest of the plant and preventingsecondary infections.The fermentation of red wine on the grape skins allows for the extraction of resveratrolfrom the skin into the wine. Red wines therefore have a significantly higher concentrationof resveratrol than white varieties, which contain little or no resveratrol at all. It is for thisreason that the moderate consumption of wine, in particular red wine, is synonymous witha healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol areimportant contributors to the cardiovascular benefits derived from the consumption of redwine. It now seems, however, that significant cardiovascular protection is derived from thesynergistic action of resveratrol, the polyphenols and the alcohol in wine.With the wholesomeness of any food or beverage being of extreme importance, theaim of this project was to manipulate wine yeast to produce resveratrol duringfermentation. This required the introduction of an entire metabolic pathway, by integratingplant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl-CoA molecules to produce one molecule of resveratrol, Saccharomycescerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following geneswere obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanineammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4-hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encodingCoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-levelexpression, the genes were subcloned under the control of the phosphoglycerate kinasegene (PGK1) promoter and terminator. Due to integration problems with these expressioncassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9,4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the controlof the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids,respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produceresveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformantsexpressing the CoA-ligase genes utilised the p-coumaric acid faster than the control,although it was not possible to determine whether p-coumaroyl-CoA was produced. Noresveratrol was produced under the assay conditions used. The results indicated that theyeast is unable to produce active resveratrol synthase, which is required to catalyse thefinal reaction in the production of resveratrol. Posttranslational modification, such as overglycosylationand disulphide formation, of the heterologous protein in yeast has beenindicated as the possible reason for the lack of enzyme activity. This introduces an excitingarea of research for the development of biotechnological tools with the ability to increasethe production of active heterologous proteins in yeast.

[发布机构] Stellenbosch University