Differential Off-line LC-NMR (DOLC-NMR) Metabolomics To Monitor

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Differential Off-line LC-NMR (DOLC-NMR) Metabolomics to Monitor Tyrosine-induced Metabolome Alterations in S. cerevisiae Richard Hammerl, Oliver Frank, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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Journal of Agricultural and Food Chemistry

1

Differential Off-line LC-NMR (DOLC-NMR) Metabolomics

2

to Monitor Tyrosine-induced Metabolome Alterations in S.

3

cerevisiae

4 5 Richard Hammerl, Oliver Frank, and Thomas Hofmann*

6 7 8 9 1

10

Chair of Food Chemistry and Molecular Sensory Science, Technische Universität

11

München, Lise-Meitner-Str. 34, D-85354 Freising-Weihenstephan, Germany

12 13 14 15 16 17 18

*

19

PHONE

+49-8161-712902

20

FAX

+49-8161-712949

21

E-MAIL

To whom correspondence should be addressed

[email protected]

22 23 24 25 26 27 28

1 ACS Paragon Plus Environment


29

ABSTRACT

30 31

A novel Differential Off-Line LC-NMR approach (DOLC-NMR) was developed to

32

capture and quantify nutrient-induced metabolome alterations in Saccharomyces

33

cerevisiae. Off-line coupling of HPLC separation and

34

supported by automated comparative bucket analyses, followed by quantitative 1H

35

NMR using ERETIC 2 (Electronic REference To access In vivo Concentrations) has

36

been successfully used to quantitatively record changes in the metabolome of S.

37

upon intervention with the aromatic amino acid L-tyrosine. Among the 33

38

metabolites identified, glyceryl succinate, tyrosol acetate, tyrosol lactate, tyrosol

39

succinate, and N-acyl-tyrosine derivatives like N-(1-oxooctyl)-tyrosine are reported

40

for the first time as yeast metabolites. Depending on the chain length, N-(1-

41

oxooctyl)-, N-(1-oxodecanyl)-, N-(1-oxododecanyl)-, N-(1-oxomyristinyl)-, N-(1-

42

oxopalmityl)-, and N-(1-oxooleoyl)-L-tyrosine imparted a kokumi taste enhancement

43

above their recognition thresholds ranging between 145 and 1432 µmol/L (model

44

broth). Finally, carbon module labelling (CAMOLA) and carbon bond labelling

45

(CABOLA) experiments with

46

biosynthetic pathway leading to the key metabolites, e.g. the aliphatic side chain of

47

N-(1-oxooctyl)-tyrosine could be shown to be generated via de novo fatty acid

48

biosynthesis from four C2-carbon modules (acetyl-CoA) originating from glucose.

13

1

H NMR spectroscopy

C6-glucose as the carbon source confirmed the

49 50

Keywords: Saccharomyces cerevisiae, yeast, quantitative NMR, qNMR, taste,

51

kokumi, CABOLA, CAMOLA, 13C-labelling, DOLC-NMR.

52 53 2 ACS Paragon Plus Environment

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54


INTRODUCTION

55 56

Due to its importance and wide application in food and beverages like bread, wine

57

and beer, Saccharomyces cerevisiae is one of the most studied microorganism in

58

the past 50 years. Catalyzed by post-genomic tools, enzymatic key processes are

59

well known and the impact of temperature, pH, oxygen level, and concentration of

60

salt and nutrients, respectively, on gene expression and metabolite profile have

61

been monitored by various imaging techniques.1–5 Targeted stress-induced

62

metabolome alterations enabled the identification of stress markers,6,7 salt stress

63

has been reported to induce the increase of trehalose levels to counteract osmotic

64

pressure while glucose, maltose and betaine levels depleted upon ethanol stress.6,7

65

Moreover, amino acid utilization by yeast has been reported to determine different

66

aroma and taste characteristics of fermented beverages.8 While the aliphatic

67

branched chain amino acids L-leucine, L-isoleucine, and L-valine were found to be

68

of prime importance in developing the typical aroma of S. cerevisiae fermented

69

beverages,8 the aromatic amino acid L-tyrosine are known to be metabolized via the

70

Ehrlich pathway giving rise to 2-(4-hydroxyphenyl)ethanol, known as tyrosol.9 This

71

fusel alcohol shows high human bioavailability,10 exhibits antioxidant11 and anti-

72

tumor activity,12 and has been reported as a quorum sensing molecule showing an

73

inhibitory effect on Candida adhesion to oral tissues.13 Besides tyrosol and the

74

biogenic amine tyramine,14 other secondary metabolites from L-tyrosine are largely

75

unkown.

76

Metabolomic studies on yeast were performed either using an intracellular

77

metabolite extraction protocol with ethanol,15 or by analysing the metabolite profile

78

secreted by the microorganism during the fermentation process.5,16 Although LC-MS 3 ACS Paragon Plus Environment


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has been frequently used for monitoring metabolite profiles due to its high resolution

80

and very low limit of detection, the unequivocal identification of key metabolites

81

increasing or decreasing upon an intervention is a rather challenging task.17 In

82

comparison, whole extract analysis by means of 1H NMR spectroscopy seems to be

83

promising as it is able to provide direct structure information as well as quantitative

84

data of previously unknown target metabolites.18–22 However, the low resolution and

85

drastic signal superimposition in one-dimensional

86

comprehensive analysis of complex natural extracts. Therefore, two different

87

approaches were followed to tackle this challenge. First, 2D-NMR experiments,

88

such

89

spectroscopy, were introduced into metabolome research to promote metabolite

90

identification on the basis of specific 1H-13C correlations recorded.7 However, the

91

incomplete transfer of the magnetization from proton to carbon lowers the suitability

92

of HSQC for accurate quantitative metabolite analysis, because the transfer step

93

may not be streamlined simultaneously for all nuclear spins in complex metabolite

94

mixtures exhibiting a range of

95

chromatographic pre-separation has been performed to de-complexify multi-

96

metabolite mixtures, such as, e.g. urine and faeces, and to increase suitability of

97

one-dimensional 1H NMR spectroscopy in metabolome research.24–26

as,

e.g.

1

H-13C

heteronuclear

1

1

H NMR spectra limits the

single-quantum

correlation

(HSQC)

H-13C coupling constants.23 Second, liquid

98

To investigate secondary metabolites from L-tyrosine in yeast, a novel

99

differential off-line HPLC-NMR approach was developed to capture metabolite

100

alterations in S. cerevisiae by comparing NMR spectral buckets that were recorded

101

from

102

intervention with the aromatic amino acid L-tyrosine. In addition, key metabolites

103

identified should be analysed in their concentrations by means of quantitative 1H

1

H NMR spectra of HPLC subfractions collected before and after an


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104

NMR spectroscopy27 and their biosynthesis monitored by means of

13

105

experiments, namely the Carbon Module Labelling (CAMOLA)28–30 and Carbon

106

Bond Labelling (CABOLA)30–34 technique with MS- and NMR-based isotopologue

107

diagnostics, respectively.

C labelling

108 109

MATERIALS AND METHODS

110 111

Chemicals. The following chemicals were obtained commercially: Glucose,

112

caffeine, monosodium L-glutamate, L-glutathione, tannic acid, quercetin 3-O-β-D-

113

glucopyranoside,

114

phosphate monobasic, alkanoyl chlorides C4 to C 18:1, tyrosol, acetic anhydride,

115

succinic anhydride, p-hydroxyphenylethyl bromide, solketal, potassium fluoride,

116

ammonium acetate, lactic acid, dioxane, diethyl ether, N,N-dimethyl formamide,

117

ethyl acetate, tetrahydrofuran, and pyridine were from Sigma-Aldrich (Steinheim,

118

Germany), amino acids, sodium hydroxide, potassium hydroxide, sodium chloride,

119

sulfuric acid, hydrochloric acid, and formic acid were from

120

Germany), 13C6-glucose from Cambridge Isotope Laboratories, Inc. (Andover, USA),

121

and dried baker´s yeast from RUF (Quakenbrück, Germany). Water used for the

122

yeast fermentation and chromatographic separations was purified with a Milli-Q

123

Gradient A10 system (Millipore, Schwalbach, Germany). Bottled water (Evian,

124

Danone, Wiesbaden, Germany) was used for sensory studies. Methanol,

125

acetonitrile and 2-propanol were from Merck (Darmstadt, Germany). Deuterium

126

oxide and methanol-d4 were supplied from Euriso-Top (Gif-sur-Yvette, France).

1,3,5-benzenetricarboxylic

acid,

maltodextrin,

potassium

Merck (Darmstadt,

127

S. Cerevisiae Fermentation with/without Tyrosine (Tyr1/Tyr0). Dry yeast (S.

128

cerevisiae, 460 mg of dried pellets) was mixed with water (200 mL), D-glucose (194 5 ACS Paragon Plus Environment


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129

mmol/L) and L-tyrosine (12 mmol/L) were added, and the suspension was incubated

130

for 96 h at 36 °C under anaerobic conditions (Tyr1). In addition, a control experiment

131

(Tyr0) was performed without the presence of L-tyrosine. Thereafter, the

132

supernatants were separated from yeast cells by filtration (0.45 µm, Sartorius

133

Stedium Biotech GmbH; Göttingen, Germany), freeze-dried, and the residue

134

obtained

135

benzenetricarboxylic acid (100 µL, 25.0 mmol/L) as recovery standard, the solution

136

was directly used for the chromatographic MPLC separation to collect a total of 34

137

fractions in 1 min intervals. After concentration of each fraction in vacuum by means

138

of a HT-12 evaporation system (Genevac Limited Ipswich, United Kingdom), the

139

individual fractions collected from Tyr1 and Tyr0, respectively, were dissolved in

140

deuterated solvents for NMR analysis.

was

dissolved

in

water

(15

mL).

After

addition

of

1,3,5-

141

S. Cerevisiae Fermentation with Fatty Acids Added. Following the same

142

protocol as described above, the tyrosine-spiked yeast fermentation broth Tyr1 was

143

spiked with either octanoic acid (12 mmol/L), decanoic acid (12 mmol/L), or oleic

144

acid (12 mmol/L), and the pH value was adjusted to 7.0 with 0.1 mol/L aqueous

145

NaOH solution. After 96 h at 36 °C under anaerobic conditions, the supernatant was

146

obtained by filtration and then used for LC-MS/MS analyses.

147

Stable Isotope Labelling Experiments. To perform a carbon module

148

labelling (CAMOLA) experiment, a mixture of dry yeast (460 mg of dried pellets),

149

glucose (97 mmol/L), 13C6-glucose (97 mmol/L), and L-tyrosine (12 mmol/L) in water

150

(200 mL) was incubated for 96 h at 36 °C under anaerobic conditions. After

151

fermentation, the supernatant was obtained by filtration and, then, directly used for

152

UPLC-ESI-TOF/MS analysis. The obtained results were analysed with MassLynx

153

4.1 software (Waters).


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154

For the carbon bond labelling (CABOLA) experiment, a mixture of dry yeast

155

(460 mg of dried pellets), glucose (184.3 mmol/L), 13C6-glucose (9.7 mmol/L), and L-

156

tyrosine (12 mmol/L) in water (200 mL) was incubated for 96 h at 36 °C under

157

anaerobic conditions. After filtration (0.45 µm; Sartorius Stedium Biotech GmbH,

158

Göttingen, Germany), the supernatant was extracted three times with ethyl acetate

159

(300 mL) to give an aqueous phase and an organic phase. The organic phase was

160

separated from solvent in vacuum and the residue was further fractionated using

161

the preparative HPLC system and stationary phase I. The gradient separation was

162

performed with aqueous formic acid (0.1% in water, pH 2.5) as solvent A and

163

acetonitrile as solvent B. After isocratic elution for 3 min at 2% B, the content of

164

solvent B was increased to 7% within 7 min, increased to 12% within 10 min, then

165

increased to 72% within 20 min and, finally, raised to 100% B within 5 min. The

166

aqueous phase remaining after ethyl acetate extraction of the fermentation

167

supernatant was fractionated on the preparative HPLC system using stationary

168

phase II. The gradient separation was performed with aqueous ammonium acetate

169

buffer (5mM in water, pH 3.2) as solvent A and acetonitrile/ammonium acetate

170

buffer (5mM in water, pH 3.2; 9/1, v/v) as solvent B. After isocratic elution for 1 min

171

at 100% B, the content of solvent B was decreased to 95% within 10 min, then,

172

reduced to 86% within 5 min, kept isocratically for 6 min, then reduced to 50%

173

within 3 min and, finally, kept constant for 3 min. The eluting peaks were collected

174

separately, solvents were removed in vacuum, the purified substances were taken

175

up deuterated solvent (D2O, methanol-d4) and analyzed by means of

176

spectroscopy to read out 13C coupling patterns of the isolated target compounds.

177 178

Medium

Pressure

Liquid

Chromatography

(MPLC)

13

C NMR

and

High

Performance Liquid Chromatography (HPLC). The MPLC system was comprised



Page 8 of 46

179

of a Spot Prep II (Gilson, Limburg, Deutschland) equipped with a preparative 250 ×

180

21.2 mm, 5µm, PhenylHexyl Luna column (Phenomenex, Aschaffenburg, Germany)

181

as the stationary phase. The effluent (21.0 mL/min) was monitored at 230 nm. The

182

gradient separation was performed with aqueous formic acid (solvent A, 0.1% in

183

water, pH 2.5) and acetonitrile (solvent B). After isocratic elution for 6 min at 2% B,

184

the content of acetonitrile was increased to 15 % B within 8 min, then, raised to 50

185

% B within 14 min and, finally, raised to 100% B within 5 min.

186

The preparative HPLC system (Jasco, Groß-Umstadt, Germany) was

187

comprised of a binary pump (PU-2087 Plus), degasser (DG 2080-53), DAD detector

188

(MD 2010 Plus), ELSD detector (Sedex LT-ELSD Model 85, Sedere, Alfortville,

189

France), and a sample loop (2000 µL). Chrompass 1.9. software was used for data

190

analyses. Chromoatography was performed either with a preparative 250 × 21.2

191

mm, 5µm, LUNA PhenylHexyl column (Phenomenex, Aschaffenburg, Germany;

192

stationary phase I), or a preparative 250 × 21.2 mm, 5µm, LUNA HILIC column

193

(Phenomenex, Aschaffenburg, Germany; stationary phase II). The effluent (21.0

194

mL/min) was monitored at 230 nm.

195

Nuclear Magnetic Resonance (NMR) Spectroscopy.

1

H/13C NMR and

196

HSQC experiments of the supernatant of the fermentation broth (Figure 1) and

197

purified compounds, respectively, were performed on a Bruker AVANCE III 500

198

MHz System equipped with a cryo-TCI Probe (300 K) and Topspin 3.0 software.27

199

NMR experiments were performed after solubilizing the test samples in the following

200

NMR-buffer solution: KH2PO4 (10.2 g) was solved in D2O (40 mL), then, KOH (1.5

201

g), TMSP (50 mg), and NaN3 (5 mg) were added, the pH-value adjusted to 7.0 with

202

a KOH solution (4.0 mol/L in D2O) and, finally, made up to 50 mL with D2O.


Page 9 of 46


203

NMR Analysis of Fractions. For NMR analysis of the MPLC fractions collected,

204

fractions no. 3-19 were dissolved in D2O (1 mL) and aliquots (540 µL) were then

205

mixed with an aliquot (60 µL) of the NMR-buffer solution prior to spectroscopy. The

206

more hydrophobic fractions no. 20-34 were taken up in CD3OD (1 mL) and aliquots

207

(600 µL) used for NMR analysis. All fraction solutions were placed in 5 mm x 7’’

208

NMR tubes (Z107374 USC tubes, Bruker, Faellanden, Switzerland) and a 1H NMR

209

spectrum was acquired using the Bruker standard water suppression 1D

210

noesygppr1d pulse sequence.35 The 90° pulse length (P1), PL9 and O1 were

211

adjusted individually on each sample and 16 scans (NS) with 4 prior dummy scans

212

(DS) were collected into 64K data points using a spectral width of 10273.97 Hz. The

213

relaxation time (T1) was set to 20 s to allow all excited nuclei to re-establish their

214

equilibrium z-magnetization prior to the application of the next pulse.27 To ensure

215

high-quality spectra, the NMR probe was manually tuned and matched to 50 Ω

216

resistive impedance to minimize radio frequency (RF) reflection, with the sample in

217

place. After automatic optimization of the lock phase, each sample was shimmed

218

(z1 − z5, xyz, z1 − z5), the 90° pulse width was determined individually for each

219

sample using the AU program pulsecal sn. All spectra were acquired without

220

spinning the sample and referenced to TMSP (0.0 ppm). The free induction decay

221

(FID) was multiplied with a 0.3 Hz exponential line-broadening factor and zero-filled

222

prior to Fourier transformation using the command apk0.noe. In case that the result

223

of the automatic phase correction was not satisfying, a careful manual zero- and

224

first-order phase correction was performed. Baseline correction was done

225

automatically with the command absn. Integration was carried out manually and

226

whenever required; adjustment of the integrals was executed by the software

227

functions SLOPE and BIAS.27



228

NMR Bucketing. The NMR-Buckets were calculated with the Amix Viewer

229

V3.9.13 Software (Bruker, Rheinstetten, Germany). Each spectrum was referenced

230

to TMSP (0.0 ppm). After checking the baseline offset and using the underground

231

removal tool, the spectra were used to determine the buckets. Covering the

232

chemical shift region from -1 to 11 ppm, the range of each bucket was set to 0.1

233

ppm. The area between 4.5 and 5 ppm was excluded from bucketing due to the

234

water signal in the spectra. The calculation of the absolute integral value for each of

235

the 115 buckets was performed successfully when the signal to noise ratio was

236

larger 10. The noise was calculated in the region from 10 to 11 ppm, where no

237

signals appeared. From the yeast fermentation with tyrosine (Tyr1) and the control

238

(Tyr0), the corresponding buckets showing an integral ratio (Tyr1/Tyr0) of >2 or 2000

n.d.a

Tyrosol lactate (8)

n.d.a

> 2000

n.d.a

Tyrosol acetate (6)

n.d.a

> 2000

n.d.a

Tyrosol succinate (7)

bitter

851

n.d.a

N-(1-Oxobutyl)-L-tyrosine (10)

bitter

647

n.d.a

N-(1-Oxohexyl)-L-tyrosine (11)

bitter

343

n.d.a

N-(1-Oxooctyl)-L-tyrosine (9)

bitter

631

1432

N-(1-Oxodecanyl)-L-tyrosine (12)

bitter

627

537

N-(1-Oxododencanyl)-L-tyrosine

bitter

480

145

N-(1-Oxomyristyl)-L-tyrosine (14)

bitter

672

160

N-(1-Oxopalmityl)-L-tyrosine (15)

bitter

627

183

N-(1-Oxooleoyl)-L-tyrosine (16)

bitter

446

217

compound

784

a

not detected.

785


Page 37 of 46

786


Table 3. Results of the CAMOLA and CABOLA labelling experiments.

787 Metabolite

CAMOLA experiment

CABOLA experiment

m/z of pseudomolecular

signal

carbon atom assignment

ions ([M-1]-)

ratio

of 13C-labelled moduls

12

C-glc

12

Carbon atom

C-glc/13C-glc

[chem. shift (ppm);

(50/50) Succinic acid (2)

117

119, 121

multiplicity; 1/2JC-C (Hz)] 1:2:1

C(2,3) [28; d; 1J=55.2] C(1,4) [180; d; 1J =55.2]

Glycerol (3)

91

94

1:1

C(1,3) [62; d; 1J =39.4] C(2) [72; dd; 1J =39.4, 39.4]

Lactic acid (4)

89

92

1:1

C(1) [179; d; 1J =58.9] C(2) [67; dd; 1J =36.5, 58.9] C(3) [19; d; 1J =36.5]

Glycerol succinate (5)

191

193, 194, 195,

1:2:1:1:2:1

196, 198

C(1) [65; dd; 1J =41.5, 2J=2.7] C(2) [72; dd; 1J =41.5, 42.5] C(3) [68; dd; 1J =42.5, 2J=2.7] C(1´) [178; d; 1J =55.4] C(2´) [33; d; 1J =55.4] C(3´) [32; d; 1J =55.3] C(4´) [182; d; 1J =55.3]



Tyrosol (1)

137

139, 141, 143,

1:2:2:2:1

Page 38 of 46

C(1) [66; d; 1J =37.3] C(2) [40; d; 1J =37.3]

145

C(1´) [134; d; 1J =56.8] C(2´) [133; d; 1J =56.8] C(3´) [118; ddd; 1J =65.0, 2

J=6.3, 3J=3.5]

C(4´) [157; dd; 1J =65.0, 65.0] C(5´) [118; ddd; 1J =65.0, 58.1 2

J=1.3]

C(6´) [134; ddd; 1J =58.1, 2

Tyrosol acetate (6)

179

181

1:1

J=6.0, 3J=3.5]

C(1´) [178; d; 1J =55.4] C(2´) [33; d; 1J =55.4]

Tyrosol lactate (8)

209

212

1:1

n.a.a

Tyrosol succinate

237

239, 241

1:2:1

C(1´´) [178; d; 1J =58.1] C(2´´) [34; d; 1J =58.1]

(7)

C(3´´) [33; d; 1J =55.0] C(4´´) [183; d; 1J =55.0] N-(1-Oxooctyl)-L-

306

tyrosine (9)

308, 310, 312,

1:4:6:4:1

314

C(1´´) [178; d; 1J =49.9] C(2´´) [39; d; 1J =49.9] C(3´´) [29; d; 1J =34.6] C(4´´) [30; d; 1J =34.6] C(5´´) [31; d; 1J =34.5] C(6´´) [34; d; 1J =34.5] C(7´´) [25; d; 1J =34.6] C(8´´) [16; d; 1J =34.6]

788

a

Not analyzed by 13C NMR.

789 790 791


Page 39 of 46

792


Hammerl et al. (Figure 1)

793 794 795

796 797 798 799 800 801 802 803 804 805 806 807



808


809 810 811

812 813 814 815 816 817 818 819


Page 40 of 46

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820



821 822 823

824 825 826 827 828 829 830 831 832 833



834


835 836 837 838

839 840 841 842 843 844 845 846


Page 42 of 46

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847



848 849 850

851 852 853 854 855 856 857 858 859



860


861 862 863 864 865

866 867 868 869 870 871 872 873 874 44 ACS Paragon Plus Environment

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875



876 877

878 879



TOC Graphic 176x115mm (150 x 150 DPI)

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Differential Off-line LC-NMR (DOLC-NMR) Metabolomics To Monitor

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