Thesis (MSc)--Stellenbosch University, 2001. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52557 |
| Date | 03 1900 |
| Creators | Armstrong, Gareth Owen |
| Contributors | Pretorius, I. S., Lambrechts, M. G., Vivier, M. A., Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 79 p. : ill. |
| Rights | Stellenbosch University |
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