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

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2.4 Micro-fermentations 292.3.3 Anaerobic yeast growth mediaSynthetic grape medium (SGM)Since the constitution of natural grape juice is not fully defined and is prone to seasonalchanges, chemically defined SGM was used in some fermentation experiments. Additionally,to reduce the osmotic pressure and final ethanol concentration, SGM withoutglucose and fructose was mixed in a ratio of 1 to 4 with natural grape juice. The basiccomposition of SGM was from Henschke and Jiranek (1993), but the amino acid and diammoniumphosphate supplementation, were adjusted to reflect Sauvignon Blanc juicefrom the Marlborough region in New Zealand (2004-2006). All stock solutions wereautoclaved and kept at room temperature except for vitamins and iron(II) sulfate, whichwere filter sterilized and stored at 4 ◦ C. Table 2.4 defines the ingredients of SGM.Sauvignon Blanc (SB) grape juiceSB grape juice from the Marlborough region in New Zealand was provided by PernodRicard NZ. It was commercially harvested and stored at -20 ◦ C prior to use. The juice ofthe year 2004 was sourced from the Squire vineyard (Rapaura, Marlborough), whereas thejuices of the years 2005 and 2006 were a blend of three different sites in the Marlboroughregion: Awatere, Brancott and Rapaura. One litre of juice was sterilized with 200 µLdimethyl dicarbonate (DMDC) and incubated at 25 ◦ C with 100 rpm shaking for 12 hprior to start of fermentation. The basic properties of the grape juices are listed in Table2.5.2.4 Micro-fermentationsFerments were conducted in triplicate or duplicate unless stated otherwise.2.4.1 Pre-culture and determination of yeast countsYeast was streaked out from a -80 ◦ C stock solution and grown for two days at 28 ◦ C onYPD plates. Single yeast colonies were then used to inoculate liquid YPD medium andgrown at 28 ◦ C, 150 rpm for 24 h. Since ergosterol is synthesized by yeast only in thepresence of oxygen, YPD culture volumes were no more than 20 % of the flask volumes.
30 MATERIALS AND METHODSTable 2.4: Composition of synthetic grape medium (SGM) with a YAN of 300 mg/LCarbon source and major salts g/L Amino acids and nitrogen source mg/LD-glucose 105 L-alanine 100D-fructose 105 L-arginine 302CaCl 2 x2H 2 O 2 0.44 L-aspartic acid 50Citric acid 0.20 L-cysteine 5L-malic acid 3 L-glutamic acid 75MgSO 4 x7H 2 O 1.23 L-glutamine 125K 2 HPO 4 1.14 L-glycine 5Trace mineralsµg/LCoCl 2 x6H 2 O 24 L-histidine 20CuSO 4 x5H 2 O 50 L-isoleucine 25FeSO 4 x7H 2 O 70 L-leucine 25H 3 BO 3 6 L-lysine 6KIO 3 11 L-methionine 10MnCL 2 x4H 2 O 248 L-phenylalanine 37.5NaMoO 4 x2H 2 O 24 L-proline 75ZnSO 4 x7H 2 O 288 L-serine 50Vitaminsmg/LBiotin 0.125 L-threonine 75Calcium pantothenate 1 L-tryptophan 10Folic acid 0.2 L-tyrosine 10Myo-inositol 100 L-valine 25Nicotinic acid 2 (NH 4 )2HPO 4 612p-aminobenzoic acid 0.2Pyridoxine HCl 2Riboflavin 0.2Thiamine HCl 0.5
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IIIAbstractThe grape variety Sauvig
Page 6: VIn my Blakean yearsI was so dispos
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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
Page 53 and 54: 26 MATERIALS AND METHODSNameTable 2
Page 55: 28 MATERIALS AND METHODSSynthetic d
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 =
Page 85 and 86: 58 OPTIMISED FERMENTATION IN GRAPE
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80 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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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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