Fungal Spores Promote the Glycerol Production of Saccharomyces

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Food and Beverage Chemistry/Biochemistry

Fungal Spores Promote the Glycerol Production of Saccharomyces cerevisiae by Upregulating the Oxidative Balance Pathway Chunmei Jiang, Xianqing Chen, Shuzhen Lei, Dongyan Shao, Jing Zhu, Yanlin Liu, and Junling Shi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00205 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 10, 2018

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Journal of Agricultural and Food Chemistry

Fungal Spores Promote the Glycerol Production of Saccharomyces cerevisiae by Upregulating the Oxidative Balance Pathway Chunmei Jiang1, Xianqing Chen1, Shuzhen Lei1, Dongyan Shao1, Jing Zhu3, Yanlin Liu2, Junling Shi1* 1

Key Laboratory for Space Bioscience & Space Biotechnology, School of Life

Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an, Shaanxi Province 710072, China 2

College of Enology, Northwest A&F University, 23 Xinong Road, Yangling,

Shaanxi Province 712100, China 3

School of Food Science, Xinyang Agriculture and Forestry University, New 24 street

of yangshan new district, Xinyang, Henan Province 464000, China

*

Corresponding author. Tel. +86-29-88460541; Fax. +86-29-88460541; E-mail:

[email protected]

1

ACS Paragon Plus Environment


1

Abstract

2

Fungal contamination is prevalent in grape berries and unavoidable during the

3

winemaking process. In the botrytised wine, Botrytis cinerea contamination of grape

4

berries beneficially promotes the wine flavor, which is desirable especially with high

5

glycerol content. To investigate the underlying mechanism, Aspergillus carbonarius

6

and B. cinerea spores were separately co-cultured with two different Saccharomyces

7

cerevisiae strains in both grape juice and synthetic nutrient media. The results showed

8

that both A. carbonarius and B. cinerea promoted glycerol accumulation and the

9

consumption of sugars in the co-culture systems, but could not synthesize glycerol by

10

themselves. The metabolites produced by fungal spores triggered these reactions.

11

RT-PCR analysis showed that the presence of A. carbonarius spores regulated the

12

expression of GPP1 and GPD2, indicating that the reaction was triggered by

13

regulating the oxidative balance pathway. The study revealed the beneficial impact of

14

fungal contamination on wine quality by influencing the yeast metabolism.

15

Keywords: Glycerol, Botrytis cinerea, Aspergillus carbonarius, Saccharomyces

16

cerevisiae, co-culture, interaction

17

2


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INTRODUCTION

19

The occurrence of fungi in grapes and during the winemaking process is

20

ubiquitous and unavoidable. Most of these, such as Aspergillus sp., Penicillum sp.

21

Botrytis sp., Alternaria sp., Cladosporium sp., and Rhizopus sp. need to be controlled

22

since they may cause the bunch rot disease of grapes, produce mycotoxin, and bring

23

undesirable flavor to the wine.1-6 However, one of the highest grades of sweet wine

24

forms an exception: botrytised wine is fermented by using grapes that have

25

specifically been infected with Botrytis cinerea. B. cinerea is generally considered as

26

the most common cause of bunch rot of grapes;3 however, under special conditions, it

27

can cause the “noble rot” of grapes and endow the special aroma and flavor of

28

botrytised wine.7 Here, it is worth mentioning that high glycerol content is an

29

important feature of botrytised wines, and that this is responsible for the unique and

30

elegant taste characteristics that are valued in botrytised wine. In numerous surveys of

31

botrytised wine, the glycerol levels have been reported to be higher in superior-grade

32

wines than in ordinary wines.8,9 An understanding of the chemistry behind this

33

reaction will facilitate its application in wine-making.

34

Glycerol is a viscous polyalcohol with a slightly sweet taste that affects the body

35

and fullness of the fermented beverage.10-12 It is a major by-product produced by S.

36

cerevisiae and is synthesized at an early stage during alcoholic fermentation due to a

37

lack of alcohol dehydrogenase and with an excess of NADH, which causes an

38

imbalance in the redox equivalent; 13 this imbalance induces yeast to produce glycerol

39

and consume NADH to rebalance the intracellular redox potential.14 The high levels

40

of initial sugar concentration in grape juice also results in high osmotic pressure

41

within the cells, indicating that the intracellular production of glycerol is necessary to

42

offset this osmotic stress.15,

16

Two pairs of isogenes are involved in the glycerol 3



43

synthesis: GPD1/GPD2 encoding the glycerol phosphate dehydrogenase (GPD) and

44

GPP1/GPP2, encoding the paralogs of glycerol-3-phosphatase (GPP). Numerous

45

studies have indicated that GPD1 and GPP2 are essential for cellular growth under

46

osmotic stress conditions. The expressions of GPD1 and GPP2 are normally regulated

47

via a high osmolarity glycerol (HOG) response pathway, while GPD2 and GPP1

48

genes induce expression under anaerobic conditions to regulate the redox balance.15-17

49

During the winemaking process, the synthesis of glycerol is normally influenced

50

by the amount of sulfur dioxide, fermentation temperature, time, pH value, nitrogen

51

source, and alcohol concentration.9, 18 Artificial inoculation with B. cinerea has been

52

conducted in past studies to simulate natural noble rot, elevate glycerol levels, and

53

improve the flavor.19-21 B. cinerea and other grape-contaminating fungi indeed have

54

the ability to increase the glycerol production.

55

widely accepted and is difficult to repeat because the essential mechanism of the

56

reaction still remains unclear.

22

However, this technique is not

57

According to current opinions, water loss and concentrated sugar content caused

58

by the fungal infection are assumed as one of the principle components contributing

59

to the positive effect of B. cinerea contamination on the improvement of wine

60

flavor.23 Many studies have aspired to explain the mechanism of botrytised wine

61

formation from the point view of fungal metabolism or from the chemical

62

transformations that occur in both juice and wine constituents.3,24,25 Nevertheless, the

63

interaction between B. cinerea and yeast, the principle factor that contributes to the

64

formation of wine flavors, has been neglected in most studies. More importantly, the

65

influence of Aspergillus on wine flavors has not been mentioned before.

66

Furthermore, the interaction between fungi and yeasts exist both on the surface

67

of the grapes in the vineyards and during the wine-making process. It is reasonable to 4


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assume that fungal spores, remaining in the musts would influence the yeast

69

metabolism and thus affect wine flavors; however, these filamentous fungi are unable

70

to tolerate an ethanol concentration above about 3%, which is why they lose viability

71

after 24-48 h of fermentation.26 In preliminary experiments, we found that the

72

glycerol content increased when the wine was fermented with grapes that were

73

contaminated with Aspergillus carbonarius in a concentration-dependent manner

74

during wine-making (Figure S1). Therefore, it is reasonable to assume that fungi,

75

especially those that enter the wine-making process, would affect the glycerol

76

production of yeast. This mechanism might be compliant for all types of fungi,

77

including Aspergillus and Botrytis.

78

This study was conducted to: 1) evaluate the effects of A. carbonarius and B.

79

cinerea on the glycerol metabolism of different S. cerevisiae strains, and 2)

80

investigate the mechanism responsible for this phenomenon in terms of the levels of

81

glycerol synthesis. Unraveling the interaction between yeast and these filamentous

82

fungi during the wine-making process would provide useful guidance to further

83

improve the wine quality via the control of fungal contamination.

84

MATERIALS AND METHODS

85

Microorganisms. Two S. cerevisiae strains were used during fermentation in

86

this study: A commercial strain called S. cerevisiae SP (SP) (Lamothe-Abiet, France)

87

and an isolated strain called M114 (M114, Genebank accession number EU386722.1)

88

obtained from the surface of Cabernet Sauvignon grape in Shaanxi province, China. B.

89

cinerea (BC) and A. carbonarius (AC) CCTCC AF 2011004 (China Center for Type

90

Culture Collection, Wuhan, China) were used as different types of fungi, both of

91

which were previously isolated from grapes.

92

Media. Grape juice medium (GJM) was prepared from Cabernet Sauvignon 5



93

grape juice (pH 3.75), containing 244.55 g/L reducing sugar and with 3.57 g/L

94

titratable acidity. Synthetic nutrient media containing 200 g/L (SNM200) and 100 g/L

95

total sugar (SNM100) were used to simulate grape juice with different sugar contents,

96

corresponding to the grape maturation and veraison stage,27 to investigate the

97

influence of sugar on the glycerol production of yeast. The SNM200 medium was

98

composed of 100 g/L d-(+)-glucose, 100 g/L d-(−)-fructose, 7 g/L l-(+)-tartaric acid,

99

10 g/L l-(−)-malic acid, 0.67 g/L (NH4)2SO4, 0.67 g/L (NH4)2HPO4, 1.5 g/L KH2PO4,

100

0.75 g/L MgSO4.7H20, 0.15 g/L NaCl, 0.15 g/L CaCl2, 0.0015 g/L CuCl2, 0.021 g/L

101

FeSO4.7H2O, 0.0075 g/L ZnSO4, and 0.05 g/L (+)-catechin. The pH was adjusted to

102

3.5 using 10 M NaOH.28 The composition of SNM100 was similar to that of SNM200,

103

with the exception that the contents of glucose and fructose were 70 g/L and 30 g/L,

104

respectively. All media were autoclaved at 100°C for 30 min prior to use.

105

Preparation of yeasts and fungal spore suspensions. SP and M114 were

106

cultured in yeast peptone dextrose medium (YPD, containing yeast extract 10 g,

107

peptone 20 g, glucose 20 g, and 1L distilled water), and cultivated at 28oC and

108

agitated at 180 rpm for 48 h. The cells were collected and adjusted to 1×108 cells/mL

109

using a hemocytometer. AC and BC were prepared as spore suspensions by flushing

110

the surface of 7 or 14-day-old cultures, respectively, grown at 25°C on potato

111

dextrose agar (PDA) with sterile water and filtering through sixteen layers of sterile

112

cheesecloth. The suspensions of AC and BC were finally adjusted to 1×108 and 1×107

113

spores/mL, respectively. All spore suspensions were kept at 4°C until use.

114

Inoculation and co-cultivation of yeast together with fungal spores. 1 mL of

115

the prepared suspensions of SP or M114 was inoculated in a 250 mL Erlenmeyer flask

116

containing 100 mL of GJM, SNM200, or SNM100 medium at a final concentration of

117

1×106 cells/mL. Next, the prepared spore suspensions of AC and BC were 6


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individually co-cultured with SP or M114. The final spore concentration of AC was

119

ca

120

SP/M114-AC-106, and SP/M114-AC-107, respectively. The final spore concentrations

121

of BC for the inoculation were ca 1×105 and 1×106 spores/mL, which were denoted as

122

SP/M114-BC-105 and SP/M114-BC-106, respectively. Media that were only

123

inoculated with SP or M114 were used as controls, and the media inoculated with AC

124

or BC alone were also analyzed for comparison. All treatments were incubated at

125

25°C via static culture method and conducted in triplicate. Samples were taken at

126

days 1, 3, 5, 7, 9, and 11 to analyze their respective glycerol, glucose, and fructose

127

contents.

1×105,

1×106,

and

1×107

spores/mL,

denoted

as

SP/M114-AC-105,

128

Mechanisms of the effects of A. carbonarius on the glycerol production of S.

129

cerevisiae SP. Three different cultures were conducted: (1) co-culture of AC and SP;

130

(2) Co-culture of AC and SP so that cells of the two organisms were not in contact

131

with each other as described below; (3) co-culture of pre-sterilized AC spores and SP.

132

During and after the cultivation, both the morphology and the amount of yeasts, and

133

the gene expression related to glycerol metabolism of SP in different cultures were

134

detected and compared. All cultures were performed in the SNM100 medium, as

135

detailed in the following.

136

Co-culture, but separated AC spores and SP. AC spores were placed in a

137

dialysis bag (interception of 8-14 kDa) and immersed in a co-culture medium that had

138

been inoculated with SP to evaluate their effects on glycerol production. First, the

139

prepared SP and AC spore suspensions were separately inoculated in a 250 mL

140

Erlenmeyer flask with 100 mL SNM100 medium and the final concentrations of SP

141

were adjusted to 1×106 cells/mL and that of AC to 1×105 and 1×106 spores/mL. The

142

medium inoculated with AC was immediately transferred to a dialysis bag that had 7



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previously been sterilized with ethanol. The dialysis bags were sealed and placed into

144

media inoculated with SP, denoted as SP-AC-105-D and SP-AC-106-D, corresponding

145

to final fungal spore concentrations of 1×105 and 1×106 spores/mL in the co-cultures,

146

respectively. In this way, only the metabolites of AC spores with a molecular weight

147

below 8-14 kDa were able to pass through the bag and into the medium to contact the

148

SP. Media inoculated with SP alone were used as control samples. Co-cultures of SP

149

and AC spores in the same media without separation by dialysis bags were tested for

150

comparison.

151

Co-culture of sterilized AC spores and SP. To test the effect of dead AC spores

152

on the SP metabolism, AC spore suspensions were sterilized at 121°C for 30 min prior

153

to co-cultured with SP at the final concentrations of 1×105 and 1×106 spores/mL,

154

denoted as SP-AC-105-S and SP-AC-106-S, respectively. Co-cultures of SP and live

155

AC spores were tested for comparison.

156

Micromorphology of the co-cultured fungi and yeasts. The micromorphology

157

of the yeasts and AC spores were observed using a light microscope (Motic BA 400)

158

and a scanning electron microscope (SEM, S-4800, Japan) to determine whether the

159

increase of glycerol production by the yeast was related to morphological changes.

160

SEM samples were prepared according to the methods described by Bo et al..29 The

161

dried samples were mounted onto copper stubs sputter-coated in gold (thickness of 20

162

nm) in an EMS-550 and observed under SEM at 10 kV.

163

Calculation of yeast amount. The amount of yeast was detected using the plate

164

count

method

in

dichloran

rose

bengal

chloramphenicol

165

(BectoneDickenson, Franklin Lakes, NJ, USA), as previously described.4 The

166

inoculated plates were incubated at 28°C for 2-3 days and the number of colonies was

167

recorded and expressed as cfu/mL sample. Each sample was tested in triplicate, and 8


agar

(DRBC)

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the mean results are reported with standard deviations.

169

RT-PCR analysis of the gene expression related to glycerol metabolism of SP.

170

RNA was extracted from the SP cells collected from different co-culture systems and

171

the culture of SP alone using a commercial kit (Sangon Biotech, Shanghai, China)

172

following the manufacturer’s instructions. Both purity and quality of the prepared

173

RNA were analyzed using 1% agarose gel electrophoresis. The extracted RNA was

174

treated with a PrimeScript

175

(TaKaRa, Japan) to remove contaminating DNA and to synthesize the cDNA. In total,

176

1 µg of RNA was used for cDNA synthesis in the 20 µL reaction system. To normalize

177

the target genes, the three reference genes TAF10, TFC1, and UBC6 were used as

178

described by Teste et al.30 Six target genes involved in glycerol metabolism (GPD1,

179

GPD2, GPP1, and GPP2) and flux (STL1 and FPS1) were selected, consistent with a

180

previous study.31 The primers and their sequences are shown in Table 1.

181

TM

RT reagent Kit with gDNA Eraser (Perfect Real time)

Real-time PCR was performed on 96-well plates with an ABI qRT-PCR 7500

182

system (Applied Biosystems, Foster City, CA) using SYBR PremixEx TaqII (TaKaRa)

183

as fluorophore. Reactions were conducted in a total volume of 25 µL that contained

184

2.0 µL cDNA, 1.0 µL forward and reverse primers (10 µM each), 12.5 µL of 2

185

×SYBR Premix Ex TaqII, and 8.5 µL dH2O. Each sample was run in triplicate with no

186

template control for each primer included in all real-time plates. Amplifications were

187

performed under the following conditions: 95°C for 5 min, 40 cycles of 94°C for 30 s,

188

54°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. At the

189

end of the amplification cycle, a melting analysis was conducted to verify the

190

specificity of the reaction. The expression level of a given gene was reported as the

191

quantification cycle (Cq), corresponding to the number of cycles required to reach a

192

predetermined fluorescence threshold. In the calculation of the relative copy number 9



193

from raw Cq, amplification efficiencies were considered and the final results were

194

expressed as relative expression. All data obtained with the target genes were

195

normalized relative to the geometric mean of TAF10, TFC1, and UBC6.

196

Determination of glycerol, glucose, and fructose concentration by HPLC.

197

Glycerol, glucose, and fructose concentrations were determined by high performance

198

liquid chromatography (HPLC) using a Shimadzu HPLC chromatograph equipped

199

with an amino column (NH2 column, 250 x 4.6 mm, 5 µm), a LC-15C pump, a

200

CTO-15C oven at 35oC, and an RID-15C refractive index detector The mobile phase

201

was 85% acetonitrile in deionized water (15% water) at a flow rate of 1.0 mL/min; the

202

sample injection volume was 20 µL. Glycerol, glucose, and fructose concentrations

203

were calculated according to their standard curves.

204

Data statistics. All statistical analyses were performed using the Statistic

205

Package for Social Sciences (SPSS version 13.0; IBM, Armonk, NY, USA). Student’s

206

t-test was used to evaluate the significance of differences (P＜0.05) between different

207

treatments.

208

RESULTS

209

Effects of AC spores on the glycerol metabolism of S. cerevisiae in grape

210

juice medium (GJM). Cabernet Sauvignon grapes are widely used in wine

211

production. Grape juice was prepared from Cabernet Sauvignon grapes to investigate

212

the influence of fungal spores on the glycerol metabolism of S. cerevisiae. In

213

co-cultures of different concentrations of AC spores and S. cerevisiae, glycerol

214

production and the consumption of both glucose and fructose were monitored. As

215

shown in Figure 1, no glycerol production was detected in cultures that only contained

216

AC, although a small amount of glycerol was detected in the grape juice medium (Fig.

217

1A). A significant increase of glycerol content was detected in all co-cultures of AC 10


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spores and SP or M114. Among the co-cultures, SP-AC-107 showed the highest rate

219

of glycerol accumulation, peaking at 1.3-fold of the controls without AC at day 7,

220

followed by SP-AC-106 and SP-AC-105 (Fig. 1A). This was also true for the

221

co-cultures containing M114 (Fig. 1B). AC promoted glycerol production in the two

222

different S. cerevisiae strains in a similar spore concentration-dependent manner.

223

As evidenced by the varying levels of both glucose and fructose content during

224

cultivation (Figs. 1C, 1E), the consumption of glucose and fructose decreased slightly

225

in the presence of AC alone; however, in the co-culture systems, it decreased greatly

226

with the increase of AC spore concentration. Glucose and fructose were almost

227

exhausted in the co-cultures of SP and AC at day 7, while there some sugars still

228

remained in the co-cultures of M114 and AC (Figs. 1D, 1F). These results indicated

229

that both yeast strains examined in this study had different abilities to use sugar in the

230

co-culture systems, and that the presence of AC promoted glycerol production by both

231

yeast strains.

232

Effects of AC spores on glycerol metabolism of S. cerevisiae in SNM media

233

with different sugar contents. Figures S2 and 2 showed the changes of glycerol,

234

glucose, and fructose content in the co-cultures carried out in SNM200 and SNM100

235

media. Compared to cultures containing only SP, a significant increase of glycerol

236

production was found in co-cultures containing different concentrations of AC spores

237

and SP or M114 (Figs. S2A, S2B, 2A, 2B); however, no glycerol production was

238

detected in cultures containing only AC (Fig. 2A). These results were consistent with

239

that found in GJM media, confirming that AC spores could not synthesize glycerol,

240

but could promote the glycerol production of different S. cerevisiae strains regardless

241

of the initial sugar content of the media. In addition, the co-cultures in SNM100

242

showed a higher rate of glycerol accumulation than that in SNM200. Among these 11



243

co-cultures, M114-AC-107 showed the highest rate of glycerol accumulation, peaking

244

at 2.4-fold compared to the controls without AC at day 1, followed by SP-AC-107 and

245

SP-AC-106 in SNM100 (Figs. 2A, 2B). However, glycerol production decreased after

246

5-7 days when glucose and fructose were almost exhausted, especially in co-cultures

247

of M114-AC-106. According to a report by Ferreira et al.,32 yeasts tend to use ethanol

248

or glycerol as a carbon source when all glucose is consumed during the stationary

249

phase. This might be the cause for the observed decrease in glycerol production of

250

co-cultures 5-7 days post-inoculation.

251

Furthermore, the rates of glucose and fructose consumption were also accelerated

252

in the co-cultures of AC with SP or M114 in SNM200 and SNM100 media, indicating

253

that the presence of AC enhanced the consumption of glucose and fructose by S.

254

cerevisiae strains. In particular, this acceleration was more apparent in low sugar

255

containing medium (SNM100). The glucose content in the co-culture of SP and AC

256

was exhausted at days 3, 5, and 7 in SP-AC-107, SP-AC-106, and SP-AC-105,

257

respectively. However, the fructose content decreased to 0 between days 5 and 7,

258

corresponding to the time at which glucose was already exhausted, suggesting that S.

259

cerevisiae preferred to consume glucose, followed by fructose.33

260

Effect of AC metabolites on the glycerol metabolism of SP. When AC spores

261

were separated from SP by the dialysis bag, only the metabolites with a molecular

262

weight < 8-14 kDa could reach SP. As shown in Figure 3A, during the first 4 days, the

263

glycerol production in the co-cultures with dialysis bag were higher than in cultures

264

containing only SP and co-cultures without dialysis bags. Among the co-cultures with

265

dialysis bags, SP-AC-105-D showed the highest glycerol production followed by

266

SP-AC-106-D. After 4 days, the promotion of glycerol production in the co-cultures

267

with dialysis bags decreased. Moreover, in SP-AC-105 and SP-AC-106 co-cultures, 12


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glycerol production significantly increased without dialysis bags at day 5 and 6 (Fig.

269

3A). However, the glucose and fructose consumption rates in the co-cultures with

270

dialysis bags were much faster than the co-cultures without dialysis bags, shifting

271

their time of depletion to nearly one day earlier (Figs. 3B, 3C). These results indicate

272

that AC might release some metabolites that promote the consumption of sugars by SP

273

and increase glycerol production.

274

Effects of sterilized AC spores on the glycerol metabolism of SP. Figure 4A

275

shows that sterilized AC spores also promoted glycerol production by SP in our

276

experiment. Among the different spore concentrations, SP-AC-106-S resulted in the

277

highest glycerol production at the end of the first 4 days; however, this production

278

declined during later cultivation stages. This was particularly true when fewer AC

279

spores were used (SP-AC-105-S), which may be due to the incapability of sterilized

280

spores to release new metabolites to stimulate SP glycerol metabolism.

281

also implied that some kinds of substances released from the sterilized spores had the

282

ability to promote SP glycerol production - said substances may or may not be similar

283

to substances released by living spores. Similar sugar consumption was observed in

284

the co-cultures using living and sterilized AC spores. In our preliminary experiments,

285

sterilized fungal spores could not grow and consume sugars (data not shown).

286

Therefore, it is reasonable to deduce that it was the component of AC spores that

287

promoted the consumption of glucose and fructose by SP (Figs. 4B, 4C).

These results

288

Changes of SP amount and micromorphology of AC and SP. The amount of

289

SP increased significantly in co-cultures of AC and SP, especially in the co-cultures

290

with high concentration of AC spores (Fig. 5A). In particular, SP-AC-106-S resulted

291

in the highest yeast amount, indicating that sterilized AC spores could release more

292

metabolites, which promoted yeast growth and glycerol production. Here, it is worth 13



293

mentioning that glycerol production increased significantly (Figs. 1A, 2A, 3A) and

294

even the amount of viable yeast cell was decreased at day 5 (Fig. 5A). This is because

295

the yeast entered the stationary phase after 5 days of inoculation. During the

296

stationary state, glycerol was excreted via a fermentative mechanism to ensure energy

297

for cell maintenance as reported by Djelal et al.. 34 The authors reported that the major

298

portion (76%) of glycerol continued to be produced to ensure sufficient energy for cell

299

maintenance during the stationary state, although the amount of yeast did not

300

increases any further. However, when all glucose and fructose were consumed, yeast

301

tended to utilize glycerol as carbon source, 32 thus leading to a decline in glycerol

302

content at day 7(Figs. 1B, 2B, 3B, 4B).

303

Furthermore, the micromorphology of spores in the co-cultures changed

304

significantly compared to that in the culture with AC spores alone. When AC spores

305

were cultured alone, they showed normal germination (Fig. 5-B1), formation of

306

mycelia, and production of a large number of conidia (Fig. 5-B3). The spores were

307

similarly shaped and the surface of each spore was verrucous (Fig. 5-B3). In contrast,

308

in the co-culture of SP and AC, AC spores were surrounded by SP and could not

309

germinate (Fig. 5-B2). The verrucous appearance disappeared and holes formed on

310

the spores (Fig. 5-B4). The disruption of AC spores might be the major reason that

311

caused metabolite release and the promotion of yeast growth and glycerol production.

312

However, the micromorphology of SP was not changed significantly in the co-cultures

313

with AC. This indicated that the improvement of glycerol synthesis in co-culture

314

systems was not related to the morphology of SP.

315

Effects of AC spores on the gene expression related to the glycerol

316

metabolism of SP. Figure 6 shows the expression level of genes related to the

317

glycerol metabolism of SP. We found the expression levels of GPD1, GPD2, GPP1, 14


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GPP2, STL1, and FPS1 were significantly increased in the co-culture systems

319

compared TO SP-only cultures. Among these genes, the STL1 gene was most

320

significantly up regulated. STL1 is responsible for encoding the proton transport

321

protein in the plasma membrane and is mainly related to the absorption of glycerol.

322

The glucose level of a given culture sample had an effect on the STL1 expression: It

323

appears that yeasts tend to use ethanol or glycerol as a carbon source at the stationary

324

phase when glucose availability is low; this is a stress response that tends to stimulate

325

the expression of STL1.33 This theory adequately explains why the expression of STL1

326

in AC spore co-cultures at the SP-AC-105 level was almost twice that of SP-only

327

cultures at days 1, 3, and 5 and even 12.5 -fold of that at day 7 (Fig. 6A), which

328

corresponds to the time when both the glucose and fructose were nearly exhausted

329

(Figs. 3B, 3C). The expression of STL1 increased when more AC spores were used

330

(SP-AC-106), and reached a peak of 17.2 and 16.4-fold that of SP-only cultures at

331

days 5 and 7, respectively (Fig. 6B), corresponding to the days when glucose (day 5)

332

(Fig. 3B) and fructose (day 7) were exhausted (Fig. 3C). These findings are also

333

consistent with the decrease in glycerol content and up-regulated STL1 gene

334

expression at the later stage of cultivation as SP utilized glycerol as a carbon source

335

for growth (Fig. 3A).

336

The GPP1 gene is one of the principle genes related to the cellular redox balance

337

of the glycerol metabolism in SP. Because it can be induced under anaerobic

338

conditions, the expression level of GPP1 in the co-culture systems was higher than

339

that in SP-only cultures. Co-cultures with fewer AC spores (SP-AC-105) exhibited an

340

increased GPP1 expression level after the first 5 days post-inoculation. At day 5, the

341

gene expression level was 10.7-fold of that of SP-only cultures (Fig. 6A) and the

342

expression level of GPP1 in the co-cultures at the fungal spore level of SP-AC-106 15



343

was 6.6-fold of that of SP-only cultures, despite remaining almost identical during

344

other periods. These results indicated that AC spores regulated glycerol production of

345

SP through the redox balance pathway in yeasts.

346

The relative expressions of GPD1, GPD2, GPP2, and FPS1 genes were also

347

up-regulated in all co-culture systems. The GPD1 gene expression was up-regulated

348

at day 5 in both the co-culture systems and the SP-only cultures. The expression of

349

GPD2 and GPP2 was up-regulated at days 1 and 3 in the presence of AC spores, but

350

down-regulated at days 5 and 7. The expression of FPS1 (the gene that encodes the

351

glycerol water channel protein and is responsible for intracellular glycerol outflow

352

and inflow control) remained lower or equal to that in SP-only cultures during most of

353

the cultivation. These results indicated that the effects of AC spores on glycerol

354

production by SP might not be related to the glycerol channel protein.

355

In summary, we concluded that the genes related to the metabolism of glycerol

356

changed at day 5 and that STL1 and GPP1 were significantly up-regulated. The

357

changes in the expression of these genes were likely related, in most cases, to the

358

extremely high glycerol accumulation in the co-culture systems at day 5.

359

Effects of Botrytis cinerea (BC) on glycerol metabolism of S. cerevisiae.

360

Changes of glycerol, glucose, and fructose contents were monitored in the co-cultures

361

of BC with SP or M114 (Fig. S3). Being consistent to that found for AC, BC did not

362

produce glycerol when it was cultivated alone, but significantly promoted the glycerol

363

production and the consumption of glucose and fructose by yeast in co-cultures in a

364

concentration-dependent manner. Compared to AC, BC showed more obvious

365

promotion of glycerol production by different yeast strains, especially when SP was

366

used. The presence of BC resulted in about 2.6-fold and 1.9-fold higher amounts of

367

glycerol accumulation compared to culture with SP alone at days 3 and 5, respectively. 16


Page 16 of 36

Page 17 of 36


368

Moreover, sterilized BC spores also showed promotion of glycerol production and

369

sugar consumption rate of SP (Fig. S4). The amount of SP cells also increased

370

significantly in the co-cultures with BC (Fig. S5). This was consistent with the results

371

obtained for AC (Fig.5A).

372

The consistency in the results obtained for AC and BC indicated that the

373

capability of fungal spores to promote the glycerol production of yeast might be a

374

common feature of most fungi.

375

DISCUSSION

376

Fungal spores promoted glycerol accumulation of S. cerevisiae. It is generally

377

accepted that glycerol accumulation in wine is related to S. cerevisiae metabolism

378

during winemaking. Ravji et al. 22 reported that fungi contaminated in grapes, such as

379

A. niger, Penicillium italicum, Rhizopus nigricans, and B. cinerea could produce a

380

high amount of glycerol in healthy Golden Chasselas and Black Hamburg grape juice.

381

Hong et al.

382

botrytized berries. In their study, grape juices were obtained via artificial rubbing and

383

were machine-homogenized without sterilization after washing the grapes with sterile

384

water, then inoculated with the tested fungi and tested for glycerol content after 26-29

385

days of incubation.

386

produced by yeasts and not by the fungi on the surface of the grapes. In the present

387

study, the capability of AC and BC to produce glycerol was measured in GJM and

388

SNM media after sterilization to minimize the influence of other fungi that

389

contaminated the cultivation. As a result, neither the AC nor BC used in this study

390

proved capable of producing glycerol when they were cultivated alone. However, is

391

remain uncertain whether these differences were caused by the different strains used.

392

35

also reported that high amounts of glycerol were only found in

22

However, without sterilization, glycerol was most likely

We also found that AC and BC spores promoted the production of glycerol by SP 17



Page 18 of 36

393

in a spore concentration-dependent manner: The production of glycerol increased with

394

increasing spore concentration. This was also true with regard to the accelerated

395

consumption of glucose and fructose by fungal spores.

396 397

The glycerol production of S. cerevisiae was stimulated by substances

398

released from AC spores. Glycerol production of yeast was influenced by many

399

environmental factors, such as temperature, pH and chemical contaminants, etc..

400

Sugar content has been reported to positively affect the glycerol formation of yeasts:

401

in the range of osmotic tolerance, higher sugar concentration tends to result in higher

402

of glycerol yield produced by S. cerevisiae.36,39,40 However, in the present study, we

403

found that AC spores could promote glycerol production of S. cerevisiae in media

404

with different sugar contents. Different from that in cultures with yeast alone, the

405

co-cultures conducted in lower sugar content medium (SNM100) showed a higher

406

increase of glycerol production and sugar consumption rate than in higher sugar

407

medium (SNM200 or GJM). Meanwhile, both sterilized and live AC spores had a

408

similar effect on the promotion of glycerol production by SP when co-cultured with

409

SP, suggesting that substances in the fungal spores might stimulate the glycerol

410

production of S. cerevisiae.

36-38

411

Substances in the spore suspensions stimulated the expression of genes

412

related to glycerol biosynthesis in S. cerevisiae. According to previous studies, the

413

glycerol synthesis is mainly confined to glycerol 3-phosphate dehydrogenase (GPD)

414

and glycerol 3-phosphatase (GPP) in yeast cells. 16 GPD is a rate-limiting enzyme due

415

to the activity of GPP in yeast cells being much higher than that of GPD under the

416

same culture conditions. 41 The GPD and GPP encoding genes we examined here were

417

GPD1, GPD2, GPP1, and GPP2, respectively, each of which are associated with 18


Page 19 of 36


418

different glycerol metabolic regulation pathways; GPD1 and GPP2 are related to

419

osmotic pressure while GPD2 and GPP1 are related to the intracellular redox balance.

420

When yeast cells were in an anaerobic environment, the expression of GPD2 and

421

GPP1 promoted the cellular synthesis of glycerol and the intracellular accumulation

422

of excess NADH was oxidized into NAD+, which maintains the equilibrium of

423

intracellular redox potential.42 The expression levels of GPD2 and GPP1 were

424

up-regulated in the presence of AC early in the experiment (3-5 days), suggesting that

425

the promotion of glycerol accumulation was regulated by the oxidative balance

426

pathways of S. cerevisiae.

427

Furthermore, in contrast to SP-only cultures, there was a large increase in the

428

expression of the STL1 gene from 5 to 7 incubation days in the presence of AC which

429

may have been related to glucose and fructose consumption. When all glucose was

430

consumed, the yeast culture entered a diauxic shift during which major changes in

431

gene expression altered the fermentative oxidative metabolism, allowing the yeast to

432

utilize the produced ethanol and glycerol before entry into the stationary phase.

433

During incubation from 5 to 7 days, glucose and fructose were successively depleted

434

with treatments of SP-AC-106 and SP-AC-105, which accelerated the absorption of

435

glycerol and stimulated the expression of the STL1 gene.

32, 43

436

The effects of BC on the glycerol synthesis in different types of S. cerevisiae

437

were also investigated to verify the hypothesis that BC had similar promotion of

438

glycerol production by yeast. Others species of fungi, such as Penicillium

439

Chrysogenum and Aspergillus ochraceus were also used and they also promoted

440

glycerol production by S. cerevisiae (Figure S6). All the results indicated that the

441

ability of fungal spores to promote the glycerol biosynthesis by yeast might be a

442

universal phenomenon for most fungal spores. Furthermore, some compounds 19



443

released from or metabolites produced by the fungal spores were found to be the

444

major factor that stimulates glycerol production of yeasts. Such compounds may

445

widely exist in fungal spores. Therefore, further research is required to clarify the

446

nature of these possible compounds.

447

In conclusion，this study revealed that the effect of fungal spores on the

448

promotion of glycerol production of S. cerevisiae might widely exist in different kinds

449

of fungi. It was confirmed that fungal spores affected the yeast metabolism by

450

promoting the glycerol biosynthesis pathway. The influence of fungal contamination

451

on the metabolism of yeast and wine quality is inevitable and needs to be further

452

explored in depth, since the winemaking process involves a mixture of yeasts and

453

different types of fungi.

454

provide a strong support for the improvement and control of the wine quality in view

455

of the interaction between fungal spores and yeast.

456

ABBREVIATIONS USED

457

26

Understanding the mechanism of such interactions would

AC, A. carbonarius; BC, B. cinerea; SP, S. cerevisiae SP; M114, S. cerevisiae

458

M114.

459

ACKNOWLEDGEMENTS

460

The authors would like to thank Professor Qing Ma who works at the College of

461

plant protection of Northwest A&F University for providing the strain of Botrytis

462

cinerea.

463 464

FUNDING This work has been granted financial support from the Agriculture Department

465

of China (CARS-30), the National Natural Science Foundation of China (No.

466

31701722), the China Postdoctoral Science Foundation (2017M620471), the

467

Fundamental Research Funds for the Central Universities (3102016QD089), the

468

National Key Technology R&D Program (2015BAD16B02), the Shaanxi Provincial 20


Page 20 of 36

Page 21 of 36


469

Natural Science Foundation (No. 2015JQ3083), and partly from the universities key

470

scientific research project of henan (18A550013).

471

SUPPORTING INFORMATION

472

Glycerol content of wine at different levels of A. carbonarius contamination is

473

shown in Figure S1. Changes of glycerol, glucose, and fructose levels and yeast

474

amount in co-cultures of SP with AC spores in SNM200 medium are shown in Figure

475

S2, and live or dead BC spores are shown in Figure S3-S5. Production of glycerol in

476

co-cultures of SP with different concentration of Aspergillus ochraceus and

477

Penicillium chrysogenum spores are shown in Figure S6.

478

NOTES

479

The authors declare no competing financial interest.

480 481

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27



Page 28 of 36

Table 1 Reference and target genes and their relative primers used in RT-PCR Genes

Description

Forward and reverse primer

PCR product size (bp)

References

TAF10

RNA Pol II transcription factor activity/Transcripti on initiation and chromatin modification RNA Pol III transcription factor activity/Transcripti on initiation on Pol III promoter Ubiquitin-protein ligase activity/ER-associ ated protein catabolic process Glycerol-3-phosph ate dehydrogenase (NAD (+)) Glycerol-3-phosph ate dehydrogenase (NAD (+)) Glycerol-1-phosph atase RHR2 Glycerol-1-phosph atase HOR2 Glycerol proton symporter of the plasma membrane

F:ATATTCCAGGATCAGGTCTTCCGT AGC R:GTAGTCTTCTCATTCTGTTGATGT TGTTGTTG

141

30

F:GCTGGCACTCATATCTTATCGTTT CACAATGG R:GAACCTGCTGTCAATACCGCCTG GAG

223

F:GATACTTGGAATCCTGGCTGGTC TGTCTC R:AAAGGGTCTTCTGTTTCATCACC TGTATTTGC

272

F: GCGAGGGCAAGGACGTCGAC R:TGGATGGCAGCAGAAGCGTTGT

184

F: TTTCCCAGAATCCAAAGTCG R: CTGAGCAGGTGGTGATCAGA

74

F:TGCTTTGAACGCCTTGCCAAAGG R: ACGGGTACCAGAGGTGGCGA F: CAGCAGGTATTGCCGCCGGA R: CGGCATTGTAGCCGCCAACT F: ACGCAAGAGGTGCTGCCGTC R: AGCAACCCCAACCGGACTGT

55

Aquaglyceroporin, plasma membrane channel

F: AAGTGCGCGGCCTACTCCCA R: CTTGCACTCGGCGGACCGTT

TFC1

UBC6

GPD1

GPD2

GPP1 GPP2 STL1

FPS1

28


145 210

141

44

Page 29 of 36


Figure captions Figure 1 Changes in glycerol (A and B), glucose (C and D), and fructose (E and F) levels in co-cultures of SP (A, C, and E) and M114 (B, D, and F) with AC in grape juice medium (GJM). Figure 2 Changes in glycerol (A and B), glucose (C and D), and fructose (E and F) levels in co-cultures of SP (A, C, and E) and M114 (B, D, and F) with AC in SNM100 medium. Figure 3 Changes in glycerol (A), glucose (B), and fructose (C) levels in the co-cultures of SP and AC with dialysis bags in SNM100 medium. Figure 4 Changes in glycerol (A), glucose (B), and fructose (C) levels in the co-culture of SP and sterilized spores of AC without dialysis bags in SNM100 medium. Figure 5 Changes of yeast amount in co-cultures of SP with live and sterilized AC spores (A) and morphology changes of AC and SP when they were co-cultured (B) in SNM100 medium. Micromorphology of AC and SP observed under both optical microscope and SEM (B1 and B3 are AC cultivated alone at days 2; B2 and B4 are the co-culture of AC and SP at day 4 , B1 and B2 are observed under optical microscope (40 folds); B3 and B4 are observed under SEM). Figure 6 Relative expression of genes related to the glycerol metabolism of SP in the presence of 1×105 (A) and 1×106 spores/mL (B) AC in SNM100 medium.

29



A

B

C

D

E

F

Figure 1

30


Page 30 of 36

Page 31 of 36


A B

C D

E F

Figure 2

31



A

B

C

Figure 3

32


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Figure 4

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(A)

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B1

B2

B3

B4

Figure 5

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Figure 6

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Fungal Spores Promote the Glycerol Production of Saccharomyces

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