Journal: Applied Microbiology and Biotechnology

Title: MET2 affects production of hydrogen sulfide during wine fermentation

Authors: Chien Huang, Miguel Roncoroni, Richard C Gardner

Corresponding author: Dr Richard Gardner, School of Biological Sciences,

University of Auckland, Private Bag 92019, Auckland, New Zealand;

r.gardner@auckland.ac.nz

Appendix S1: Detailed methods for quantification of aroma compounds

(a) Quantification of reductive sulfur compounds using HS-SPME and GC-MS

HS-SPME / GC-MS controlled by AGILENT MSD ChemStation D.01.02.16 software

(Agilent Technologies, Santa Clara, CA 95051, http://agilent-msd-chemstation-g1701da-d-

01-02.software.informer.com/) was used to analyse the reductive sulfur compounds in wines

produced by yeast strains. The method was initially developed by Nguyen et al. (2010) which

was developed further by Mandy Herbst-Johnstone, at The University of Auckland. The

analysis required 10 mL of wine sample, which was added into an Agilent 20 mL amber

Headspace screw vial contained 2.5 g of magnesium sulfate. The vial was purged with argon,

sealed with an Agilent screw cap then 50 μl of a standard mix solution containing 2H6-

dimethyl sulfide (30 μg/L), isopropyl disulfide (2 μg/L), and 3-(methylthio)-1-hexanol (154

μg/L) was injected using a SGE gas-tight syringe. The sample then placed onto the Gerstel

MultiPurpose Sampler MPS2 tray (VT32-20).

Each sample was incubated for 5 min in the Gerstel Agitator/Stirrer AS at 45°C with

agitation at 500 rpm prior to extraction. A 2-cm, 23-gauge, 50/30 m, DVB/CAR/PDMS

fibre for Automated Holder, Gray, Notched (Supelco, Bellefonte, Pen. USA), which was pre-

baked for 5 min (60 mm bakeout penetration) at 250°C (pressure at 50 kPa, total flow rate of

14.022 mL per min, and a septum purge flow of 2 mL per min) in the front injection port

(FrontS/S), was exposed in the 20-ml capped vial (30 mm vial penetration) for 30 min at

45°C. During this exposure period the quantity of analyte extracted by the fibre was

proportional to its concentration in the sample as long as equilibrium was reached. After

extraction, the fibre was transferred to the rear injection port (Back PTV Inlet) of an Agilent

7890A GC System coupled to a mass selective detector model 5975C inert XL (Santa Clara,

CA, USA), where desorption of the analyte in splitless mode (pressure 56.92 kPa, total flow

rate of 91.5 mL per min, and a septum purge flow of 10 mL per min) takes place for 10 min

at 250°C (60 mm injection penetration). Helium was used as the carrier gas (56.92 kPa) at a

flow rate of 1.5 mL per min. Volatiles were separated on a tandem column composed of a

HP-1ms (30 m, 0.320 mm ID, 0.25 µm film) and HP-INNOWax (30 m, 0.320 mm ID, 0.25

µm film) (Agilent, CA, USA). A CTS 2 (cryogenic trap system; Gerstel, Mülheim, Germany),

mounted on the inside of GC oven, was used to try to improve the detection of H2S with an

initial temperature set to -40°C for 2 min. Low pressure liquid nitrogen with an initial

temperature set to -40°C for 2 min was used for cryo cooling (to improve the detection of

H2S) the head of the column using a CTS 2 (cryogenic trap system; Gerstel), mounted on the

inside of GC oven. The oven program was as follow: the initial oven temperature of 40°C

was held for 5 min, then ramped to 60°C at a rate of 1.5°C per min, then at 4°C per min to

150°C held for 5 min, raised to 240°C at 60°C per min and held for 5 min, giving a total run

time of 52.333 min. The temperature of the interface line was set to 250°C. The ion source,

operating in electron impact mode at 70 eV, was held at 230°C. The quadrupole temperature

was set at 150°C. The MassHunter software (Agilent, CA, USA) was used to analyse the

results.

(b) Quantification of esters, terpenes, norisoprenoids, cinnamate, phenol, fatty

acids, alcohols and aldehydes using headspace solid-phase microextraction and

gas chromatography-mass spectrometry (HS-SPME and GC-MS)

HS-SPME / GC-MS controlled by AGILENT MSD ChemStation D.01.02.16 software was used

to analyse the aroma compounds in wines produced by different yeast strains. The method was

developed by Mandy Herbst-Johnstone at The University of Auckland. The analysis required 10

mL of wine sample, which was added into an Agilent 20-mL amber Headspace screw vial

contained 3.5 g of sodium chloride, 30 μL of deuterated standard solution (Table S2 below) and

50 μL of a non-deuterated standard mix solution containing DL-3-octanol (414 μg/L), 4-decanol

(639 μg/L), and 3,4-dimethylphenol (158 μg/L). The vial was purged with argon and sealed with

an Agilent screw cap then placed onto the Gerstel MultiPurpose Sampler MPS2 tray (VT32-20).

Each sample was incubated for 10 min in the Gerstel Agitator/Stirrer AS at 45°C with agitation at

700 rpm prior to extraction. A 2-cm, 23-gauge, 50/30 μm, DVB/CAR/PDMS fibre for automated

Holder, Gray, Notched (Supelco, Bellefonte, Pen. USA), which was pre-baked for 5 min (54 mm

bake out penetration) at 250°C (pressure at 50 kPa, total flow rate of 14.022 mL per min, and a

septum purge flow of 2 mL/min) in the front injection port (FrontS/S), was exposed in the 20-ml

capped vial (30 mm vial penetration) for 60 min at 45°C. During this exposure period, the

quantity of analyte extracted by the fibre was proportional to its concentration in the sample as

long as equilibrium was reached. After extraction, the fibre was transferred to the rear injection

port (back PTV Inlet) of an Agilent 7890A GC system coupled with a mass selective detector

model 5975C inert XL (Santa Clara, CA, USA), where desorption of the analyte in splitless mode

(pressure 27.881 kPa, total flow rate of 15 mL per min, and a septum purge flow of 2 mL per took

place for 10 min at 250°C (54 mm injection penetration). Helium was used as the carrier gas

(27.881 kPa) at a flow rate of 1 mL per min. Volatiles were separated on a tandem column

composed of a HP-1ms (30 m, 0.320 mm ID, 0.25 μm film) and HP-INNOWax (30 m, 0.320 mm

internal diameter and 0.25 μm film) (Agilent, CA, USA). The oven program was as follow:

initially 40°C for 5 min then ramped to 200°C at a rate of 2°C per min and held for 5 min, raised

to 240°C at 80°C per min and held for 5 min, giving a total run time of 95.5 min. The temperature

of the interface line was set to 250°C. The ion source, operating in electron impact mode at 70 eV,

was held at 230°C. The quadrupole temperature was set at 150°C. The MassHunter software

(Agilent, CA, USA) was used to analyse the results.

Table S1: PCR primers and sequences

Name Sequence (5'–3

') Use

Met2F TATCCGGTAGAGGAGCAACC Combine with Met2R: amplification of

MET2

Met2R CGCAGGCTTTATGTCCTTCA See above

MET2KF CATGTTAATCGTTTGGATTT Integration of kanMX into MET2 of F15,

M2

MET2KR GTATGATTATTGCGATATGC

Matα GCACGGAATATGGGACTACTTCG Combine with MatR and MatA; indicates

mating type, ploidy

MatA ACTCCACTTCAAGTAAGAGTTTG Combine with MatR and Mat; indicates

mating type, ploidy

MatR AGTCACATCAAGATCGTTTATGG See above

Met5testF ATGACGCGGAATTCAGTAGC

Combine with Met5R and Met5delR:

amplification of MET5

Met5delR ATTACCAATATTAGTCAACACTAC See above

Met5R ACGCTGGGCTTCTTGTTTTA See above

MET-II test F TTAATGAATTGACTGGATTGAGA Combine with METestR: amplification of

MET10

METest R TTGGGGACTAAGACAACATC See above

MET10 SEQ1-F CCCATAGTCATCTTCGAGCA Combine with MET10 SEQ1-R:

amplification of MET10 for sequencing

MET10 SEQ1-R

CGGAGTCACGACACCATACT See above

MET10 SEQ2-F AACCGTCTCTCATGATGGTAAGT Combine with MET10 SEQ2-R:

amplification of MET10 for sequencing

MET10 SEQ2-R GTGCGGCCGTGGTAAAATAA See above

MET10 SEQ3-F GTCACTCACGTTCCATCCACT Combine with MET10 SEQ3-R:

amplification of MET10 for sequencing

MET10 SEQ3-R ACGGCTTGGATAAACAAGGACA See above

MET10 SEQ4-F TCAGTCGTTGAAGATGTCAGTTT Combine with MET10 SEQ4-R:

amplification of MET10 for sequencing

MET10 SEQ4-R AGCAGGCGTAACACGTCTAT See above

MET10 SEQ5-F ACCATTGAAGAAATACCATTACCTG Combine with MET10 SEQ5-F:

amplification of MET10 for sequencing

MET10 SEQ5-R TCTGAACCGACAGCAAGGTC See above

MET10 SEQ6-F ATCATTGAACCATTGAAGAGAAGAG Combine with MET10 SEQ6-F:

amplification of MET10 for sequencing

MET10 SEQ6-R GCAGCCAATAGAAAAGCTTGC See above

Table S2: Deuterated standard mix solution

Internal standards Concentrations in final sample (μg/L) ethyl butyrate-4,4,4-d3 494

ethyl hexanoate-d11 1178

ethyl octanoate-d15 1168

3-methylbutyl acetate-d3 1194

n-hexyl acetate-d3 659

2-phenylethyl acetate-d3 292

(±)-linalool-d3 76

3-methyl-1-butyl-1,1-d2-alcohol 29531

n-hexyl-2,2,3,3,4,4,5,5,6,6,6-d11-alcohol 1189

2-phenyl-d5-ethanol 9166

hexanal-d12 67

Table S3 Average concentrations (μg L-1

) of reduced sulfur compounds, esters, higher

alcohols, terpenes and fatty acids quantified in SB wines fermented at 25°C

(A) Wines fermented by E4 and by six E4xF15 F1 progeny with the MET10 allele from

the parent E4.

(B) Wines fermented by F15 and six E4xF15 F1 progeny with the MET10 allele from the

parent F15.

A.

μgL-1

Threshold 1 E4

1 32 11 8*** 2 19 28

Aroma (EEE)

3 (EEE) (EEF) (EFE) (EFE) (EFF)

Reduced sulfur

compounds

Methionol

4

1500–3200 1940.9

e 5 2012.7

e 1192.2

e 2632.0

e 10721.8

b 2116.9

e 2092.5

e

Cooked potato,

cauliflower

Carbon disulfide 2>38 0.08

e 0.12

de 0.15

cde 0.17

cd 0.22

bc 0.18

cd 0.17

cd Rubber, sulfidy

Methylthioacetate 250 2.64

c 2.75

c 2.58

c 3.92

abc 2.98

bc 3.92

abc 3.14

bc Sulfurous, cheesy

Diethyl sulfide 20.9 5.84

b 7.43

ab 8.16

ab 8.24

ab 5.72

b 8.1

ab 8.08

ab Garlic, rubbery

S-ethyl thioacetate 210 1.45

g 1.9

fg 3.79

a 2.57

de 2.41

e 2.87

cde 3.31

abc Sulfurous, garlic

Dimethyl trisulfide – 1.52a 1.35

cd 1.44

abc 1.42

abcd 1.28d 1.55

a 1.44

abc

Methanethiol 21.8–3.1 nd

f nd

f 0.14

a 0.05

de 0.03

e 0.06

cd 0.08

bc Rotten cabbage

Dimethyl sulfide C 1.41

ab 1.43

ab 1.44

ab 1.2

b 1.23

b 1.27

b 1.74

a Cooked cabbage

Dimethyl disulfide 229 0.35

de nd

e 0.51

bcd 0.52

abcd 0.39

cde 0.52

abcd 0.55

abc Cabbage, onion

Esters

Hexylacetate

3700 328.6

ab 232.0

cd 253.7

bcd 263

bcd 141

e 347.2

a 265.8

bcd Floral, fruity, pear

Isobutyl acetate 41600 28.6

abcde 30.8

abcd 16.8

e 18.7

de 40.9

a 31.2

abcd 19.4

de Apple, floral, fruity

Isoamyl acetate 430 4586.7

ab 2424.7

d 2380.8

d 3104.2

bcd 3240.9

bcd 4214.6

abc 3177.3

bcd Banana, fruity

Cis-3-hexenyl acetate – 33.8ab

18de

23.6bcde

25.5bcd

15.2e 36.5

a 25.4

bcd

β-phenylethyl acetate 4250 573.1

d 200.3

f 193.5

f 374.9

e 1348.8

b 505.8

de 819.2

c

Ethyl isobutyrate 415 10.8

cd 26.6

ab 17.9

bcd 14.9

bcd 24.1

abc 9.3

cd 9.9

cd Apple, fruity, sweet

Ethyl butanoate 420 227.1

abc 178.8

defg 205.3

bcd 157.2

fg 148.2

g 230.7

abc 186.9

def Apple, fruity, peach

Ethyl isovalerate 53 1.7f

fg 2.3

cdef 1.6

g 2.0

efg 2.8

bcd 2.0

efg 2.3

cdef Fruit

Ethyl hexanoate 414 684.2

abc 678.6

abc 748.2

ab 593.6

abcd 264.0

e 712.6

ab 579.1

abcd Apple, fruity, sweet

Ethyl octanoate 2580 285.6

c 405.1

bc 450

b 425.4

b 333.8

bc 408.7

bc 388.4

bc Floral, fruity, sweet

Ethyl decanoate 2200 96.6

de 218.4

cde 308.6

abc 351.8

ab 225.2

bcd 305.1

abc 272.4

abc Floral, fruity, sweet

Ethyl dodecanoate – 13.7ef 47.3

def 75.8

bcd 71.5

bcd 48.5

de 92.1

bc 57.4

bcd

Methyl octanoate – 0.8cd

1.0bcd

1.1ab

1.0abc

0.7d 1.0

abc 0.9

bcd

Higher alcohols

1-butanol

5150 3316.7

a 891.0

ef 1343.0

cd 1107.1

de 1458.0

cd 980.3

ef 2279.3

b Fusel, spiritous

Isobutanol 440000 21932.4

de 36386.1

bcde 21011.7

de 26970.7

cde 80097.0

a 25713.9

cde 13853.0

e Nailpolish, solvent

Isoamyl alcohol 430000 110349.1

gh 153131.1

ef 104414.4

h 146447.0

ef 201421.2

bcd 128889.0

fgh 137064.1

efgh Smoky, solvent

Benzyl alcohol 4200000 68.2

bc 68.3

bc 63.8

c 69.8

bc 77.0

a 66.7

bc 68.1

bc Floral, jasmine

Phenylethyl alcohol 410000 18262.6

fg 20473.7

fg 11966.0

g 27758.2

ef 88740.9

b 24182.8

f 41218

d Floral, honey, spice

Terpenes

β-damascenone

120.05–0.14 3.2

cde 3.6

bcd 4.9

ab 3.2

cde 2

e 3.6

cd 5.1

a

β-ionone – 0.3a 0.3

a 0.3

a 0.3

a 0.3

a 0.3

a 0.3

a

Cis/trans-rose-oxide – 0.2a 0.2

a 0.6

a 0.2

a 0.2

a 0.2

a 0.4

a

Linalool 525.2 5.4

e 5.6

e 8.3

bc 6.2

e 7.8

cd 6.1

e 7.6

cd Fruity, floral

α-terpineol 5250 0.4

e 0.5

de 1.2

c 0.7

d 0.6

de 0.9

c 1.4

b Floral, sweet

β-citronellol 5100 2.8

cdef 3.4

cde 2.9

cdef 1.8

f 2.7

def 4.1

cd 4.3

c Rose

Nerol (cis-geraniol) 420 2

d 2.4

bcd 3.0

abc 2.6

bcd 3.1

ab 2.3

bcd 2.5

bcd Fruity, floral, sweet

Fatty acids

Isobutyric acid 4

173 516.9e 1205.8

a 812.1

cd 823.5

cd 1023.6

abc 665.5

de 741.5

de

Hexanoic acid 4420 2960.0

bc 3374.6

ab 2953.1

bc 2891.8

bc 2392.7

c 2949.4

bc 3130.5

b Cheese, spicy

Octanoic acid 4500 2812

cd 3967.1

abcd 3553

bcd 3401.1

bcd 2804.6

cd 3550.2

bcd 3478

bcd Cheese, goaty

B

μg L-1

Threshold F15 18* 4 6 9 26 29**

Aroma (FEE) (FEF) (FEF) (FFE (FFF) (FFF)

Reduced sulfur

compounds

Methionol 1500–3200 7873.2

c 4695.4

d 12781.1

a 8213.7

c 8553.7

c 8344.5

c 11878.2

ab

Cooked potato,

cauliflower

Carbon disulfide 2>38 0.3

a 0.33

a 0.33

a 0.29

ab 0.3

ab 0.32

a 0.35

a Rubber, sulfidy

Methylthioacetate 250 6.28

a 4.91

abc 6.56

a 5.17

abc 5.69

ab 5.69

ab 4.21

abc Sulfurous, cheesy

Diethyl sulfide 20.9 8.02

ab 8.29

ab 9.58

ab 9.23

ab 10.3

a 8.93

ab 6.83

ab Garlic, rubbery

S-ethyl thioacetate 210 1.84

g 2.97

cd 2.38

ef 2.5

de 2.49

de 3.16

bc 3.64

ab Sulfurous, garlic

Dimethyl trisulfide – 1.46abc

1.4abcd

1.53a 1.51

ab 1.51

ab 1.49

abc 1.37

bcd

Methanethiol 21.8–3.1 nd

f 0.13

a nd

f nd

f nd

f 0.11

b 0.06

cd Rotten cabbage

Dimethyl sulfide 225 1.12

b 1.22

b 1.15

b 1.07

b 1.09

b 1.2

b 1.36

ab Cooked cabbage

Dimethyl disulfide 229 0.33

de 0.72

a 0.26

e 0.33

de 0.33

de 0.62

ab 0.43

bcde Cabbage, onion

Esters

Hexylacetate 3700 231.5

cd 210.7

de 303.1

abc 311.6

abc 269.9

abcd 257.5

bcd 194.0

de Floral, fruity, pear

Isobutyl acetate 41600 39.2

ab 33.8

abc 32.2

abc 36.5

abc 27.3

bcde 26.2

cde 31.2

abcd Apple, floral, fruity

Isoamyl acetate 430 3384.8

bcd 2637

cd 5469.6

a 3725.4

bcd 3139.5

bcd 3046.3

bcd 4286.6

ab Banana, fruity

Cis-3-hexenyl acetate – 23.1cde

18.6de

30.8abc

31.7abc

23.2cde

29.5abc

16.5de

β-phenylethyl acetate 4250 556.5

d 362.8

ef 1190.9

b 456.7

de 2240.5

a 590

d 599.3

d

Ethyl isobutyrate 415 18.9

bcd 26.5

ab 14.2

bcd 8.5

d 9.4cd 19.1

bcd 37.9

a Apple, fruity, sweet

Ethyl butanoate 420 232.7

ab 168.8

efg 196.5

cde 254.4

a 163.3

efg 166.8

efg 239.7

ab Apple, fruity, peach

Ethyl isovalerate 53 3.1

b 2.1

defg 4.0

a 2.4

bcdef 2.7

bcde 2.8

bc 4.5

a Fruit

Ethyl hexanoate 414 560.6

bcd 465.5

de 618.6

abcd 777.4

a 487.3

cd 600.1

abcd 509.2

cd Apple, fruity, sweet

Ethyl octanoate 2580 384.6

bc 327.8

bc 364.2

bc 609.5

a 427.5

b 434.3

b 365.1

bc Floral, fruity, sweet

Ethyl decanoate 2200 210.7

cde 240

abc 265.7

abc 363.2

a 355.7

ab 336.1

abc 91.5

e Floral, fruity, sweet

Ethyl dodecanoate – 71.8bcd

54.1cd

63.4bcd

142a 94.9

b 69.7

bcd 8.9

f

Methyl octanoate – 0.9bcd

0.8bcd

1.0bc

1.2a 1.0

abc 1

ab 0.9

bcd

Higher alcohols

1-butanol

5150 691.6

f 791.9

ef 848.0

ef 1356.0

cd 978.6

ef 1113.4

de 1476.0

c Fusel, spiritous

Isobutanol 440000 55947.0

ab 51024.2

bc 53582.6

abc 35050.9

bcde 33050.4

bcde 43519.6

bcd 56412.7

ab Nailpolish, solvent

Isoamyl alcohol 430000 208956.5

bc 143528.8

efg 258019.2

a 141182.2

efg 186903.8

cd 167980.0

de 229117.7

ab Smoky, solvent

Benzyl alcohol 4200000 72.4

ab 68.2

bc 65.9

c 65.8

c 69.2

bc 68.4

bc 68.8

bc Floral, jasmine

Phenylethyl alcohol 410000 40135.5

d 24634.0

f 62784.2

c 21535.4

fg 107716.2

a 36682.2

de 54330.5

c Floral, honey, spice

Terpenes

β-damascenone

120.05–

0.14 3.0

de 3.5

cd 3.6

bcd 3.3

cde 3.5

cd 4.3

abc 3.0

de

β-ionone – 0.3a 0.3

a 0.3

a 0.3

a 0.3

a 0.3

a 0.4

a

Cis/trans-rose-oxide – 0.2a 0.3

a 0.2

a 0.2

a 0.2

a 0.2

a 0.3

a

Linalool 525.2 7.6

cd 7.6

cd 8.0

bc 8.0

bc 6.5

de 10.2

a 9.3

ab Fruity, floral

α-terpineol 5250 0.5

de 1.0

c 1.0

c 1.0

c 0.7

d 1.9

a 1.0

c Floral, sweet

β-citronellol 5100 3.1

cdef 2.4

ef 2.1

ef 1.7

f 6.0

b 2.8

cdef 8.7

a Rose

Nerol (cis-geraniol) 420 2.4

bcd 3

abc 2.8

abcd 3.7

a 2.8

abcd 3.1

abc 2.2

cd Fruity, floral, sweet

Fatty acids

Isobutyric acid 4

173 1107.9ab

1030.4abc

803.6cd

836.7cd

874.9bcd

684.8de

707.6de

Hexanoic acid 4420 3461.8

ab 3056.0

b 3221.3

ab 3788.2

a 3496.0

ab 3014.1

bc 3088.7

b Cheese, spicy

Octanoic acid 4500 4511.5

abc 3233.9

bcd 4981.3

ab 5781.7

a 5009.2

ab 3659.5

bcd 2498.3

d Cheese, goaty

1 Perception thresholds and descriptors were obtained from the references listed: 1 Moreira et al. (2010),

2Siebertet al. (2010),

3Swiegers et

al. (2005), 4Escudero et al. (2007),

5 Benkwitzet al. (2012).

2 Asterisks indicate flasks (from each triplicate) in which the H2S tubes were blown off at one point during maximum fermentation, so that

there was a failure to collect H2S for a small part of the ferment, and the ferments were also exposed to the air for part of one night.

3 The genotypes of each colony are indicated in the order MET10, MET5, MET5, with E representing the E4 parental allele and F

representing the F15 parental allele.

4 Bolding of the first three compounds indicates a significant difference between the A and B results; nd = not detected.

5 Samples not connected by the same letter among each set of 14 analysed are significantly different (ANOVA, Tukey’s HSD); n = 3.

Table S4 LOD scores for intensity of colony color scored from photgraphs of six

different indicator agar plates and assessed with Colorpic software (ICONICO, New

York, NY) using three different hues or the sum of all three hues (nd=no peak detected)

LOD score (intensity of color)

Indicator plate Locus

RED GREEN BLUE R+G+B

BIGGY agar MET2 nd nd nd nd

MET5 21 24 20 23

HGJ-Bi agar (m19 juice)

MET2 12.6 8.7 6.0 9.4

MET5 nd 5.0 6.9 5.0

HGJ-Bi agar (m403 juice)

MET2 3.6 nd nd nd

MET5 nd 7.2 8.9 6.6

HGJ-Bi agar (m1005 juice)

MET2 6.8 4.6 3.5 5.4

MET5 nd 7.1 7.3 6.0

HGJ-Bi agar (m1008 juice)

MET2 4.9 nd nd 3.7

MET5 nd 4.8 5.6 4.1

YPD-Cu agar

PTR2 5.9 5.7 8.6 7.6

CUP1 3.8 4.9 3.7 4.4

MET2 nd nd 3.5 nd

MET5 nd nd nd nd

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