Î³-Dodecelactone Production from Safflower Oil via 10-Hydroxy-12(Z

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#-Dodecelactone Production from Safflower Oil via 10Hydroxy-12(Z)-octadecenoic Acid Intermediate by Whole Cells of Candida boidinii and Stenotrophomonas nitritireducens Ye-Seul Jo, Jung-Ung An, and Deok-Kun Oh J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501081z • Publication Date (Web): 26 Jun 2014 Downloaded from http://pubs.acs.org on June 30, 2014

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Journal of Agricultural and Food Chemistry

γ-Dodecelactone Production from Safflower Oil via 10-Hydroxy-12(Z)octadecenoic Acid Intermediate by Whole Cells of Candida boidinii and Stenotrophomonas nitritireducens

Ye-Seul Jo, Jung-Ung An, Deok-Kun Oh

ACS Paragon Plus Environment


1

γ-Dodecelactone Production from Safflower Oil via 10-

2

Hydroxy-12(Z)-octadecenoic Acid Intermediate by

3

Whole Cells of Candida boidinii and Stenotrophomonas

4

nitritireducens

5

6

Ye-Seul Jo, Jung-Ung An, and Deok-Kun Oh*

7 8

Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701,

9

Republic of Korea

1


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ABSTRACT: Candida boidinii was selected as a γ-dodecelactone producer because of

11

the highest production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid

12

among the 11 yeast strains tested. Under the reaction conditions of pH 5.5 and 25 °C

13

with 5 g/L 10-hydroxy-12(Z)-octadecenoic acid and 30 g/L cells, whole C. boidinii cells

14

produced 2.1 g/L γ-dodecelactone from 5 g/L 10-hydroxy-12(Z)-octadecenoic acid after

15

6 h, with a conversion yield of 64% (mol/mol) and a volumetric productivity of 350

16

mg/L/h. The production of γ-dodecelactone from safflower oil was performed by lipase

17

hydrolysis reaction and two-step whole-cell biotransformation using Stenotrophomonas

18

nitritireducens and C. boidinii. γ-Dodecelactone at 1.88 g/L was produced from 7.5 g/L

19

safflower oil via 5 g/L 10-hydroxy-12(Z)-octadecenoic acid intermediate by these

20

reactions after 8 h of the reaction time, with a volumetric productivity of 235 mg/L/h

21

and a conversion yield of 25% (w/w). To the best of our knowledge, this is the highest

22

volumetric productivity and conversion yield reported to date for the production of γ-

23

lactone from natural oils.

24 25 26

KEYWORDS: safflower oil, two-step biotransformation, γ-dodecelactone, Candida

27

boidinii, Stenotrophomonas nitritireducens, 10-hydroxy-12(Z)-octadecenoic acid

2



28

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INTRODUCTION

29 30

Lactones are molecules characterized by a ring derived and are from the cyclic

31

esterification between a hydroxyl group and a carboxylic group of a hydroxy fatty acid.1

32

These compounds exude the characteristic buttery, coconut-like, creamy, sweet, peach-

33

like, and/or apricot-like smells.2,3 Lactones are naturally distributed as aroma

34

compounds in many beverages, food products, and dairy products and are used as

35

flavoring agents in many foods, cosmetics, and drugs. Lactones are also used as

36

fragrance compounds in food additives, chewing gums, tooth pastes, cosmetic powders,

37

medicinal products, hair preparation, detergents, smoking tobaccos, and perfumed

38

goods.4

39

monounsaturated penta-ring lactone, has the characteristic butter flavor.5 This lactone is

40

present in the Swiss Gruyere cheese and the peri-anal gland hormone of crested

41

porcupines.6,7 γ-Dodecelactone has been used with γ-decelactone and γ-dodecalactone

42

to make a flavoring solution for bran pickles.8

Among

all

the

lactones,

γ-dodecelactone

(cis-6-dodecen-4-olide),

a

43

Lactones have been produced by microbial conversion, chemical synthesis, and

44

natural extraction from fruits. This natural extraction method is relatively expensive

45

because of the low concentration in the fruits and the complex purification process

46

owing to the many compounds present in the fruits. Although lactones have been

47

mainly manufactured by chemical synthesis, the consumer prefers natural flavors. Thus,

48

a biological process for the economic production of lactone is required.9,10 Lactone

49

production by microbial conversion has been mainly attempted using the yeast

50

Yarrowia lipolytica.11-13

3


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Yeast strains are known to produce high concentrations of lactone from hydroxy fatty

52

acids as substrates,9,14 however, yeast cells produce no or very low concentrations of

53

lactones from free fatty acids. Moreover, the production of γ-dodecelactone from

54

hydroxy fatty acid has not been reported. Castor oil has been used in the production of

55

lactone, especially γ-decalactone, using yeasts15-18 because castor oil contains the

56

hydroxy fatty acid, ricinoleic acid, as a major component (about 80 %).1,19 Other oils

57

that do not contain any hydroxy fatty acids have little been used in lactone production.

58

Quantitative production of γ-dodecelactone has been reported only for the fermentation

59

of soybean oil or copra oil by Penicillium roqueforti.20 However, the conversion yield

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and productivity of lactone from oils including castor, soybean, copra oils were too low.

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Therefore, γ-dodecelactone production from oil via 10-hydroxy-12(Z)-octadecenoic acid

62

intermediate, can be regarded as a potentially efficient bioprocess.

63

In the present study, the reaction conditions were determined for the increased

64

production of γ-dodecelactone from the substrate 10-hydroxy-12(Z)-octadecenoic acid

65

by whole Candida boidinii cells; 10-hydroxy-12(Z)-octadecenoic acid was converted

66

from linoleic acid by whole Stenotrophomonas nitritireducens cells. Under the reaction

67

conditions, γ-dodecelactone was produced from 10-hydroxy-12(Z)-octadecenoic acid.

68

To attain higher productivity and conversion yield of γ-dodecelactone using oil, lipase-

69

treated safflower oil hydrolyzate was used as the substrate and a two-step whole-cell

70

biotransformation process via hydroxy fatty acid intermediates was established (Figure

71

1).

72 73

MATERIALS AND METHODS

74

4



75

Materials. Safflower oil and γ-dodecelactone were purchased from Santa Cruz

76

Biotechnology (Santa Cruz, CA) and Penta International (West Caldwell, NJ),

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respectively. γ-Dodecalactone, lipase from Candida rugosa, and the fatty acids,

78

including linoleic acid, oleic acid, and palmitic acid, were purchased from Sigma (St.

79

Louis, MO).

80 81

Preparation of hydroxy fatty acids. 10-Hydroxystearic acid was produced by

82

the reaction of whole recombinant Escherichia coli cells expressing oleate hydratase

83

from Stenotrophomonas maltophilia.21 The reaction was performed in 50 mM

84

citrate/phosphate buffer (pH 6.5) containing 50 g/L oleic acid, 10 g/L of E. coli cells,

85

and 0.05% (w/v) Tween 40 at 35 °C for 4 h. 10-Hydroxy-12(Z)-octadecenoic acid was

86

produced by the reaction of whole S. nitritireducens cells.22 The reaction was performed

87

in 50 mM Tris-HCl buffer (pH 7.5) containing 20 g/L linoleic acid, 20 g/L of S.

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nitritireducens cells, and 0.02% (w/v) Tween 80 at 30 °C for 2 h. 10-Hydroxystearic

89

acid and 10-hydroxy-12(Z)-octadecenoic acid were prepared from the obtained reaction

90

products by the following low-temperature solvent fractional crystallization.9 An equal

91

volume of ethyl acetate was added to the reaction products, and the ethyl acetate from

92

the reaction mixture was removed using a rotary evaporator. The extract was mixed with

93

30% acetonitrile and 70% acetone at room temperature. The solution obtained was

94

incubated at −80 °C for 24 h in a deep freezer. The liquid fraction containing the

95

unsaturated fatty acid was separated at room temperature and the remained solid

96

fraction containing hydroxy fatty acid was obtained. The acetonitrile and acetone in the

97

solid fraction removed using a rotary evaporator. This fractionization procedure was

98

repeated thrice, and the obtained products of 10-hydroxystearic acid and 10-hydroxy5


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12(Z)-octadecenoic acid with high purity (>99%) were identified by GC and

100

GC/MS.21,23 The two hydroxy fatty acids were used as the standard compounds for

101

product analysis, and 10-hydroxy-12(Z)-octadecenoic acid was also used as a substrate

102

for γ-dodecelactone production.

103 104

Microorganisms. The isolated strain S. nitritireducens22 was used for 10-hydroxy-

105

12(Z)-octadecenoic acid production from linoleic acid. E. coli ER2566 expressing oleate

106

hydratase from S. maltophilia KCTC 1773 was used for the production of 10-

107

hydroxystearic acid from oleic acid. C. boidinii KTCT 17776, Candida palmioleophila

108

KTCT 17452, Candida tropicalis KTCT 7221, Citeromyces matritensis KTCT 17714,

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Cryprococcus curvatus KTCT 7225, Saccharomyces cerevisiae KTCT 7704,

110

Schizosaccharomyces pombe KTCT 7167, Sporobolomyces odorus KTCT 17242,

111

Starmerella bombicola KTCT 17652, Waltomyces lipofer KTCT 17657, and Y. lipolytica

112

KTCT 17170 were used for γ-dodecelactone production from 10-hydroxy-12(Z)-

113

octadecenoic acid.

114 115

Media. S. nitritireducens was cultivated in nutrient broth containing 5 g/L peptone

116

and 3 g/L beef extract. Recombinant E. coli cells were cultivated in Luria-Bertani (LB)

117

agar. The growth medium was yeast malt (YM) broth containing 10 g/L glucose, 3 g/L

118

malt extract, and 5 g/L peptone. The induction medium contained 5 g/L glucose, 7 g/L

119

oleic acid, 0.1 g/L yeast extract, 2.1 g/L potassium phosphate monobasic, 4.51 g/L

120

potassium phosphate dibasic, 0.1 g/L sodium chloride, 0.2 g/L magnesium sulfate, 9.14

121

mg/L iron sulfate heptahydrate, 0.5 mg/L zinc chloride, and 1.56 mg/L copper sulfate.

122

The reaction medium contained 50 mM citrate/phosphate buffer (pH 5.5), 3.4 g/L yeast 6



123

nitrogen base, 0.5 g/L 10-hydroxy-12(Z)-octadecenoic acid, and 0.05 % (w/v) Tween 80.

124 125

Culture and reaction conditions. The cells were cultivated on the YM agar plate

126

containing. After 1 day, a single colony in the plate was inoculated into 15 mL of the

127

growth medium and cultivated at 30 °C with agitation at 200 rpm for 12 h. The seed

128

culture was then transferred into a 2-L baffled flask containing 500 ml of the induction

129

medium and was cultivated at 30 °C with agitation at 200 rpm for 15 h. The cells from

130

the culture medium were harvested by centrifugation at 13000g for 20 min at 4 °C and

131

then were washed twice with 50 mM citrate/phosphate buffer (pH 5.5). The harvest cells

132

were used for γ-dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acid.

133

Unless otherwise stated, the biotransformation reaction was performed in a 500-mL

134

baffled flask containing 100 ml of the reaction medium at pH 5.5 and 25 °C with

135

agitation of 200 rpm for 3 h. For lactonization, the pH of the reaction solution was

136

adjusted to 2.0 by adding M HCl and then incubated at 100 °C for 30 min.9,10,14

137 138

Selection of inducer and determination of induction process. Fatty acids

139

and hydroxy fatty acids, including lauric acid, stearic acid, oleic acid, erucic acid,

140

linoleic acid, 10-hydroxydecanoic acid, and 10-hydroxystearic acid, were used as

141

inducers for the β-oxidation pathway. To select inducer, C. boidinii cells were cultivated

142

in the induction medium supplemented with 7 g/L inducer. The effect of inducer on γ-

143

dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acid was investigated

144

using whole C. boidinii cells, and thus the best inducer oleic acid was selected. To

145

determine the concentration of oleic acid for increased γ-dodecelactone production, the

146

concentration of oleic acid was varied from 0 g/L to 15 g/L in the induction medium 7


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containing 5 g/L glucose. To determine the optimal concentration of glucose, the

148

concentration of glucose was varied from 1 g/L to 15 g/L in the induction medium

149

containing 7 g/L oleic acid. The cells were cultivated at 30 °C with agitation at 200 rpm

150

for 15 h. To determine the induction time for the increased activity of β-oxidation, the

151

cells were cultivated in the induction medium containing 5 g/L glucose and 7 g/L oleic

152

acid at 30 °C with agitation at 200 rpm for 24 h. Samples were withdrawn at several

153

intervals within 24 h. C. boidinii cells obtained from the cultures were harvested, and γ-

154

dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acid using the harvested

155

cells was conducted at 30 °C and pH 5.5 for 3 h.

156 157

Effects of pH and temperature on γ-dodecelactone production. The effect

158

of pH on γ-dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acid by

159

whole C. boidinii cells was evaluated by varying the pH from 4.0 to 6.5 in 50 mM

160

citrate/phosphate buffer, while a constant temperature of 25 °C was maintained. In order

161

to evaluate the effect of temperature, the temperature was varied from 15 °C to 35 °C,

162

while the pH was constantly maintained at 5.5. The reactions were performed under

163

above-described standard reaction conditions, only the pH and temperature were varied.

164 165

Effects of cells and substrate concentrations on γ-dodecelactone

166

production. The optimal concentrations of cells and substrate for the increased

167

production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid by whole C.

168

boidinii cells was determined by varying the cell concentration from 5 g/L to 40 g/L at a

169

constant 10-hydroxy-12(Z)-octadecenoic acid concentration of 5 g/L, and varying the

170

substrate concentration from 2.5 g/L to 15 g/L at a constant cell concentration of 30 g/L. 8



171

The reactions were performed under above-described standard reaction conditions, only

172

the cell and substrate concentrations were varied.

173 174

Production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic

175

acid. γ-Dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acids by whole

176

C. boidinii cells were performed in 50 mM citrate/phosphate buffer (pH 5.5) containing

177

3.4 g/L yeast nitrogen base, 5 g/L 10-hydroxy-12(Z)-octadecenoic acid, 30 g/L cells,

178

and 0.05% (w/v) Tween 80 at 25 °C for 8 h.

179 180

Production of γ-lactones from safflower oil. The hydrolysis of fatty acids

181

from safflower oil by lipase from Candida rugosa were carried out in 50 mM Tris-HCl

182

buffer (pH 7.5) containing 7.5 g/L safflower oil, 1 g/L lipase, and 0.02% (w/v) Tween

183

80 at 37 °C for 30 min. To inactivate the lipase and arrest the reaction, the reaction

184

solution was boiled at 100 °C for 50 min.24 The production of hydroxy fatty acids by

185

whole cells of S. nitritireducens was conducted in 50 mM Tris-HCl buffer (pH 7.5)

186

containing the hydrolyzate obtained from 7.5 g/L safflower oil, 20 g/L cells, and 0.02%

187

(w/v) Tween 80 at 30 °C with agitation at 200 rpm for 90 min, under anaerobic

188

conditions.22 An equal volume of ethyl acetate was added to the cell reaction solution,

189

and the ethyl acetate from the reaction mixture was removed using a rotary evaporator,

190

and the concentration of 10-hydroxy-12(Z)-octadecenoic acid in the reaction solution

191

was adjusted to 5.0 g/L by dissolving the residue for the production of γ-lactones. The

192

time course reactions for the production of γ-lactones by whole C. boidinii cells were

193

carried out in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen

194

base, 5.0 g/L 10-hydroxy-12(Z)-octadecenoic acid, 0.85 g/L 10-hydroxystearic acid, 9


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0.27 g/L linoleic acid, 0.07 g/L oleic acid, 0.31 g/L palmitic acid, 30 g/L cells, and 0.05%

196

(w/v) Tween 80 at 25 °C for 7 h.

197 198

Analytical methods. The cell mass was determined by calibrating of the optical

199

density at 600 nm to the dry cell weight. The concentration of glucose in the medium

200

was analyzed using a glucose assay kit (Asan Pharm, Seoul, South Korea).

201

Heptadecanoic acid at 50 mM was used as an internal standard. The silylation of

202

linoleic acid, oleic acid, palmitic acid, 10-hydroxy-12(Z)-octadecenoic acid, and 10-

203

hydroxystearic

204

(trimethylsilyl)trifluoroacetamide with a ratio of 3:1. Silylated fatty acids, silylated

205

hydroxy fatty acids, and γ-lactones were analyzed by a gas chromatography (Agilent

206

7890 N, Santa Clara, CA) with a flame ionization detector and a Supelco SPB-1

207

capillary column. The column temperature was increased from 100 to 220 °C at the rate

208

of 5 °C/min for 24 min, and then it was maintained at 220 °C. The injector and detector

209

temperatures were maintained at 260 °C and 250 °C, respectively. γ-Dodecelactone

210

(ring form), γ-dodecalactone, γ-dodecelactone (open form), palmitic acid, linoleic acid,

211

oleic acid, 10-hydroxy-12(Z)-octadecenoic acid, and 10-hydroxystearic acid were

212

detected on the basis of their retention times of 6.1 min, 6.7 min, 10.8 min, 14.3 min,

213

17.3 min, 17.9 min, 21.1 min, and 21.8 min, respectively. The γ-lactones, fatty acids,

214

and hydroxy fatty acids in the reaction samples after hydrolysis of safflower oil; and γ-

215

dodecelactone and 10-hydroxy-12(Z)-octadecenoic acid in the reaction samples of

216

whole C. boidinii cells containing 5 g/L 10-hydroxy-12(Z)-octadecenoic acid were

217

identified to have the same retention times as those of their corresponding standards.

218

The amounts of the products were calculated by calibrating of the peak areas to the

acid

were

conducted

by mixing

pyridine

10


and

N-methyl-N-


219

concentrations of γ-lactones, fatty acids, and hydroxy fatty acid standards.

220 221

RESULTS AND DISCUSSION

222 223

Selection of an efficient γ-dodecelactone-producing strain. In the GC

224

profiles, the peaks for the substrate and the product obtained from the reaction solutions

225

of whole C. boidinii cells were detected at the same retention times as those of the 10-

226

hydroxy-12(Z)-octadecenoic acid and γ-dodecelactone standards respectively (Figure 2).

227

The open and ring forms of γ-dodecelactone in the reaction solutions of whole C.

228

boidinii cells before and after acidification were analyzed by the GC. The open and ring

229

forms of γ-dodecelactone before acidification were detected (Figure S1A). The open

230

and ring forms of γ-dodecelactone were distinguishable each other, showing different

231

retention time in the GC profile. However, after acidification, the open form of γ-

232

dodecelactone was converted to the ring form, and then it disappeared (Figure S1B).

233

The yeast Sporobolomyces odorus25 and the fungi Fusarium poae5,26 and P.

234

roqueforti20 have been reported to produce γ-dodecelactone. However, the yield of γ-

235

dodecelactone produced by these microorganisms was very low. To obtain an effecient

236

γ-dodecelactone-producing strain, 11 yeast strains were screened for γ-dodecelactone

237

production from 10-hydroxy-12(Z)-octadecenoic acid. Seven out of the 11 yeast strains

238

exhibited γ-dodecelactone-producing activity, and the efficiency of production was in

239

following order: with the order C. boidinii > C. curvatus > C. palmioleophila > S.

240

bombicola > Y. lipolytica > S. odorus > S. cerevisiae, however, C. tropicalis, C.

241

matritensis, S. pombe, and W. lipoper did not produce γ-dodecelactone (Figure 3). The

242

activity of C. boidinii, which was approximately 10-fold higher than that of the known 11


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γ-dodecelactone-producing stain S. odorus, was the highest among the 11 yeast strains.

244

Thus, C. boidinii was selected as an efficient γ-dodecelactone-producing yeast and was

245

used for γ-dodecelactone production. C. boidinii has been known to produce the

246

saturated lactones such as γ-dodecalactone and δ-dodecalactone.27,28 However, the

247

production of the monounsaturated lactone γ-dodecelactone by C. boidinii has not been

248

reported yet.

249 250

Induction of C. boidinii cells for the increased production of γ-

251

dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid. C. boidinii was

252

cultivated in the induction medium supplemented with 7 g/L inducer, and then the

253

induced whole cells were used in the reactions for γ-dodecelactone production from 10-

254

hydroxy-12(Z)-octadecenoic acid for 3 h. The inducers tested included lauric acid,

255

stearic acid, oleic acid, linoleic acid, erucic acid, 10-hydroxydecanoic acid, and 10-

256

hydroxystearic acid. The activity of C. boidinii cells for γ-dodecelactone production

257

followed the order oleic acid > stearic acid > erucic acid > control (with no addition of

258

inducer) > linoleic acid > 10-hydroxystearic acid > 10-hydroxydecanoic acid (Figure

259

4A).

260

The maximum production of γ-lactones by W. lipofer was observed at 5 g/L oleic acid

261

as an inducer.9 Thus, the concentration of oleic acid was varied from 0 g/L to 15 g/L for

262

γ-dodecelactone production using C. boidinii cells. The γ-dodecelactone-producing

263

activity was the highest when oleic acid was used as an inducer at a concentration of 7

264

g/L (Figure 4B). γ-Dodecelactone production by the cells induced with 7 g/L oleic acid

265

was 2.2-fold higher than that by the non-induced cells. The concentration of glucose as

266

an additional carbon source for the maximum production of γ-lactones by W. lipofer was 12



267

1 g/L,9 and the growth medium of C. boidinii, which was yeast malt (YM) broth,

268

contained 10 g/L glucose. Thus, glucose at various concentrations from 1 g/L to 15 g/L

269

was added to the induction medium containing 7 g/L oleic acid for increasing the

270

activity for γ-dodecelactone production. The optimal concentration of glucose for γ-

271

dodecelactone production was 5 g/L (Figure 4C). To decide the induction time, C.

272

boidinii cells were cultivated in the induction medium for 24 h. A sample was

273

withdrawn at regular time points and γ-dodecelactone-producing activity of C. boidinii

274

was determined. The maximum γ-dodecelactone-producing activity was observed at 15

275

h of induction time (Figure 4D). Therefore, C. boidinii cells grown on the induction

276

medium containing 7 g/L oleic acid and 5 g/L glucose for 15 h of induction time were

277

used for γ-dodecelactone production. Under these induction conditions, γ-dodecelactone

278

production by the induced cells was 14-fold higher than that by the non-induced cells.

279

Fatty acids have been used as inducers of the β-oxidation pathway.29-31 Among the

280

fatty acids, oleic acid has been determined to be the most effective inducer9,32,33 of the

281

enzymes related to the β-oxidation pathway of yeast.34,35 High glucose concentrations

282

repress the enzymes related to the β-oxidation pathway,36,37 whereas low glucose

283

concentrations stimulate the β-oxidation pathway.9 When W. lipoper cells were induced,

284

the induction time (12 h) in the medium containing oleic acid and glucose for the

285

increased activity of β-oxidation was 4 h of progress time after glucose exhaustion (8

286

h).9 In the induction of C. boidinii cells, glucose was exhausted at 12 h, and induction

287

time for the increased activity of β-oxidation (15 h) was 3 h of progress time after

288

glucose exhaustion. The activity of C. boidinii cells for γ-dodecelactone production

289

appeared to be maximal at 3−4 h of progress time after glucose exhaustion.

290 13


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291

Determination of the reaction conditions for the increased production of

292

γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid by induced C.

293

boidinii cells. The reaction conditions for saturated γ-lactone production have been

294

investigated.9,14,38,39 However, to the best of our knowledge, the reaction conditions for

295

unsaturated γ-lactone production have been not investigated. Therefore, the pH,

296

temperature, and the concentrations of cells and substrate for γ-dodecelactone

297

production from 10-hydroxy-12(Z)-octadecenoic acid were investigated using induced

298

whole C. boidinii cells. The maximal activity for γ-dodecelactone production from 10-

299

hydroxy-12(Z)-octadecenoic acid was observed at pH 5.5 and 25 °C (Figure 5A and B).

300

γ-Lactone production was performed at pH 6.5 and 35 °C by W. lipofer40; and at pH 7.0

301

and 25 °C by baker’s yeast.41

302

The concentrations of whole W. lipofer cells for the maximum production of γ-

303

dodecalactone14 and γ-lactones9 were 30 g/L and 20 g/L, respectively. To determine the

304

concentration of whole C. boidinii cells for the maximal production of γ-dodecelactone,

305

the concentration of the induced cells was varied from 5 to 40 g/L using 5 g/L 10-

306

hydroxy-12(Z)-octadecenoic acid as a substrate for 3 h (Figure 6A). Below 30 g/L cells,

307

10-hydroxy-12(Z)-octadecenoic acid production increased with increasing the cell

308

concentration, however, above 30 g/L, it reached a plateau. As the cell concentration

309

was higher than 30 g/L, the high viscosity of the reaction solution interfered with mass

310

transfer in the aqueous phase, resulting in the conversion rate decreased. These results

311

indicated that the cell concentration for the maximal production of γ-dodecelactone was

312

30 g/L.

313 314

The concentrations of saturated hydroxy fatty acids, including 10-hydroxystearic acid, 12-hydroxystearic acid, and 12-hydroxydodecanoic acid, for the maximum production 14



315

of saturated lactones, including γ-dodecalactone, γ-decalactone, and γ-butyrolactone,

316

respectively, were 60 g/L.9,14 The antimicrobial activities of monounsaturated hydroxy

317

fatty acids were significantly higher than those of saturated hydroxy fatty acids.42 Thus,

318

the production of the monounsaturated lactone γ-dodecelactone was investigated with

319

the range of low concentrations for 10-hydroxy-12(Z)-octadecenoic acid as a substrate

320

from 2.5 to 15 g/L using 30 g/L cells for 3 h (Figure 6B). At concentrations below 12

321

g/L 10-hydroxy-12(Z)-octadecenoic acid, γ-dodecelactone production increased with

322

increasing the concentration of 10-hydroxy-12(Z)-octadecenoic acid. However, the

323

conversion yield decreased as the concentration of substrate increased. This may be due

324

to antimicrobial activity of 10-hydroxy-12(Z)-octadecenoic acid to cells.42 Therefore, a

325

substrate concentration of 5 g/L 10-hydroxy-12(Z)-octadecenoic acid for γ-

326

dodecelactone production was selected as a suitable concentration to improve the

327

performance in terms of both conversion yield and product concentration.

328 329

Production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic

330

acid by induced C. boidinii cells. The reaction conditions for the increased

331

production of γ-dodecelactone by whole C. boidinii cells were pH 5.5 and 25 °C with 30

332

g/L cells and 5 g/L 10-hydroxy-12(Z)-octadecenoic acid. Under these conditions,

333

induced C. boidinii cells produced 2.1 g/L (10.7 mM) γ-dodecelactone from 5 g/L (16.7

334

mM) 10-hydroxy-12(Z)-octadecenoic acid after 6 h, with a molar conversion yield of 64%

335

(corresponding to 42%, w/w) and a volumetric productivity of 350 mg/L/h (Figure 7A).

336

After 8 h, the production of γ-dodecelactone was observed to decrease, because γ-

337

dodecelactone was degraded by the cells.43 Yeast cells consume γ-lactones as the carbon

338

source by extensive β-oxidation degradation,43 and γ-lactones are thus converted to 15


Page 16 of 47

Page 17 of 47

339


viable biomass for longer cell maintenance.44,45

340 341

Production of γ-dodecelactone from lipase-treated safflower oil by two-

342

step whole-cell biotransformation. γ-Dodecelactone production from safflower oil

343

as a substrate via 10-hydroxy-12(Z)-octadecenoic acid intermediate was performed by

344

an enzymatic hydrolysis reaction and a two-step whole-cell biotransformation process.

345

The conversion yield and productivity at each step during the conversion reactions are

346

presented in Table 1. Natural safflower oil was hydrolyzed by lipase from C. rugosa

347

because S. nitritireducens could not perform the hydroxylation of safflower oil. The

348

reactions for the oil hydrolysis were performed using 7.5 g/L safflower oil and 1 g/L

349

lipase at pH 7.5, 35 °C, and 200 rpm for 40 min (Figure 7B). At 30 min, safflower oil

350

hydrolyzate contained 5.3 g/L linoleic acid, 0.93 g/L oleic acid, and 0.37 g/L palmitic

351

acid. After 30 min, the contents of these fatty acids decreased with increasing the

352

reaction time because lipase could catalyze the reverse reaction.46 Therefore, the

353

reactions were terminated at 30 min.

354

The first cell-biotransformation step for γ-dodecelactone production from lipase-

355

treated safflower oil hydrolyzate was the conversion of unsaturated fatty acids into

356

hydroxy fatty acids by hydrating the double bond in the unsaturated fatty acids from

357

water.47 The maximum production of hydroxy fatty acid by whole S. nitritireducens

358

cells were observed at pH 7.5, 30 °C, and 20 g/L cells, under anaerobic conditions.22

359

Under these conditions, S. nitritireducens cells converted 5.3 g/L linoleic acid and 0.93

360

g/L oleic acid in the hydrolyzate obtained from 7.5 g/L safflower oil into 5 g/L 10-

361

hydroxy-12(Z)-octadecenoic acid and 0.85 g/L 10-hydroxystearic acid after 90 min,

362

respectively (Figure 7C), with molar conversion yields of 89 and 86%; and volumetric 16



363

productivities of 3333 and 567 mg/L/h, respectively (Table 1).

364

The second cell-biotransformation step was the conversion of hydroxy fatty acids

365

into γ-lactones by the induced whole C. boidinii cells. Under the conditions of pH 5.5

366

and 25 °C with 30 g/L cells and 5 g/L 10- hydroxy-12(Z)-octadecenoic acid, the induced

367

C. boidinii cells produced 1.88 g/L γ-dodecelactone and 0.54 g/L γ-dodecalactone from

368

5 g/L 10-hydroxy-12(Z)-octadecenoic acid and 0.85 g/L 10-hydroxystearic acid after 6 h,

369

respectively (Figure 7D), with conversion yields of 57 and 96% (mol/mol); and

370

volumetric productivities of 313 and 90 mg/L/h, respectively (Table 1). The cells well

371

consumed oleic acid and linoleic acid, however, they poorly consumed palmitic acid.

372

The total concentration of the two γ-lactones produced from 7.5 g/L safflower oil by an

373

enzymatic hydrolysis reaction and a two-step whole-cell biotransformation process was

374

2.42 g/L for 8 h of the reaction time, with a volumetric productivity of 303 mg/L/h and a

375

conversion yield of 32% (w/w). Several rounds of biotransformation reusing whole S.

376

nitritireducens and C. boidinii cells were performed (Figure S2). The activities of the

377

cells were decreased with increasing the number of reuse, and then showed 11% and 24%

378

after the 4th batch, respectively. Thus, the hydration activity of S. nitritireducens and the

379

β-oxidation activity C. boidinii cells are unstable.

380

The conversion yield and productivity of γ-lactones from oil are summarized in Table

381

2. The reported volumetric productivities of γ-decalactone from castor oil were 4

382

mg/L/h,48 10 mg/L/h,15 and 6 mg/L/h.16 The conversion yield and productivity of γ-

383

dodecelactone using soybean oil were 0.00036% (w/w) and 0.025 mg/L/h, respectively,

384

and those using copra oil were 0.0003% (w/w) and 0.0125 mg/L/h, respectively.20 These

385

yields and productivities were very low. In the present study, 1.88 g/L γ-dodecelactone

386

was produced from 7.5 g/L safflower oil for 8 h of the reaction time, with a conversion 17


Page 18 of 47

Page 19 of 47


387

yield of 25% (w/w), a volumetric productivity of 235 mg/L/h. The time to prepare

388

whole S. nitritireducens and C. boidinii cells was 21 h and 15 h, respectively. Thus, the

389

total time to produce γ-dodecelactone from safflower oil, including the preparation time

390

of whole cells, was 44 h. The volumetric productivity calculated from the total time was

391

43 mg/L/h. These conversion yield and productivity of γ-dodecelactone from oil were

392

69400- and 4.3-fold higher than the previously reported highest conversion yield and

393

productivity, respectively. These results indicate that the two-step whole cell

394

biotransformation process via hydroxy fatty acid intermediate is an efficient method for

395

increasing γ-lactone production from oil. A process developed in this study for the

396

production of γ-dodecelactone from safflower oil contained an enzymatic reaction plus

397

two-step biotransformation. The first step was the hydrolysis of safflower oil, the

398

second step was the hydration of fatty acid, and the third step was the latonization of

399

hydroxy fatty acid. In the second step, the reaction was performed using oil hydrolyzate

400

obtained from the first step by adding S. nitritreducens cells without additional

401

extraction or purification. In the third step, the reaction was performed using the

402

extracted solution after the reaction solution obtained from the first step by adding C.

403

boidinii cells with extraction to adjust the concentration of hydroxy fatty acid and

404

without purification. In the industrial and large-scale production, this extraction can be

405

omitted. The process containing an enzymatic reaction plus two-step biotransformation

406

can be performed without extraction and purification by only adding enzyme and cells.

407

Therefore, the process is not too complex for the industrial and large-scale production.

408

In conclusion, the production of the flavor γ-dodecelactone from safflower oil was

409

demonstrated via an enzymatic hydrolysis reaction and a two-step whole-cell

410

biotransformation process. Lipase-treated safflower oil hydrolyzate containing linoleic 18



was

converted

into

10-hydroxy-12(Z)-octadecenoic

Page 20 of 47

acid

by

whole

S.

411

acid

412

nitritireducens cells and was subsequently converted to γ-dodecelactone by induced

413

whole C. boidinii cells. To the best of our knowledge, our bioprocess exhibits the

414

highest productivity and yield of γ-lactone produced from natural oil reported to date.

415

Thus, the two-step whole cell biotransformation process via the intermediate hydroxy

416

fatty acid may be useful in lactone production from oils.

417 418

AUTHOR INFORMATION

419 420

Corresponding Author

421

*Phone: (822) 454-3118. Fax: (822) 444-5518. E-mail: [email protected]

422 423

Funding source

424 425

This study was supported by a grant from the Bio-industry Technology Development

426

Program, Ministry for Ministry for Agriculture, Food and Rural Affairs (No. 112002-3),

427

Republic of Korea.

19


Page 21 of 47

428


References

429 430

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431

lactones and peroxisomal b-oxidation in yeasts. Crit. Rev. Biotechnol. 1996, 16, 301-

432

329.

433

(2) Okamoto, K.; Chimori, M.; Iwanaga, F.; Hattori, T.; Yanase, H., Production of g-

434

lactones by the brown-rot basidiomycete Piptoporus soloniensis. J. Biosci. Bioeng.

435

2002, 94, 182-185.

436 437

(3) Longo, M. A.; Sanroman, M. A., Production of food aroma compounds: microbial and enzymatic methodologies. Food Technol. Biotechnol. 2006, 44, 335-353.

438

(4) Farbood, M. I.; Morris, J. A.; Mclean, L. B. Fermentation process for preparing 10-

439

hydroxy-C18-carboxylic acid and g-dodecalactone derivatives. EP Patent, 0578388,

440

1998.

441 442

(5) Guichard, E.; Mosandl, A.; Hollnagel, A.; Latrasse, A.; Henry, R., Chiral g-lactones from Fusarium poae. Z. Lebensm. Unters. Forsch. 1991, 193, 26-31.

443

(6) Mariaca, R. G.; Imhof, M. I.; Bosset, J. O., Occurrence of volatile chiral

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compounds in dairy products, especially cheese - A review. Eur. Food Res. Technol.

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(7) Massolo, A.; Dani, F. R.; Bella, N., Sexual and individual cues in the peri-anal

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gland secretum of crested porcupines (Hystrix cristata). Mamm. bio. 2009, 74, 488-496.

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Yashiro, A.; Yokoi, A. Process for producing a bran pickless flavoring solution. US

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Patent, 7037672, 2002.

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452

by whole Waltomyces lipofer cells induced with oleic acid. Appl. Microbiol. Biotechnol.

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(10) Feron, G.; Dufosse, L.; Pierard, E.; Bonnarme, P.; Quere, J. L.; Spinnler, H. E.,

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Production, identification, and toxicity of g-decalactone and 4-hydroxydecanoic acid

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from Sporidiobolus sp. Appl. Environ. Microbiol. 1996, 62, 2826-2831.

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(11) Wache, Y.; Aguedo, M.; Nicaud, J. M.; Belin, J. M., Catabolism of hydroxyacids

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and biotechnological production of lactones by Yarrowia lipolytica. Appl. Microbiol.

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Biotechnol. 2003, 61, 393-404.

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(12) Wache, Y.; Aguedo, M.; Choquet, A.; Gatfield, I. L.; Nicaud, J. M.; Belin, J. M.,

461

Role of b-oxidation enzymes in g-decalactone production by the yeast Yarrowia

462

lipolytica. Appl. Environ. Microbiol. 2001, 67, 5700-5704.

463

(13) Aguedo, M.; Gomes, N.; Garcia, E. E.; Wache, Y.; Mota, M.; Teixeira, J. A.; Belo,

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I., Decalactone production by Yarrowia lipolytica under increased O2 transfer rates.

465

Biotechnol. Lett. 2005, 27, 1617-1621.

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(14) An, J. U.; Joo, Y. C.; Oh, D. K., New biotransformation process for production of

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the fragrant compound g-dodecalactone from 10-hydroxystearate by permeabilized

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Waltomyces lipofer cells. Appl. Environ. Microbiol. 2013, 79, 2636-2641.

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(15) Gomes, N.; Braga, A.; Teixeira, J. A.; Belo, I., Impact of lipase-mediated

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hydrolysis of castor oil on g-decalactone production by Yarrowia lipolytica. J. Am. Oil

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(16) Neto, R. S.; Pastore, G. M.; Macedo, G. A., Biocatalysis and biotransformation producing g-decalactone. J. Food Sci. 2004, 69, C677-C680. (17) Lee, S. L.; Chou, C. C., Effects of various fatty acid components of castor oil on

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Bioeng. 1996, 82, 42-45.

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(18) Piazza, G. J.; Farrell Jr, H. M., Generation of ricinoleic acid from castor oil using

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the lipase from ground oat (Avena sativa L.) seeds as a catalyst. Biotechnol. Lett. 1991,

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13, 179-184.

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of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia

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coli containing oleate hydratase from Stenotrophomonas maltophilia. J. Biotechnol.

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2012, 158, 17-23.

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(22) Yu, I. S.; Kim, H. J.; Oh, D. K., Conversion of linoleic acid into 10-hydroxy-

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12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens.

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Biotechnol. Prog. 2008, 24, 182-186.

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(23) Yu, I. S.; Yeom, S. J.; Kim, H. J.; Lee, J. K.; Kim, Y. H.; Oh, D. K., Substrate

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specificity of Stenotrophomonas nitritireducens in the hydroxylation of unsaturated

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fatty acid. Appl. Microbiol. Biotechnol. 2008, 78, 157-163.

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(24) Gordillo, A.; Obradors, N.; Montesinos, J. L.; Valero, F.; Lafuente, F. J.; Salo, C.,

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Stability studies and effect of the initial oleic acid concentration on lipase production by

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Candida rugosa. Appl. Microbiol. Biotechnol. 1995, 43, 38-41.

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(25) Lee, S. L.; Chou, C. C., Growth and production of g-decalactone and cis-6-

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dodecen-4-olide by Sporobolomyces odorus in the presence of fatty acids and oils. J. 22



499 500 501 502 503 504 505

Ferment. Bioeng. 1994, 78, 114-116. (26) Latrasse, A.; Guichard, E.; Piffaut, C.; Fournier, N.; Dufosse, L., Chirality of the g-lactones formed by Fusarium poae INRA 45. Chirality 1993, 5, 379-384. (27) Boog, A. L. G. M.; Peters, A. L. J.; Roos, R. Process for producing d-lactones from 11-hydroxy fatty acids. US patent 5215901, 1993. (28) Boog, A. L. G. M.; van Grinsven, A. M.; Peters, A. L. J.; Roos, R.; Wieg, A. J. Process for producing g-lactones. US Patent 5789212, 1998.

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(29) Endrizzi, A.; Awade, A. C.; Belin, J. M., Presumptive involvement of methyl

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ricinoleate b-oxidation in the production of g-decalactone by the yeast Pichia

508

guilliermondii. FEMS Microbiol. Lett. 1993, 114, 153-159.

509

(30) Pagot, Y.; Le Clainche, A.; Nicaud, J. M.; Wache, Y.; Belin, J. M., Peroxisomal b-

510

oxidation activities and g-decalactone production by the yeast Yarrowia lipolytica. Appl.

511

Microbiol. Biotechnol. 1998, 49, 295-300.

512 513

(31) Veenhuis, M.; Mateblowski, M.; Kunau, W. H.; Harder, W., Proliferation of microbodies in Saccharomyces cerevisiae. Yeast 1987, 3, 77-84.

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(32) Dommes, P.; Dommes, V.; Kunau, W. H., b-Oxidation in Candida tropicalis

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partial purification and biological function of an inducible 2,4-dienoyl coenzyme a

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reductase. J. Biol. Chem. 1983, 258, 10846-10852.

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(33) Valenciano, S.; Lucas, J. R. D.; Pedregosa, A.; Monistrol, I. F.; Laborda, F.,

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Induction of b-oxidation enzymes and microbody proliferation in Aspergillus nidulans.

519

Arch. Microbiol. 1996, 166, 336-341.

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(34) Einerhand, A. W.; Kos, W.; Smart, W. C.; Kal, A. J.; Tabak, H. F.; Cooper, T. G.,

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The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A

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thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A binding

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sites that participate in its regulation by glucose repression. Mol. Cell. Biol. 1995, 15,

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3405-3414.

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(35) Dmochowska, A.; Dignard, D.; Maleszka, R.; Thomas, D. Y., Structure and

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transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding

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acylcoenzyme A oxidase. Gene 1990, 88, 247-252.

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(36) Wilson, W. A.; Hawley, S. A.; Hardie, D. G., Glucose repression/derepression in

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budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing

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conditions, and this correlates with a high AMP:ATP ratio. Curr. Biol. 1996, 6, 1426-

531

1434.

532

(37) Stanway, C. A.; Gibbs, J. M.; Berardi, E., Expression of the FOX1 gene of

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Saccharomyces cerevisiae is regulated by carbon source, but not by the known glucose

534

repression genes. Curr. Genet. 1995, 27, 404-408.

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(38) Gomes, N.; Aguedo, M.; Teixeira, J. A.; Belo, I., Oxygen mass transfer in a

536

biphasic medium: Influence on the biotransformation of methyl ricinoleate into g-

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decalactone by the yeast Yarrowia lipolytica. Biochem. Eng. J. 2007, 35, 380-386.

538 539

(39) Gomes, N.; Teixeira, J. A.; Belo, I., Empirical modelling as an experimental approach to optimize lactone production. Catal. Sci. Technol. 2010, 1, 86-92.

540

(40) An, J. U.; Joo, Y. C.; Oh, D. K., New biotransformation process for production of

541

the fragrant compound g-dodecalactone from 10-hydroxystearate by permeabilized

542

Waltomyces lipofer cells. Appl. Environ. Microbiol. 2013, 79, 2636-2641.

543

(41) Gocho, S.; Tabogami, N.; Inagaki, M.; Kawabata, C.; Komai, T.,

544

Biotransformation of oleic acid to optically active g-dodecalactone. Biosci. Biotechnol.

545

Biochem. 1995, 59, 1571-1572. 24



546

(42) Black, B. A.; Zannini, E.; Curtis, J. M.; Ganzle, M. G., Antifungal hydroxy fatty

547

acids produced during sourdough fermentation: microbial and enzymatic pathways, and

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antifungal activity in bread. Appl. Environ. Microbiol. 2013, 79, 1866-1873.

549 550

(43) Welsh, F. W.; Murray, W. D.; Williams, R. E., Microbiological and enzymatic production of flavor and fragrance chemicals. Crit. Rev. Biotechnol. 1989, 9, 105-169.

551

(44) Nago, H.; Matsumoto, M.; Nakai, S., 2-Deceno-d-lactone-producing fungi,

552

strains of Fusarium solani, isolated by using a medium containing decano-d-lactone as

553

the sole carbon source. Biosci. Biotech. Biochem. 1993, 57, 2107-2110.

554 555 556 557

(45) Spinnler, H. E.; Dufosse, L.; Souchon, I.; Latrasse, A.; Piffaut Juffard, C.; Voilley, A.; Delest, P. Production of g-decalactone by bioconversion. FR Patent, 2705971, 1994. (46) Rajendran, A.; Palanisamy, A.; Thangavelu, V., Lipase catalyzed ester synthesis for food processing industries. Braz. Arch. Biol. Technol. 2009, 52, 207-219.

558

(47) Koritala, S.; Hosie, L.; Hou, C. T.; Hesseltine, C. W.; Bagby, M. O., Microbial

559

conversion of oleic acid to 10-hydroxystearic acid. Appl. Microbiol. Biotechnol. 1989,

560

32, 299-304.

561 562

(48) Farbood, M. I.; Willis, B. J. Production of g-decalactone. WO Patent, 1983001072, 1983.

563 564

25


Page 26 of 47

Page 27 of 47


Table 1 Biotransformation of Safflower Oil to γ-Lactones

step

hydrolysis (lipase from Candida

substrate (g/L) [mM]

product (g/L) [mM]

safflower oil (7.50)

rugosa)

(Stenotrophomonas nitritireducens) lactonization (Candida boidinii)

step yield

total yield

(g/g) [mM/mM]

(%, g/g)

linoleic acid (5.30)

10.6 ± 0.09

0.71

0.71

oleic acid (0.93)

1.86 ± 0.89

0.12

0.12

0.740 ± 0.005

0.05

0.05

palmitic acid (0.37) hydration

productivity (g/L/h)

linoleic acid (5.30) [18.90]

10-hydroxy-12-(Z)-octadecenoic acid (5.00) [16.78]

4.27 ± 0.09

0.94 [0.89]

0.67

oleic acid (0.93) [3.20]

10-hydroxystearic acid (0.85) [2.82]

0.73 ± 0.03

0.91 [0.88]

0.12

10-hydroxy-12-(Z)-octadecenoic acid (5.00) [16.78]

γ-dodecelactone (1.88) [9.60]

0.310 ± 0.004

0.38 [0.57]

0.25

10-hydroxystearic acid (0.85) [2.82]

γ-dodecalactone (0.54) [2.7]

0.090 ± 0.002

0.64 [0.96]

0.08

26



Page 28 of 47

Table 2 Conversion Yield and Productivity of γ-Lactones from Oil

strain

method

substrate (g/L)

product (mg/L)

conversion yield (%)

productivity (mg/L/h)

reference

Yarrowia lipolytica

fermentation

castor oil (10)

γ-decalactone (610)

0.061

4

48

Yarrowia lipolytica

fermentation

castor oil (30)


0.0613

10

15

fermentation

castor oil (50)


0.012

6

16

fermentation

soybean oil (5)

γ-docecelactone (1.84)

0.00036

0.025

20

copra oil (5)

γ-docecelactone (1.5)

0.0003

0.0125

safflower oil (7.5)

γ-docecelactone (1880)

25

235 (43a)

Geotrichum fragrans

Penicillium roqueforti

Candida boidinii

enzymatic reaction and two-step whole cell biotransformation

a

Productivity included the preparation time of whole cells.

27


this study

Page 29 of 47


Figure legends

Figure 1. Production of γ-lactones from safflower oil by lipase and whole cells of S. nitritireducens and C. boidinii via fatty acids and hydroxy fatty acids.

Figure 2. Gas chromatograms of 10-hydroxy-12(Z)-octadecenoic acid, γ-dodecelactone, and γ-dodecalactone. The reaction of whole C. boidinii cells was performed in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 5 g/L 10hydroxy-12(Z)-octadecenoic acid, 30 g/L cells, and 0.05 % (w/v) Tween 80 at 25 °C with agitation of 200 rpm. (1) Gas chromatogram of 10-hydroxy-12(Z)-octadecenoic acid standard. The retention time of 10-hydroxy-12(Z)-octadecenoic acid was 21.1 min. (2) Gas chromatogram of γ-dodecelactone standard. The retention time of γdodecelactone was 6.1 min. (3) Gas chromatogram of γ-dodecalactone standard. The retention time of γ-dodecalactone was 6.7 min. (4) Gas chromatogram of the reaction solution at 3 h. (5) Gas chromatogram of the reaction solution at 6 h.

Figure 3. γ-Dodecelactone production from 10-hydroxy-12(Z)-octadecenoic acid by whole cells of the yeast strains. The reactions were performed in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 5 g/L cells, 0.5 g/L 10-hydroxy-12(Z)-octadecenoic acid, and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 3 h. The data represent the means of 3 separate experiments, and error bars represent the standard deviation.

Figure 4. Induction of C. boidinii cells for increasing γ-dodecelactone production from 28



10-hydroxy-12(Z)-octadecenoic acid. After induction, the reactions were performed in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 5 g/L cells, 0.5 g/L 10-hydroxy-12(Z)-octadecenoic acid, and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 3 h. (A) Effect of inducer type. C. boidinii cells were grown on the induction medium containing 7 g/L inducer. The control reaction was γdodecelactone production by cells that were cultivated in the growth medium without the inducer. (B) Effect of oleic acid concentration. C. boidinii cells were cultivated in the induction medium by varying the concentration of oleic acid from 0 to 15 g/L. (C) Effect of glucose concentration. C. boidinii cells were cultivated in the induction medium by varying the concentration of glucose from 1 to 15 g/L. (D) Effect of induction time. C. boidinii cells were cultivated in the induction medium containing 7 g/L oleic acid and 5 g/L glucose for 24 h. Samples were withdrawn within 24 h at several intervals. The data represent the means of 3 separate experiments, and error bars represent the standard deviation.

Figure 5. Effects of pH and temperature on the production of γ-dodecelactone from 10hydroxy-12(Z)-octadecenoic acid by whole C. boidinii cells. The data represent the means of 3 separate experiments, and error bars represent the standard deviation. (A) Effect of pH. The reactions were performed by varying the pH from 4.0 to 6.5 in 50 mM citrate/phosphate buffer containing 3.4 g/L yeast nitrogen base, 5 g/L cells, 0.5 g/L 10hydroxy-12(Z)-octadecenoic acid, and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 3 h. (B) Effect of temperature. The reactions were performed by varying the temperature from 15 °C to 35 °C in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 5 g/L cells, 0.5 g/L 10-hydroxy-12(Z)-octadecenoic acid, 29


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and 0.05 % (w/v) Tween 80 with agitation at 200 rpm for 3 h.

Figure 6.

Effects of the concentrations of cells and substrate on the production of γ-

dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid. The data represent the means of 3 separate experiments, and error bars represent the standard deviation. (A) Effect of the concentration of cells. The reactions were performed by varying the cell concentration from 5 g/L to 40 g/L in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 5 g/L 10-hydroxy-12(Z)-octadecenoic acid, and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 3 h. (B) Effect of substrate concentration. The reactions were performed by varying the concentration of 10-hydroxy-12(Z)-octadecenoic acid from 2.5 g/L to 15 g/L in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 30 g/L cells, and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 3 h. Production (◆) and conversion yield (□) of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid

Figure 7. Time-course reactions for the production of fatty acids, hydroxy fatty acids, and γ-lactones by lipase, S. nitritireducens, and C. boidinii, respectively. The data represent the means of 3 separate experiments, and error bars represent the standard deviation. (A) Production of γ-dodecelactone from 10-hydroxy-12(Z)-octadecenoic acid by whole C. boidinii cells. The reactions for the production of γ-dodecelactone (◆) were performed in 50 mM citrate/phosphate buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 30 g/L cells, 5 g/L 10-hydroxy-12(Z)-octadecenoic acid (○), and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 8 h. (B) Production of fatty acids

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from safflower oil by lipase from C. rugosa. Fatty acids were linoleic acid (●), oleic acid (▲), and palmitic acid (◇). The reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing 1 g/L lipase, 7.5 g/L safflower oil, and 0.02% (w/v) Tween 80 at 37 °C with agitation at 200 rpm for 40 min. (C) Production of hydroxy fatty acids from fatty acids by whole S. nitritireducens cells. Linoleic acid (●) and oleic acid (▲) were converted to 10-hydroxy-12(Z)-octadecenoic acid (○) and 10-hydroxystearic acid (△), respectively, by whole S. nitritireducens cells. However, palmitic acid (◇) was not converted. The reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing safflower oil hydrolyzate, 20 g/L cells, and 0.02% (w/v) Tween 80 at 30 °C with agitation at 200 rpm for 90 min under anaerobic conditions. (D) Production of γlactones from hydroxy fatty acids by whole C. boidinii cells. The cells well consumed oleic acid (▲), linoleic acid (●), however, they poorly consumed palmitic acid (◇). The reactions for the production of γ-dodecelactone (◆) and γ-dodecalactone (■) were performed in 50 mM Tris-HCl buffer (pH 5.5) containing 3.4 g/L yeast nitrogen base, 30 g/L cells, 5 g/L 10-hydroxy-12(Z)-octadecenoic acid (○), 0.85 g/L 10-hydroxystearic acid (△), and 0.05 % (w/v) Tween 80 at 25 °C with agitation at 200 rpm for 7 h.

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Figure 1

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Figure 2

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Figure 3

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A

Figure 4-continued

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B

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Figure 4-continued

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Figure 4

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Figure 5-continued

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Figure 5

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Figure 6-continued

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Figure 6

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0 16

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g-Lactones (g/L)

Hydroxy fatty acids, fatty acids (g/L)

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Î³-Dodecelactone Production from Safflower Oil via 10-Hydroxy-12(Z

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