Engineering yeast for the production of optimal levels of volatile phenols in wine

Smit, Annel (2002-12)

Thesis (MSc)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with tartaric acid, are natural constituents of grape must and wine, and can be released as free acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms present in wine. These volatile phenols contribute to the aroma of the wine. The Bretfanomyces yeasts are well known for their ability to form volatile phenols in wine. However, these species are associated with the more unpleasant and odorous formation of the ethylphenols and the formation of high concentrations of volatile phenols. Other organisms, including some bacterial species, are responsible for the formation of volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the organoleptic properties of the wine. The enzymes responsible for the decarboxylation of phenolic acids are called phenolic acid decarboxylases; and several bacteria and fungi have been found to contain the genes encoding these enzymes. The following genes have been characterised: PAD1 from Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified. S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast. Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not show any higher enzyme activity than the control strain. Both bacterial genes, under the control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the SMR1 gene as selective marker. The cloning and transformation of pdc and padc into industrial wine yeast strains can therefore be used to detect the effect of phenolic acid decarboxylase genes in the winemaking process for the possible improvement of wine aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1 disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile phenols in the wine. The manipulated strains also produced enzymes capable of producing large amounts of favourable monoterpenes in the wine. This study paves the way for the development of wine yeast starter culture strains for the production of optimal levels of volatile phenols, thereby improving the sensorial quality of wine.

AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige fenole bekend en kan tot die aroma van wyn bydra. Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die 4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n aanvullende effek op die wyn aroma kan hê. . Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer. S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag. Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen (PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is. Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene, onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In verhoogde mate gedurende hierdie proses gevorm. Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van die wyn te verbeter.

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