Channelling metabolic flux away from ethanol production by modification of gene expression under wine fermentation conditions

Heyns, Eva Hutton (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: There is a global demand for technologies to reduce ethanol levels in wine without compromising wine quality. While several chemical and physical methods have been developed to reduce ethanol in finished wine, the target of an industrially applicable biological solution has thus far not been met. Most attempted biological strategies have focused on developing new strains of the main fermentative organism, the yeast Saccharomyces cerevisiae. Gene modification approaches have primarily focused on partially redirecting yeast carbon metabolism away from ethanol production towards glycerol production. These techniques have met with some moderate success, thus the focus of the current study was to re-direct carbon flux towards trehalose production by moderate over-expression of the TPS1 gene. This gene encodes trehalose-6-phosphate synthase, which converts glucose 6-phosphate and UDPglucose to α,α-trehalose 6-phosphate. Previous data have shown that the overproduction of trehalose restricts hexokinase activity reducing the amount of glucose that enters glycolysis. Nevertheless, preliminary TPS1 over-expression studies using multiple copy plasmids have shown some promise, but also indicated significant negative impact on the general fermentation behaviour of strains. In order to reduce such negative impacts of excessive trehalose production, a new strategy consisting in increasing the expression of TPS1 only during specific growth phases and by a relatively minor degree was investigated. Our study employed a lowcopy number episomal vector to drive moderate over-expression of the TPS1 gene in the widely used industrial strain VIN13 at different stages during fermentation. The fermentations were performed in synthetic must with sugar levels representative of those found in real grape must. This, as well as the use of an industrial yeast strain, makes it easier to relate our results to real winemaking conditions. A reduction in fermentation capacity was observed for all transformed strains and controls. Expression profiles suggest that the DUT1 promoter certainly results in increased TPS1 expression (up to 40%) during early exponential growth phase compared to the wild type strain (VIN13). TPS1 expression under the control of the GIP2 promoter region showed increased expression levels during early stationary phase (up to 60%). Chemical analysis of the yeast and the must at the end after fermentation showed an increase in trehalose production =in line with the expression data of TPS1. Importantly, glycerol production was also slightly increased, but without affecting acetic acid levels for the transformed strains. Although ethanol yield is not significantly lower in the DUT1-TPDS1 strain, s statistically significantly lower ethanol yield is observed for over-expression under the GIP2 promotor. Increasing trehalose production during stationary phase appears therefore to be a more promising approach at lowering ethanol yield and redirecting flux away from ethanol production. This controlled, growth phase specific over expression suggests a unique approach of lowering ethanol yield while not impacting on the redox balance.

AFRIKAANSE OPSOMMING: Wêreldwyd is daar ‘n aanvraag na tegnologie wat die etanol vlakke in wyn kan verminder sonder om wyngehalte te benadeel. Terwyl verskeie chemiese en fisiese metodes ontwikkel is om etanol in die finale wynproduk te verminder, is die soeke na 'n industrieel gebaseerde biologiese oplossing tot dusver nie gevind nie. Meeste biologiese strategieë fokus op die ontwikkeling van nuwe rasse van die primêre fermentatiewe organisme, naamlik Saccharomyces cerevisiae. Geen modifikasie benaderings het hoofsaaklik gefokus op die gedeeltelike kanalisering van koolstof metabolisme weg van etanol produksie na gliserol produksie. Hierdie benadering is net matiglik suksesvol, dus is ons huidige fokus om koolstof te kanaliseer na trehalose produksie deur gematigde oor-uitdrukking van die TPS1 geen. Hierdie geen kodeer vir trehalose-6-fosfaat sintase, wat glukose-6-fosfaat en UDP-glukose omskakel na α, α-trehalose-6-fosfaat. Vorige data het getoon dat die oorproduksie van trehalose hexokinase aktiwiteit beperk en die hoeveelheid glukose wat glikolise binne gaan. Voorlopige TPS1 ooruitdrukking studies met behulp van multi-kopie plasmiede toon matige sukses, maar het ook ‘n negatiewe impak op die algemene fermentasie kapasiteit van die gis. Ten einde so 'n negatiewe impak van oormatige trehalose produksie te oorkom, is 'n nuwe strategie gevolg wat bestaan uit die verhoogde uitdrukking van die TPS1 geen slegs gedurende spesifieke groei fases met baie lae vlakke van oor-uitdrukking. Ons studie gebruik 'n lae-kopie episomale vektor met matige oor-uitdrukking van die TPS1 geen in die industriële ras VIN13 op verskillende stadiums tydens fermentasie. Die fermentasie is uitgevoer in sintetiese mos met suiker vlakke verteenwoordigend van dié van werklike wyn mos. Hierdie, sowel as die gebruik van 'n industriële gisras, maak dit makliker om ons resultate te vergelyk met regte wyn fermentasie kondisies. Verlaagde fermentasie kapasiteit is waargeneem vir alle getransformeerde stamme en hul kontroles. Geen uitdrukkings profiele dui op verhoogde TPS1 uitdrukking (tot 40%) onder beheer van die DUT1 promotor gedurende die vroeë eksponensiële groeifase wanneer vergelyk word met die wilde tiepe (VIN13). TPS1 uitdrukking onder die beheer van die GIP2 promotor het verhoogde uitdrukking van tot 60% gedurende die vroeë stasionêre fase. Chemiese analise van die gis aan die einde van fermentasie dui op ‘n toename in trehalose produksie wat korreleer met die uitdrukking profiele van TPS1. Gliserol produksie is ook effens verhoog, maar sonder ‘n toename in asynsuur vlakke vir die getransformeerde rasse. Alhoewel etanol opbrengs nie aansienlik laer vir die DUT1-TPS1 ras is nie, is etanol opbrengs vir die oor-uitdrukking onder beheer van die GIP2 promotor wel laer. Toenemende trehalose produksie gedurende stasionêre fase blyk dus 'n meer belowende benadering op die verlaging van etanol opbrengs en her-kanaliseering weg van etanol produksie. Hierdie benadering met die fokus op groeifase spesifieke oor-uitdrukking dui op 'n unieke strategie vir die verlaging van etanol opbrengs sonder om die redoks balans te beinvloed.

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