The production of resveratrol by wine yeast

Armstrong, Gareth Owen (2001-03)

Thesis (MSc)--Stellenbosch University, 2001.

Thesis

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 of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes 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 control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing 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. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast.

AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

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