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

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1.2 Yeast in winemaking 7pletion and can lower hydrogen sulfide production (Bely et al., 1990). Both high (> 480mg/L) and low (< 140 mg/L) nitrogen concentrations in grape must are associated withthe production of undesirable aromas (Bell and Henschke, 2005). The addition of nitrogenshould therefore be carefully planned (e.g. sequential supplementation during thefermentation) in order to maximise desirable aromas. In any case, nitrogen addition atany stage of the fermentation is thought to result in a shorter fermentation (Beltran et al.,2005).Yeast assimilable nitrogen is rapidly taken up during the first part of the yeast growthphase, stored in the vacuole, and used for protein synthesis and growth when required(Beltran et al., 2005). Nitrogen permeases, responsible for ammonium transport into theyeast cell, include Mep1p, Mep2p (high affinity) and Mep3 (low affinity) (Marini et al.,1997). Gap1p is a general amino acid permease with high capacity and low affinity,whereas numerous other amino acid permeases transport specific amino acids with lowcapacity and high affinity (Magasanik and Kaiser, 2002). Ammonium ions are preferentiallyutilised and stimulate yeast growth, leading to an overall increase in nitrogenutilisation (Jiranek et al., 1995a). Beltran et al. (2005) confirmed the findings by Jiraneket al. (1995a) and grouped amino acids according to their consumption preferences by theyeast cell: (a) glutamine and tryptophan were the most consumed; (b) followed by histidine,isoleucine, leucine, methionine, phenylalanine, valine, threonine; and (c) alanine,arginine, aspartate, glycine and tyrosine, which were hardly consumed, especially whenammonium was not deficient.Nitrogen catabolite repressionAs outlined in the previous section, yeast has a choice of different nitrogen sources duringfermentation, but not all nitrogen sources support growth equally well. A mechanismcalled nitrogen catabolite repression (NCR) enables yeast to select nitrogen sources thatprovide the best growth (Wiame et al., 1985). All pathways for metabolising the variousnitrogen sources present in the grape feed into the core pathway for the biosynthesis ofglutamate and glutamine. The ammonia ions resulting from the various nitrogen sourcesreact with α-ketoglutarate (a key intermediate from the Krebs cycle) to form glutamate,or with glutamate to form glutamine (Magasanik and Kaiser, 2002). The amino group ofglutamate supplies 85 % total cellular nitrogen and the amide group of glutamine is thesource of the remaining 15 % (Cooper, 1982). NCR allows yeast to utilize “preferred”
8 INTRODUCTIONnitrogen sources for glutamate and glutamine synthesis, by repressing the transcriptionof genes encoding for enzymes and permeases for the utilization for “non-preferred” nitrogensources (Magasanik and Kaiser, 2002). Once preferred nitrogen sources becomelimited or are depleted in the media, genes responsible for the utilisation of non-preferredsources are gradually derepressed and NCR is lifted. A study by Godard et al. (2007)revealed that a range of different nitrogen sources in the media can affect the expressionof about 10 % of all yeast genes.The classification between preferred and non-preferred nitrogen sources is not alwaysclear as yeast strain-specific preferences do exist. Generally two criteria are applied tojudge the quality of a particular nitrogen source: (1) preferred nitrogen sources generallyallow greater growth rates; (2) preferred nitrogen sources do not derepress nitrogenregulatedgenes for the use of poor nitrogen sources, whereas nitrogen sources that doinduce derepression of these genes are considered to be non-preferred (Magasanik andKaiser, 2002). By applying these two criteria, Godard et al. (2007) classified the nitrogensources for the laboratory yeast ∑1278b as follows:• asparagine, glutamine and serine were classified as preferred, inducing strong NCRassociated with fast growth.• ammonium and aspartate induced intermediate NCR combined with fast growth,whereas alanine and arginine caused intermediate NCR combined with slower growth.• ornithine, phenylalanine, proline and valine induced weak NCR associated withslow growth, while urea caused weak NCR but induced fast growth.• isoleucine, leucine, methionine, threonine, tryptophan and tyrosine do not induceNCR but were associated with very slow growth.NCR is regulated by short-term post-transcriptional responses leading to direct enzymeinactivation and/or degradation, and longer-term transcriptional response (Beltran et al.,2005).The post-transcriptional regulation mostly involves the rapid proteolytic degradationof permeases responsible for the transport of poor nitrogen sources (e.g. Beltran et al.,2005). Phosphorylation and dephosphorylation of permeases by specific phosphatases isthe main regulatory process.The transcriptional regulation involves four members of the GATA family of transcriptionfactors, as well as the regulatory protein Ure2p (Cooper, 2002). The target promoter
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Page 4 and 5: IIIAbstractThe grape variety Sauvig
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: 6 INTRODUCTION• Sulfur-containing
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
Page 53 and 54: 26 MATERIALS AND METHODSNameTable 2
Page 55 and 56: 28 MATERIALS AND METHODSSynthetic d
Page 57 and 58: 30 MATERIALS AND METHODSTable 2.4:
Page 59 and 60: 32 MATERIALS AND METHODSTo determin
Page 61 and 62: 34 MATERIALS AND METHODSx 10 6 cell
Page 63 and 64: 36 MATERIALS AND METHODSTable 2.6 -
Page 67 and 68: 40 MATERIALS AND METHODS2.9.3 Isola
Page 73 and 74: 46 MATERIALS AND METHODSPCR product
Page 75 and 76: 48 MATERIALS AND METHODS45 bpUKP-Ge
Page 77 and 78: 50 MATERIALS AND METHODS2.12.2 Tran
Page 79 and 80: 52 MATERIALS AND METHODSTable 2.8:
Page 81 and 82: 54 MATERIALS AND METHODSGeneral set
Page 83 and 84: 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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