Identification of yeast genes involved in Sauvignon Blanc aroma ...

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IIIAbstractThe grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industryand accounted for over 75 % of the value of total wine exports in 2008. Two volatilethiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscentof grapefruit and passion fruit respectively, are critical for the main varietal charactersin New Zealand SB. These aromatic thiols are not present in the grape juice, but aresynthesized and released by the yeast during alcoholic fermentation from non-aromaticprecursors.The aim of this work was to elucidate the underlying genetics of volatile thiol synthesisin yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A genedeletionstrategy was chosen for the investigation of putative genes influencing 3MH and3MHA release.The first part of this thesis optimized fermentation conditions in grape-juice-based media,which enabled auxotrophic laboratory strains, derived from S288C, to ferment grapejuice to completion with high efficiency. Key steps to achieving this goal were the supplementationof the grape juice with higher than recommended amounts of amino acids,which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophicstrains especially benefited from this measure. In combination with the dilution of SBgrape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratoryyeast BY4743 was able to metabolize all sugars in the grape-juice-based media in a timeframe similar to that of a commercial wine yeast. The key properties of the resulting winewere comparable to wine made with a commercial wine yeast under the same conditions.In the second part, these newly developed fermentation conditions were employed toscreen 69 single-gene deletion strains in the laboratory yeast background BY4743. Thelist of the 69 candidate genes was compiled by combining existing knowledge about thiolproduction in yeast with the mining of several biological databases. Screening of thesingle-gene deletions revealed 17 genes which caused biologically relevant increases ordecreases in volatile thiol production, but none abolished it. The majority of the 17 geneswere related to the sulfur and nitrogen metabolism in yeast. A subset of these thiolinfluencinggenes were also deleted in a wine yeast, and were overexpressed in both wineyeast and laboratory yeast, to gain more insight in their regulatory effects. The findingsconfirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MHand 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape
IVmust prior to fermentation and their effect on thiol release was studied. It was foundthat nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine(SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition ofcysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 morethan doubled total thiol production.Mapping approaches to investigate thiol production in yeast were employed in the finalpart of this thesis. Genetically mapped F2 progeny of a cross between a low thiolproducingyeast strain and a high-thiol producer were screened for their thiol phenotype.The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions,indicating a quantitative trait. Subsequent mapping identified a locus on chromosome14 with a small effect on the 3MHA phenotype, but no obvious candidate geneswere evident in the region.Another approach to investigate the evolution of volatile thiols in yeast included theuse of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a solenitrogen source in a yeast growth assay. It was found that most wine yeast, European yeastisolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas variousother S. cerevisiae isolates could not. Crosses between three pairs of Sec − and Sec +yeast strains strongly indicated that this trait was monogenically inherited. However, nodirect correlation between the SEC phenotype and volatile release could be observed.Genetic mapping experiments in one SEC-segregating yeast population linked this SECphenotype to the leu2-∆0 deletion in a cross between a Leu + and Leu − yeast strain. Itwas shown that leucine auxotrophy most likely caused the Sec − phenotype.In a second F2 population of a cross between prototrophic Sec + and Sec − strains,strong linkage was established to a region on chromosome 6 containing two candidategenes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype,whereas IRC7 remains a strong candidate gene.
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Page 6: VIn my Blakean yearsI was so dispos
Page 9 and 10: VIII• Kien Ly for letting me “b
Page 11 and 12: XTABLE OF CONTENTS1.8.2 Winemaking
Page 13 and 14: XIITABLE OF CONTENTS3.1 Introductio
Page 15 and 16: XIVTABLE OF CONTENTS5.4 S-ethyl-L-c
Page 18 and 19: XVIIList of figures1.1 Model of NCR
Page 20 and 21: XIX5.3 Fermentation rate and volati
Page 22 and 23: XXIList of tables1.1 Characteristic
Page 24 and 25: XXIIIAbbreviationsAbbreviations of
Page 26: XXVUUVUKPv/vVAV maxwtw/vw/wYAN2-ami
Page 29 and 30: 2 INTRODUCTIONfactor to enable this
Page 31 and 32: 4 INTRODUCTIONThis work is entirely
Page 33 and 34: 6 INTRODUCTION• Sulfur-containing
Page 35 and 36: 8 INTRODUCTIONnitrogen sources for
Page 37 and 38: 10 INTRODUCTION1.3 An overview of t
Page 39 and 40: 12 INTRODUCTIONH 2 S into organic s
Page 41 and 42: 14 INTRODUCTIONTable 1.1: Character
Page 43 and 44: 16 INTRODUCTIONThe current model of
Page 45 and 46: 18 INTRODUCTIONThe C6 compound (E)-
Page 47 and 48: 20 INTRODUCTIONThe only indication
Page 49 and 50: 22 INTRODUCTIONgrape-bunch pressing
Page 51 and 52: 24 INTRODUCTION3. The third goal wa
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26 MATERIALS AND METHODSNameTable 2
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28 MATERIALS AND METHODSSynthetic d
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30 MATERIALS AND METHODSTable 2.4:
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32 MATERIALS AND METHODSTo determin
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34 MATERIALS AND METHODSx 10 6 cell
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36 MATERIALS AND METHODSTable 2.6 -
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40 MATERIALS AND METHODS2.9.3 Isola
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46 MATERIALS AND METHODSPCR product
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48 MATERIALS AND METHODS45 bpUKP-Ge
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50 MATERIALS AND METHODS2.12.2 Tran
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52 MATERIALS AND METHODSTable 2.8:
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54 MATERIALS AND METHODSGeneral set
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56 MATERIALS AND METHODSSD pooled =
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58 OPTIMISED FERMENTATION IN GRAPE
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834 Identification of yeast genesin
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4.2 Candidate genes for volatile th
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4.3 Screening of single-gene deleti
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4.4 Deletion of candidate genes in
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4.5 Volatile thiol release in yeast
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4.6 Supplementation of grape juice
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4.7 Discussion and conclusion 1174.
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4.7 Discussion and conclusion 1194.
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4.7 Discussion and conclusion 121Th
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4.7 Discussion and conclusion 123Ta
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4.7 Discussion and conclusion 125to
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4.7 Discussion and conclusion 127ca
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4.7 Discussion and conclusion 129me
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4.7 Discussion and conclusion 131an
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4.7 Discussion and conclusion 133ce
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136 GENETIC MAPPING APPROACHES TO A
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1676 Concluding DiscussionMy resear
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6.1 Laboratory yeast can be used in
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6.2 Sulfur metabolism plays an impo
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6.4 Volatile thiol synthesis of 3MH
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6.5 S-ethyl-L-cysteine as a model c
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6.6 Concluding thoughts 177from dif
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180 APPENDICEScompounds. However, w
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182 APPENDICESA.3 Variation of vola
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2287 bp1765 bp2103 bp2286 bp184 APP
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186 APPENDICESA.5 Overexpression of
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188 APPENDICESAUKP-GeneX-F/R3236 bp
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190 APPENDICESTable A.4: S-ethyl-L-
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192 APPENDICESTable A.5: SEC phenot
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194 APPENDICESA.9 S-ethyl-L-cystein
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196 APPENDICESA.10 Deleting DUG1 in
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198 APPENDICESA.11 Differentiation
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200 APPENDICESA.12 Overexpression o
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202 APPENDICESA.12.2 Transformation
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204 APPENDICESCumulative weight los
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207ReferencesAllen, M. S. and Lacey
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209Ciani, M., Beco, L., and Comitin
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211cerevisiae.” Mol. Cell. Biol.,
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213Kumar, C., Sharma, R., and Bachh
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215Nakagawa, S. and Cuthill, I. C.
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217Schlenk, F. and Zydek, C. R. (19
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219thesis, University of Bordeaux 2
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221expression of some stress induce
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