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

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Channelling metabolic flux away from ethanol production by modification of gene expression under wine fermentation conditions By Eva Hutton Heyns Thesis presented in partial fulfilment of the requirements
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Channelling metabolic flux away from ethanol production by modification of gene expression under wine fermentation conditions By Eva Hutton Heyns Thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Stellenbosch University Institute for Wine Biotechnology, Faculty of AgriSciences Supervisor: Prof FF Bauer Co-supervisor: Dr ME Setati Co-supervisor: Dr D Rossouw March 2013 Declaration By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: 19 December 2012 Copyright 2013 Stellenbosch University All rights reserved This thesis is dedicated to my family and friends for their continuous support Biographical sketch Eva Hutton Heyns was born in Cape Town, South Africa on 6 July She attended Park Primary School and Point High School in Mossel Bay, South Africa and obtained her matric exemption at Bergvlam High School in Nelspruit, South Africa in Hutton obtained her Bachelor of Science in Human Genetics at the University of Pretoria, South Africa in 2001, majoring in genetics, microbiology and biochemistry. She then enrolled at the University of Limpopo obtaining her Med (Hons) Molecular Genetics in In 2010, she enrolled for an MSc in Wine Biotechnology at the Institute for Wine Biotechnology at Stellenbosch University. Summary 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. 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. Acknowledgements I wish to express my sincere gratitude and appreciation to the following persons and institutions: Prof Florian Bauer, who acted as supervisor for this study. Dr Debra Roussouw, who acted as co-supervisor for this study. Dr Evodia Setati, who acted as co-supervisor for this study. All my colleagues for their support and help throughout my project My family and friends for supporting me through a personally challenging time during my studies The NRF, IWBT and the University of Stellenbosch for funding. Preface This thesis is presented as a compilation of four chapters. Each chapter is introduced separately and is written according to the style of the journal Applied Microbiology and Biotechnology. Chapter 1 Chapter 2 Chapter 3 Chapter 4 General Introduction and project aims Literature review Approaches to lowering ethanol in wine Research results Construction of a recombinant industrial Saccharomyces cerevisiae strain for low ethanol fermentation General discussion and conclusions Table of Contents Chapter 1. General introduction and project aims Introduction Project aims References 5 Chapter 2. Literature review Introduction Viticultural and physical approaches Reverse Osmosis Spinning cone column (SCC) Non-GMO based biological approaches GMO based approaches Deletion of alcohol dehydrogenase (ADH) encoding genes Alterations of glycerol metabolism Introduction of glucose oxidase (GOX) into S. Cerevisiae to reduce glucose availability NADH oxidases (NOX) over-expression to reduce intracellular NADH Diminished pyruvate decarboxylase (PDC) activity to increase glycerol production Deletion of triose phosphate isomerase (TPI) to increase glycerol production Deletion and over expression of trehalose-6-phosphate synthase (TPS) to shift carbon flux towards trehalose production Combined approaches Conclusion References 26 Chapter 3. Research results Introduction Materials and methods Strains and culture conditions DNA manipulation and plasmid construction Yeast transformation Verification of gene expression by quantitative real-time PCR analysis (QRT-PCR) Metabolite analysis Protein extraction and quantification Results Monitoring fermentation performance and biomass formation Expression of the TPS1 gene Chemical analysis Discussion References 51 Chapter 4. General discussion and conclusions General discussion and concluding remarks References 58 Addendum A 60 Chapter 1 Introduction and project aims 1.1. INTRODUCTION Over the past few decades, winemaking has changed dramatically and has had to keep up with the competitive nature of the global economy. There is a constant need for improving viticultural and oenological practices. Vine growing and wine making are biological processes, and the main contributors are grape vine and microbial organisms, in particular yeast. Many studies have focused on the improvement of wine making process and of wine quality by studying these biological systems (Pretorius, 2000). The traditional approach to wine making, and which continues to be used by some smaller and boutique wineries, was for the wine fermentation process to be carried out by the naturally occurring microbes in the vineyard and in the winery (Henschke, 1997). Today s competitive industry demands a more controlled, reliable and predictable production of wines on a larger industrial scale. This is the reason for the addition of pure yeast inocula that was introduced by Müller Thurgau in In most instances Saccharomyces cerevisiae strains are inoculated into the grape must at the start of fermentation (Henschke, 1997; Pretorius, et al., 2003). S. cerevisiae not only converts fermentable sugars into ethanol but also plays a role in producing many flavour and aroma compounds in wine. These flavour compounds formed by yeast metabolism include esters, fatty acids and higher alcohols (Scudamore-Smith and Moran 1997; Pickering et al. 1998) One of the more recent consumer and industry demands has been to lower the ethanol content of wines. One of the reasons for this is that high ethanol content can compromise the quality of wine, by creating a perception of increased hotness and viscosity and by masking other aromatic compounds (Gawel et al., 2007). Other reasons include the health risks involved in excessive alcohol consumption, and the cost to consumer as taxes are levied according to the alcohol content of beverages (de Barros, 2000; Kutyna et al., 2010). Comparative studies have shown that averarge ethanol concentrations of commercial wines have risen over the past two decades. This rise in ethanol content may be due to a number of factors including rising temperatures due to global warming (Catarino et al., 2011), as well as changes to viticultural practices aiming at increased ripeness of berries to improve flavour characteristics (Godden, 2000). The different approaches for dealing with excessive ethanol can be divided into three groups, namely viticultural, mechanical or biological. Viticultural methods could include berry picking times and vine canopy control measures which influence the exposure of grapes to light and temperature. Physical methods may include removal of alcohol at the end of fermentation by reverse osmosis, dilution or distillation (Bui et al. 1986; Pickering et al. 2 1999a; Mermelstein 2000). Fermentation management methods rely on regulation of fermentation conditions by temperature control, nutrient regulation or osmotic stress management (Attfield, 1997; d Amore et al., 1987; Hinchcliffe et al., 1985). Biological approaches focusing on the genetic modification of yeast also have the potential to address the ethanol problem, and have met with relative success in recent years (Kutyna et al., 2010). These biological approaches target various genes that impact on central carbon metabolism, with the aim to redirect carbon flux. Most focus on genes involved in redirecting flux toward glycerol production. These include GPD1 and GPD2 encoding isozymes of glycerol 3- phosphate dehydrogenase (de Barros Lopes et al., 2000; Cambon et al., 2006; Eglington et al., 2002; Michnick et al., 1997; Nevoight et al., 1996; Remize et al., 2001; Remize et al., 1999), alcohol dehydrogenase (ADH) mutants (Drewke et al., in 1990), PDC2 Pyruvate decarboxylase mutants (Nevoigt & Stahl, 1996; Schmitt & Zimmermann, 1982). Other approaches focused on the heterologous expression of genes that remove glucose from the system in order to lower ethanol, such as expression of the GOX gene from Aspergillus niger, encoding an enzyme converting glucose to gluconic acid (Pickering et al., 1999a).. Finally attempts have been made to modify the hexose transporters that facilitate the transport of glucose. The approach described in this work is based on redirecting metabolic carbon flux towards the stress and reserve carbohydrates trehalose.the TPS1 gene encodes trehalose-6- phosphate synthase, a key enzyme in the trehalose biosynthesis pathway (Francois et al., 2001). Trehalose is synthesized in two steps: First glucose 6-phosphate and UDP-glucose is converted to α,α-trehalose 6-phosphate by trehalose-6-phosphate synthase encoded by the TPS1 gene. In the second step α,α-trehalose 6-phosphate and water are converted to trehalose and phosphate by trehalose-6-phosphate phosphatase (encoded by TPS2 gene; Francois et al., 2001)(Fig1). 3 TPS1: TPS2: phosphate UDP--glucose α,α-trehalose 6- phosphate Glucose-6- trehalose-6- phosphate synthase trehalose-6- phosphate phosphatase trehalose UDP H 2O phosphate Figure 1: Trehalose synthesis from glucose 6-phophate and UDP-glucose Trehalose-6-phosphate inhibits hexokinase activity. The overproduction of trehalose may therefore restrict the amount of glucose that enters glycolysis, in turn lowering the ethanol output, but also fermentative efficiency (Hohmann et al., 1996). Preliminary studies on TPS1 deletion and overexpression mutants in our laboratory (unpublished data) have shown that both over expression and deletion of the TPS1 gene in the lab strain S288C leads to a decrease in ethanol yield, but also an overall reduction in fermentation rate (unpublished data). Both deletion and overexpression mutants produced less ethanol but had higher residual sugars at the end of fermentation (unpublished data). Glycolytic flux was impaired in the over expression strain thus accounting for the reduced fermentation efficiency and higher residual sugars. However, studies thus far have tended to use strong overexpression systems such as multiple copy plasmids and strong promoters combined to the TPS1 ORF. These excessively high expression levels may have been responsible for generating an excessive metabolic burden to the yeast, leading to the secondary effects that negatively impact on fermentation kinetics and a broad redirection of metabolic flux. Furthermore, in these studies, laboratory strains were employed for overexpression, and the fermentation conditions (low sugar levels) were not representative of real winemaking conditions. Our study therefore focuses on improving the widely used industrial Saccharomyces cerevisiae strain - VIN13 to produce less ethanol in a controlled over expression study. The aim was to increase expression of the TPS1 gene only during specific phases of growth and by a minor degree using two different promoters: The promoters of the DUT1 gene to express the gene during the exponential growth phase and of the GIP2 gene to activate gene expression during stationary phase. The aim therefore is to increase TPS1 gene expression and hopefully enzyme activity without imposing additional stress on the yeast cell and without impacting on the redox balance. Maintaining redox balance is very problematic 4 in most over-expression mutants as the production of ethanol regenerates reducing equivalents needed for the continuation of glycolysis PROJECT AIMS The following aims were set for this project: The first aim was to construct two TPS1 over-expression strains under control of different promoters. These constructs and their controls (containing only promoter sequences) were transformed into the industrial VIN13 strain of Saccharomyces cerevisiae. The second aim was to evaluate these two strains and their controls in synthetic wine, to establish the variations in ethanol yield, sugar consumption and trehalose production REFERENCES Attfield PV (1997) Stress tolerance: the key to effective strains of baker s yeast. Nature Biotechnoly 15: Bui K, Dick R, Moulin G, Galzy P (1986) A reverse osmosis for the production of low ethanol content wine. American Journal of Enology and Viticulture 37: Cambon B, Monteil V, Remize F, Camarasa C, Dequin S (2006) Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes Applied Environmental Microbiology, 72 (2006), pp Catarino M, Mendes, AV (2011) Dealcoholizing wine by membrane separation processes. Innov. Food Science & Emerging Technologies 12: Ciriacy M (1975) Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. Mutation Research 29: d Amore T, Stewart GG (1987) Ethanol tolerance of yeast. Enzyme and Microbial Technology 9: de Barros Lopes M, Rehman A, Gockowiak H, Heinrich A, Langridge P, Henschke P (2000) Fermentation properties of a wine yeast overexpressing the Saccharomyces cerevisiae glycerol 3- phosphate dehydrogenase gene (GPD2) Australian Journal of Grape and Wine Research 6: Drewke C, Thielen J, Ciriacy M (1990) Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae Journal of Bacteriology 172: Elbing K, Larsson C, Bill RM, Albers E, Snoep JL, Boles E, Hohmann S, Gustafsson L (2004) Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae. Applied and Environmental Microbiology 70: Eglinton J, Heinrich A, Pollnitz A, Langridge P, Henschke P, Lopes Mde-B (2002) Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 a
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