Investigation of resveratrol production by genetically engineered Saccharomyces cerevisiae strains
Year: 2006
Type: dissertation
Abstract: Research ResultsOptimisation of both resveratrol production in recombinant Saccharomyces cerevisiae and the required analytical tools Chapter 4General Discussion and Conclusions SUMMARYResveratrol 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 resveratrolproducing 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 endogenouslyproduced 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. OPSOMMINGResveratrol is 'n fitoaleksien wat in die blare and doppe van druiwekorrels geproduseer word as gevolg van biotiese en abiotiese faktore wat op die wingerdplant inwerk.Substitusie en polimerisasie van resveratroleenhede het 'n verskeidenheid verbindings tot gevolg, wat 'n belangrike deel van die aktiewe weerstandsmeganisme in wingerd vorm.Wyn is van die belangrikste resveratrolbronne in die menslike dieet.Resveratrol is een van 'n verskeidenheid fenoliese verbindings wat in wyn voorkom wat 'n positiewe invloed op die mens se gesondheid het.Wat dit aanbetref, is daar al bewys dat dit die ontwikkeling van kardiovaskulêre siektes, kanker en patogenese wat met inflammasie verbind word, kan voorkom.Rooiwyne se resveratrolvlakke is hoër as dié van witwyne as gevolg van die verlengde dopkontakperiodes gedurende die fermentasie van rooiwyne.Gedurende die maak van witwyne word die doppe egter voor fermentasie verwyder en die ekstraksie van resveratrol is dus minimaal in dié wyne.Die hoofdoel van hierdie navorsing is die ontwikkeling van 'n wyngis wat resveratrol gedurende fermentasie van mos kan produseer.Die navorsing is gebaseer op die veronderstelling dat beide rooi-en witwyne wat met só 'n resveratrolproduserende gis geproduseer is, verhoogde vlakke van resveratrol sal bevat en dat dit dus ook bykomende gesondheidsvoordele vir die verbruiker sal inhou.Vorige navorsing in ons labarotorium het bewys dat laboratoriumgis oor die vermoë beskik om resveratrol te produseer wanneer die gene wat die koënsiem A ligase (4CL216) en resveratrolsintase (vst1) enkodeer op multikopie-plasmiede uitgedruk word indien die groeimedia met p-koumariensuur aangevul word.Hierdie studie het op die optimisering van resveratrolproduksie in Saccharomyces cerevisiae gefokus.Dit het die integrasie en konstitutiewe uitdrukking van 4CL216 uit hibriede populier en vst1 uit wingerd behels, en die integrasie en uitdrukking van die gene in drie laboratoriumgiste is deur middel van Southern-en Northern-kladtegnieke bevestig.Die evaluering van resveratrolproduksie deur gis het dit noodsaaklik gemaak dat die analitiese tegnieke aanvanklik geoptimiseer moes word.Dit is bereik deur middel van monstervoorbereiding vanuit die intrasellulêre fraksie van gis en 'n prosedure wat uitgewerk is vir die bepaling van resveratrol in die ekstrasellulêre fraksie.Die LCMSMSmetode is verder ontwikkel om die opspoor en kwantifisering van verwante verbindings met betrekking tot resveratrolproduksie in gis ook moontlik te maak.Vergelyking van resveratrolproduksie in drie giste van verskillende genetiese agtergronde het aangedui dat die begin van produksie en die resveratrolopbrengs is gisras-afhanklik.Voorlopervoerstudies het aangedui dat die beskikbaarheid van p-koumariensuur 'n faktor was wat maksimale resveratrolproduksie beperk het.Vroeë aanduidings het getoon dat endogeen-geproduseerde resveratrol moontlik 'n impak op gis se lewensvatbaarheid gedurende verlengde groeiperiodes kan hê.Met hierdie studie is die kennis van die dinamiek van resveratrolproduksie in S. cerevisiae uitgebrei en dit het ook belangrike aanduiders verskaf watter aspekte geoptimiseer moet word om 'n wyngis vir maksimale resveratrolproduksie optimaal geneties te verbeter.
Author: Kim M. Trollope
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2139128344