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

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1.7 Genetic regulation of volatile thiol synthesis by yeast 19ing for similarities between cross-species carbon-sulfur (C-S) lyases and S. cerevisiaesequences. A single-gene deletion strategy was employed and the genes were individuallyknocked out in haploid laboratory strain BY4742 and wine yeast VL3 (GLO1 was nottested in VL3). Deletion mutants were fermented in a synthetic medium containing Cys-4MMP (200 µM). All four single-gene deleted strains exhibited at least a 40 % reductionin 4MMP production compared to their corresponding wild type strain, indicating thatall genes played an important role in the release of 4MMP. However, during the courseof this thesis Thibon et al. (2008) deleted BNA3, CYS3 and IRC7 in the same wine yeastbackground using synthetic media spiked with Cys-4MMP and Cys-3MH in amounts normallyencountered in French SB grape must (20 nM and 3000 nM respectively). It wasfound that the amount of precursor in the media had a major impact on the outcome of theexperiment. Only the irc7 mutant exhibited a significant reduction in 4MMP (∼96 %),3MH (∼40 %) and 3MHA (∼60 %).In the same study Thibon et al. (2008) also investigated the effect of NCR (see Section1.2.4) on the release of volatile thiols. It was found that wine yeast VL3, deleted in URE2,exhibited a 2.3- to 4-fold increased thiol release (4MMP, 3MH and 3MHA), whereasVL3 deleted in GLN3 showed a 3.3-fold decrease in all thiols. Additionally, the ure2mutant was found to upregulate mainly the synthesis of the R-form of 3MH. These resultsstrongly indicate that NCR is a major regulatory mechanism for volatile thiol synthesisin yeast. Furthermore, Thibon et al. (2008) demonstrated that IRC7 was transcriptionallyregulated by URE2, because an irc7/ure2 double mutant strain exhibited the same lowthiolphenotype as a single-gene irc7 deletion. These results show that in synthetic media,spiked with Cys-4MMP and Cys-3MH, IRC7 is the principal gene responsible for thiolprecursor cleavage.Swiegers et al. (2006b) was the first to show that 3MHA can be formed from 3MHby yeast during fermentation. Commercial wine yeast VIN13, overexpressing the geneATF1, was employed in the fermentation of synthetic media spiked with 1 mg/L Cys-3MH. A 6-fold increase in 3MHA synthesis could be detected in the ferments conductedwith the overexpressed ATF1 strain compared to the wild type strain. Furthermore, theoverexpression of IAH1 in the same yeast background resulted in significant decrease of3MHA, as did the the deletion of ATF1 in a laboratory strain (∼ 30 % decrease). Theenzyme Atf1p is an alcohol acetyltransferase mainly known for its role in the formationof the ester ethyl acetate during wine fermentation (see Section 1.2.3), whereas Iah1p isan esterase counteracting the function of Atf1p (Lilly et al., 2006a).
20 INTRODUCTIONThe only indication about the genetic regulation of the uptake of potential 3MH precursorswas recently provided by Subileau et al. (2008a,b). In one instance, the generalamino acid permease GAP1 (see Section 1.2.4) was deleted in a laboratory strain, whichwas employed for the fermentation of synthetic media spiked with Cys-3MH (250 µg/L).Subileau et al. (2008b) encountered a significant decrease of 3MH synthesis in fermentsconducted with the gap1-deleted strain. This indicated that the Gap1p permease was responsiblefor the uptake of the major part of the Cys-3MH. However, these results wererestricted to synthetic media spiked with Cys-3MH, as the gap1 strain showed no effecton volatile thiol production in SB grape must. In another study, Subileau et al. (2008a)investigated the effect of the main glutathione permease Opt1p on thiol synthesis in grapemust. A opt1-deleted laboratory yeast mutant exhibited a 50 % reduction in 3MH and3MHA, which indicated that G-3MH could be a potential 3MH precursor (see Section1.6.2).As mentioned in Section 1.5, the conversion yields of the cysteinylated precursors intotheir corresponding volatile thiols are very low, which means that less than 10 % of thegrape aromatic potential is converted into volatile thiols. In order to utilize this untappedaroma potential, Swiegers et al. (2007) cloned and overexpressed the bacterial tnaA gene,encoding a tryptophanase with strong cysteine-β-lyase activity, into the genome of a commercialwine yeast (Vin13). Overexpression of the heterologous tnaA gene from E. coliwas achieved by utilizing the yeast promoter of phosphoglycerate kinase I, which is abundantlyexpressed in cells growing on glucose. The overexpression resulted in ∼ 25-fold increaseof the 3MH and 4MMP concentrations in synthetic media spiked with Cys-4MMP(16 mg/L) and Cys-3MH (2 mg/L), and overpowering passion fruit aromas in SB wines.1.8 Factors influencing volatile thiol concentrations inwineThe concentration of volatile thiols in wine can be influenced by a range of parameters,starting from viticultural practices–which influence the composition of the grape must–winemaking practices like fermentation temperature and the choice of the yeast strain.
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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 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: 18 INTRODUCTIONThe C6 compound (E)-
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
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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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70 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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