Ravasio et al., SREP-13-03702-T-R1
An indirect assay for volatile compound production in yeast strains
Davide Ravasio1, Andrea Walther1, Kajetan Trost2, Urska Vrhovsek2, and Jürgen Wendland1*
1Carlsberg Laboratory; Yeast Genetics
Gamle Carlsberg Vej 10
DK-1799 Copenhagen V, Denmark
2Fondazione Edmund Mach
Research and Innovation Centre
Food Quality and Nutrition Department
Via E.Mach 1,
I-38010 S.Michele all'Adige, Italy
ONline Materials and methods
Strains and media.
The strains used in this study are listed in Table S2. Yeast strains were grown in YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose). Geneticin/G418 (200µg/ml) or Nourseothricin/clonNAT (50 µg/ml) was added for selecting plasmid transformants. For the selection of auxotrophic markers minimal medium was used (CSM-URA: 2% glucose, 6.7 g/l yeast nitrogen base w/o amino acids with ammonium sulfate, 0.77 g/l CSM-URA dropout mixture). For double selection of auxotrophic markers and antibiotics ammonium sulfate was replaced by asparagine (CSM-URA w/ clonNAT: 2% glucose, 1.7 g/l yeast nitrogen base w/o amino acids and ammonium sulfate, 0.77 g/l CSM-URA dropout mixture, 1 g/l asparagine, 50 µg/l clonNAT). Tall tube fermentations were performed in either YPD medium (140 g/l glucose, 14.6 °P) or granmalt medium (150 g/l malt granules, 5 g/l yeast extract, 13.7 °P). Escherichia coli strain DH5αserved as host for plasmid propagations and was grown at 37 °C in 2YT medium (2YT: 1.6% peptone, 1% yeast extract, 0.5 % NaCl).
Fermentation conditions
Fermentation was performed in 250ml tall tube cylinders filled with either 200 ml YPD or granmalt medium at 14 °C. Cells were inoculated with a starting OD600 of 0.2 and stirred at 190 rpm using a magnetic stirrer. Fermentation performance was monitored for 7-10 days by daily measurement of CO2 loss and sugar content using a DMA 35 Anton Paar densitometer (medium gravity in °P). The fermentation was defined as finished when the Plato value did not drop further for 2 days. 50 ml samples were taken and used for GC/MS analysis. All fermentations were carried out in biological duplicated and technical triplicates.
Plasmid constructs.
Plasmids used inthis study are shown in Table S2.Primers were obtained from IDT (Integrated DNA Technologies, Leuven, Belgium) and are listed in Table S3. For the cloning of ARO gene promoters primers with specific restriction sites to KpnI and SalI/XhoI were used to amplify these sequences from genomic DNA. After cleavage the promoter fragments were cloned into pRS416-AgTEFp-lacZ or pRS417-AgTEFp-lacZ replacing the AgTEF promoter. The ScARO80 open reading frame was amplified using the primers #6391 and #6418, cleaved then with XbaI/SpeI and cloned into pRS418-AgTEFp-lacZ replacing the lacZ open reading frame. Correct cloning of all constructs was confirmed by sequencing.
Yeast transformation.
Yeast cells were transformed by the Lithium acetate method as described24.
Quantification of β-galactosidase expression
The quantification of β-galactosidase expression was performed using a color reaction (yellow) based on the conversion of ONPG (ο-nitrophenyl galactopyranoside) to o-nitro phenole and galactose by β-galactosidase25. Yeast cultures were grown in selective media o/n. The OD600 was measured and 2 ml cell suspensions were centrifuged, washed and resuspended in Z- buffer (60 mM Na2HPO4x2H2O, 40mMNaH2PO4x2H2O, 10 mM KCl, 1 mM MgSO4x7H2O). Cells were disrupted by freezing in liquid nitrogen followed by 1min incubation at 37 °C and vortexing. To 100µl cell suspension 150 µl ONPG/Z-buffer solution (4 mg/ml) was added. Samples were incubated at 37 °C for 30 min. The enzymatic reaction was stopped by adding 400 µl 1M Na2CO3. Samples were centrifuged to remove the cell debris and OD420 was measured. Enzymatic activity was calculated using the equation: A = 1000 x [OD420 – (1, 75 x OD550)] / t [min] x V [ml] x OD600.
Analytical methods – GC-MS
Volatile compounds were measured using a Thermo Scientific TSQ Quantum GC Triple Quadrupole GC/MS. As an internal standards 2-octanol and butyl heptanoate were added to each sample. The internal standards were chosen as compounds known not to be present in beer fermentation. Headspace aqueous solutions were prepared in 20 ml vials by mixing 1ml of sample with 3.85 ml deionized water with natrium sulphate (0.1%), appropriate amounts of magnesium sulphate and ascorbic acid.All samples were incubated for 10 min at 50 °C. The volatile compounds were collected on a Divinylbenzene/Carboxen/Polydimethylsiloxane fiber (DVB-CAR-PDMS) for an extraction time of 40 min. A Solgel-wax column, 30m/I.D 0.25mm/Film 0.25μm, was used for all analyses.
The oven was kept at 40°C for 4 min then increased by 6 °C/min to 250°C and kept at the final temperature for 5 min. The injector and interface temperatures were kept at 250°C as well. Helium was used as the carrier gas with a flow rate of 1.2ml/min. The time for thermal desorption of analytes was 4min.The MS detector was operated in full scan mode at 70 eV and the scan range was from 35 to 350 m/z.
Data analysis was performed using the software ThermoExalibur (Version 1.0.1.03, Thermo scientific). Identification of compounds was based on comparison with a mass spectral database (Nist 1.0.0.23). One characteristic quantifier ion and two to three qualifier ions were selected for each compound. The peak area of the quantifier ion was used for quantification.The concentrations of esters were expressed as butyl heptanoate equivalents while alcohols and acids were expressed as 2-octanol equivalents.
Multivariate data analysis:
Principal Component Analysis (PCA) was carried using R with "pcaMethods" and "FactoMineR" packages. The Matrix containing 33 samples from 33 individuals, was preprocessed. Unit variance was scaled and mean centered. Three principal components were calculated. Finally the barycenter of the five grouped samples was calculated and ellipses (with 0.95-confidence level) were constructed around them. PCA analyses confirmed that replicate samples of the same strain grouped together23(data not shown).
References for Supplemental Material and Methods
23Ghosh, D. & Chattopadhyay, P. Application of principal component analysis (PCA) as a sensory assessment tool for fermented food products. J Food Sci Technol49, 328-334, doi:10.1007/s13197-011-0280-9 280 [pii] (2012).
24Gietz, R. D. & Schiestl, R. H. Microtiter plate transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc2, 5-8, doi:nprot.2007.16 [pii] 10.1038/nprot.2007.16 (2007).
25Rose, M. & Botstein, D. Construction and use of gene fusions to lacZ (beta-galactosidase) that are expressed in yeast. Methods Enzymol101, 167-180 (1983).
Supplemental Tables
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Table S1. VOCs produced at the end of the fermentation (μg/L)Compounds / BAY / PAR / MIK / UVA / KUD / EUB / CAR / C44 / C444 / CEN / Wine
Isoamyl alcohol / 2468±(110,53) / 2850±(54,86) / 1081,71±(32,52) / 2199±(48,59) / 2812±(171,60) / 2651±(160,86) / 1100±(67,78) / 2409±(68,17) / 2142±(147,92) / 1141±(19,06) / 2246±(135,20)
Acetic acid / 35±(5,92) / 38±(2,42) / 42,51±(4,15) / 29±(1,66) / 23±(2,43) / 41±(3,03) / 43±(1,75) / 22±(1,23) / 11±(0,45) / 47±(1,78) / 104±(3,65)
1-Octanol / 15±(1,96) / 11±(0,58) / 6,52±(0,87) / 9±(0,40) / 6±(0,57) / 8±(0,28) / 5±(0,20) / 13±(0,06) / 5±(0,29) / 3±(0,23) / 47±(5,50)
Butyric acid / 22±(0,84) / 17±(1,00) / 22,04±(2,22) / 34±(1,13) / 14±(0,25) / 23±(1,85) / / / 19±(0,55) / 15±(0,03) / / / /
Nonanol / 15±(1,48) / 8±(1,58) / / / 26±(4,29) / 8±(1,21) / 10±(0,27) / / / / / / / 8±(0,49) / /
Isovaleric acid / / / 8±(0,61) / 11,34±(1,19) / 9±(0,22) / 12±(0,52) / 13±(0,28) / 11±(0,54) / 8±(0,37) / 7±(0,26) / 11±(0,54) / /
1-Decanol / 7±(0,41) / 8±(1,00) / 8,96±(2,65) / 9±(0,28) / 6±(0,45) / 5±(0,69) / 9±(0,29) / 8±(0,00) / 9±(0,46) / 3±(0,61) / 5±(0,22)
Hexanoic acid / 51±(0,26) / 90±(6,72) / 35,48±(5,50) / 55±(0,80) / 35±(1,25) / 59±(7,35) / 31±(2,70) / 84±(7,08) / 107±(9,78) / 35±(3,20) / 224±(13,54)
2-Phenylethanol / 291±(8,52) / 416±(24,63) / 66,99±(3,91) / 496±(25,88) / 534±(7,81) / 773±(79,42) / 25±(2,01) / 225±(21,32) / 293±(7,13) / 44±(3,81) / 57±(2,14)
Heptanoic acid / 6±(0,22) / 6±(0,19) / 4,61±(0,75) / 5±(0,19) / 6±(0,69) / 6±(0,60) / 4±(0,08) / 5±(0,75) / 7±(0,35) / 4±(0,57) / 6±(0,21)
Octanoic acid / 289±(1,71) / 171±(11,10) / 122,07±(28,67) / 171±(5,99) / 133±(7,93) / 178±(0,08) / 86±(9,57) / 270±(21,97) / 439±(32,78) / 132±(0,40) / 771±(59,67)
Nonanoic acid / 297±(42,39) / 272±(32,14) / 158,53±(7,08) / 247±(22,76) / 154±(32,30) / 289±(50,11) / 130±(2,09) / 195±(17,24) / 189±(19,87) / 280±(48,40) / 298±(49,54)
4-Vinylguaiacol / 346±(1,21) / 344±(20,20) / 318,76±(2,03) / 337±(27,25) / 371±(9,10) / 495±(118,74) / 222±(35,68) / 86±(7,42) / 274±(32,48) / 234±(22,81) / 2±(1,31)
Decanoic acid / 98±(11,47) / 90±(10,70) / 12,61±(7,86) / 87±(2,08) / 211±(26,40) / 86±(4,23) / 14±(0,17) / 14±(0,41) / 112±(17,50) / 55±(4,16) / 73±(8,63)
9-Decenoic acid / 65±(9,28) / 24±(1,48) / / / 73±(2,08) / 9±(0,88) / 111±(36,18) / / / 19±(1,96) / 116±(4,69) / 8±(4,36) / 257±(26,31)
Butyl acetate / / / / / / / / / / / / / / / / / / / / / 24±(1,42)
Isoamyl acetate / 1744±(0,00) / 1912±(124,44) / 122,41±(11,44) / 143±(6,08) / 115±(26,18) / 365±(17,52) / 355±(24,89) / 21±(0,73) / 1366±(315,88) / 727±(11,33) / 25±(1,95)
Ethyl hexanoate / 41±(1,83) / 70±(2,66) / 7,26±(0,29) / 71±(3,42) / 90±(8,71) / 60±(2,38) / 5±(0,34) / 72±(1,02) / 76±(0,01) / 3±(0,37) / 194±(4,96)
2-Octanone / 3±(1,96) / 3±(0,49) / 5,84±(1,56) / 7±(0,80) / 6±(1,43) / 8±(0,29) / 6±(0,47) / 5±(0,57) / 5±(0,98) / 15±(1,27) / 3±(0,55)
Nonanal / 2±(0,11) / 2±(0,60) / 2,31±(0,35) / 2±(1,22) / 2±(1,43) / 3±(0,32) / 8±(0,02) / 12±(0,16) / 7±(1,27) / 19±(11,83) / 21±(0,63)
Ethyl octanoate / 187±(8,88) / 138±(4,97) / 28,94±(7,23) / 213±(4,53) / 294±(48,31) / 439±(28,13) / 11±(2,38) / 454±(45,95) / 476±(76,99) / 26±(3,09) / 263±(48,56)
Furfural / 4±(1,26) / 6±(1,52) / 9,32±(0,74) / 10±(0,00) / 17±(2,49) / 17±(4,71) / 11±(0,48) / 11±(2,18) / 15±(1,22) / 6±(1,18) / 9±(0,87)
Benzaldehyde / 1±(0,02) / 1±(0,04) / 1,54±(0,22) / 1±(0,08) / 2±(0,11) / 2±(0,16) / 2±(0,11) / 1±(0,10) / 2±(0,16) / 1±(0,07) / 2±(0,38)
Ethyl decanoate / 60±(1,96) / 45±(2,65) / 2,43±(0,02) / 73±(7,96) / 291±(27,01) / 89±(7,78) / 2±(0,15) / 5±(0,60) / 36±(1,33) / 6±(0,14) / 8±(1,83)
4-Methyl benzaldehyde / 2±(0,11) / 2±(0,13) / 2,39±(0,43) / 3±(0,49) / 4±(0,26) / 4±(0,37) / 2±(0,07) / 3±(0,23) / 4±(0,40) / 2±(0,17) / 3±(0,74)
Ethyl 9-decanoate / 109±(6,59) / 44±(3,47) / 0,91±(0,02) / 154±(13,25) / 39±(5,10) / 204±(19,80) / 1±(0,14) / 41±(3,28) / 94±(4,95) / 3±(0,48) / 61±(7,67)
2-Phenylethyl acetate / 188±(7,30) / 82±(3,53) / 37,46±(5,44) / 303±(1,46) / 350±(20,02) / 689±(51,77) / 3±(0,53) / 346±(38,06) / 463±(10,83) / 6±(0,74) / 121±(10,74)
Ethyl dodecanoate / 27±(1,41) / 11±(2,16) / / / 28±(3,32) / 28±(4,89) / 14±(2,53) / / / / / 6±(2,07) / 0±(0,31) / 2±(2,32)
β-phenylethyl butyrate / 8±(0,14) / / / / / 16±(2,55) / 18±(0,93) / 17±(2,43) / / / / / / / / / /
4 Ethyl-hydroxybenzoate / 2±(0,05) / 2±(0,24) / 2,57±(0,22) / 3±(0,45) / 3±(0,31) / 4±(0,45) / 2±(0,27) / 3±(0,26) / 4±(0,39) / 2±(0,11) / 2±(0,34)
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Table S2
Strains used in this study
Strain / Description / Genotype / SourceBY4741 / S. cerevisiae / his3Δ; leu2Δ;met15Δ; ura3Δ / Euroscarf
NBRC1948 / S. bayanus / Wild type / NBRC, Japan
CBS 432 / S. paradoxus / Wild type / CBS
CBS 8839 / S. mikatae / Wild type / CBS
CBS 7001 / S. uvarum / Wild type / CBS
CBS 8840 / S. kudriavzevii / Wild type / CBS
CBS 12357 / S. eubayanus / Wild type / CBS
CBS 8841 / S. cariocanus / Wild type / CBS
C44 / S. pastorianus / Weihenstephan LagerYeast 34/70 / Carlsberg
C444 / S. pastorianus / Lager yeast / Carlsberg
CEN.PK / S. cerevisiae / Prototrophic / Leiden
165 / S. cerevisiae / Bordeaux wine yeast / Carlsberg
C782 / BY4741; ∆aro80 / his3Δ1; leu2Δ0;met15Δ0; ura3Δ0; aro80::KanMX / Euroscarf
Table S3
Plasmids used and generated in this study
Plasmid / Description / Source108 / pRS416 (URA3) / Lab collection
C177 / pRS417 (GEN3) / Lab collection
C218 / pRS418 (NAT5) / Lab collection
628 / pRS416-AgTEFp-lacZ1 / Lab collection
651 / pRS417-AgTEFp-lacZ / Lab collection
C455 / pRS418-AgTEFp-lacZ / Lab collection
C814 / pRS416-ScARO8p-lacZ / This study
C815 / pRS416-ScARO9p-lacZ / This study
C816 / pRS416-ScARO10p-lacZ / This study
C817 / pRS416-ScARO80p-lacZ / This study
C858 / pRS418-AgTEFp-ScARO80 / This study
C907 / pRS417-ScARO9p-lacZ / This study
1: LacZ is derived from Streptococcus thermophilus
Table S4
Oligos used in this study
Oligo / Name / Sequence 5’-to-3’*#6281 / 5'-ScARO8p (KpnI) / AGAATAggtaccCAGGGGTTCGAGCCCCCTATG
#6282 / 3'-ScARO8p (SalI) / TAGAATgtcgacGATAGTAACGATCGGTTGTCC
#6283 / 5'-ScARO9p (KpnI) / AGAATAggtaccCGGTATGTTACAACAGTCAAAG
#6284 / 3'-ScARO9p (XhoI) / TAGAATctcgagTGAGTCGATGAGAGAGTGTAATTG
#6285 / 5'-ScARO10p (KpnI) / AGAATAggtaccGCGTACAACAACGTCTTAGCG
#6286 / 3'-ScARO10p (XhoI) / TAGAATctcgagGCTTAAGGGAGTTTCTTTGTTATC
#6287 / 5'-ScARO80p (KpnI) / AGAATAggtaccGAAGATTTAGAAATAGAGGATATTG
#6288 / 3'-ScARO80p (XhoI) / TAGAATctcgagAGAGGATAAAGCAGTGCTTAATG
#6391 / A1-ScARO80 (XhoI) / ATTACActcgagATGTCTGCTAAGAAAAGGCCTTCGGG
#6418 / A4-ScARO80 (SpeI) / ATAAGAactagtGGCAATCCACTTCTCAGCAT
* Restriction sites are in lower case.
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