Biotechnological Production of Methyl-Branched Aldehydes - Journal

Dec 1, 2016 - A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after...
2 downloads 14 Views 471KB Size
Subscriber access provided by Georgia State University Library

Article

Biotechnological production of methyl branched fatty aldehydes Marco Alexander Fraatz, Michael Goldmann, Thorsten Geissler, Egon Gross, Michael Backes, Jens-Michael Hilmer, Jakob P. Ley, Johanna Rost, Alexander Francke, and Holger Zorn J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04793 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 1, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

Journal of Agricultural and Food Chemistry

1

Biotechnological Production of Methyl-Branched Aldehydes

2 3

Marco Alexander Fraatz,† Michael Goldmann,† Torsten Geissler,‡ Egon Gross,‡

4

Michael Backes,‡ Jens-Michael Hilmer,‡ Jakob Ley,‡ Johanna Rost,† Alexander

5

Francke,§ and Holger Zorn*,†

6 7



8

Biotechnology, Heinrich-Buff-Ring 17, 35392 Giessen, Germany

9



Symrise AG, Muehlenfeldstrasse 1, 37603 Holzminden, Germany

10

§

Justus Liebig University Giessen, Institute of Organic Chemistry, Heinrich-Buff-Ring

11

17, 35392 Giessen, Germany

Justus Liebig University Giessen, Institute of Food Chemistry and Food

12

Corresponding author (Tel: +49 641 99-34900; Fax: +49 641 99-34909; E-mail:

13

*

14

[email protected])

15

1 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

16

ABSTRACT: A number of methyl-branched aldehydes impart interesting flavor

17

impressions, and especially 12-methyltridecanal is a highly sought after flavoring

18

compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For

19

the biotechnological production of 12-methyltridecanal, the literature was screened

20

for fungi forming iso-fatty acids. Suitable organisms were identified and successfully

21

grown in submerged cultures. The culture medium was optimized to increase the

22

yields of branched fatty acids. A recombinant carboxylic acid reductase was used to

23

reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell

24

catalysis was compared to that of the purified enzyme preparation. After lipase

25

catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were

26

converted to the corresponding aldehydes, including 12-methyltridecanal and 12-

27

methyltetradecanal.

28 29

KEYWORDS: biotransformation, carboxylic acid reductase, Conidiobolus

30

heterosporus, flavor, 12-methyltridecanal, 12-methyltridecanoic acid

31

2 Environment ACS Paragon Plus

Page 2 of 27

Page 3 of 27

Journal of Agricultural and Food Chemistry

32

INTRODUCTION

33

Methyl-branched fatty acids (MBFAs) are suitable precursors for the biotechnological

34

production of their corresponding aldehydes. Naturally occurring MBFAs are typically

35

saturated and mono-methyl-branched. They are common, but usually minor

36

constituents of lipids of bacteria and animals, and scarce in higher plants. Their

37

major physiological role may be to increase the fluidity of lipids as an alternative to

38

unsaturated fatty acids (FAs).1 MBFAs with a methyl branch at the penultimate

39

position (isopropyl group) are called iso- and those with a methyl branch at the

40

antepenultimate carbon (sec-butyl group) anteiso-fatty acids. For meat and milk of

41

ruminants it is assumed that rumen bacteria are the major source of iso- and

42

anteiso-FAs.2

43

While extensive research has been published on the occurrence and de novo

44

synthesis of branched-chain fatty acids (BCFAs) in bacteria,1 only a few studies

45

report on fungal sources. Most of them date back to the late 1960s and early/mid-

46

1970s and cover Conidiobolus species.3-5 Up to 73% BCFAs of total FAs have been

47

reported for these Zygomycota.5 In general, the percentage of BCFAs in

48

Basidiomycota is much lower.6 For Armillaria mellea, ~10% of BCFAs have been

49

reported.7

50

Some methyl-branched aldehydes impart interesting flavor impressions.12-

51

Methyltridecanal (1) (Figure 1) especially is a highly sought after flavoring compound

52

for savory foods. Its smell is reminiscent of cooked beef and tallow.8 By aroma

53

extract dilution analysis (AEDA), stable isotope dilution analysis (SIDA),

54

determination of the corresponding odor thresholds, and calculation of odor activity

55

values (OAVs), 1 was found to be the most important contributor to the flavor of

56

stewed beef.8 Additionally, it was identified as one of the character impact

3 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

57

compounds of stewed beef juice odor.9 1 is also present in lower concentrations in

58

other animal meat, but primarily in ruminants. For beef, concentrations of 55–150

59

µg/g lipid were quantitated.8 While raw beef does not exhibit the typical smell of 1,

60

the methyl-branched aldehyde is released during cooking and roasting. It is assumed

61

that 1 is synthesized in the ruminants’ stomachs by bacteria, and small amounts are

62

incorporated into the plasmalogens after resorption and transport to the muscular

63

tissues.8 This hypothesis is supported by investigations on the occurrence of 1 in

64

microorganisms and physiological samples isolated from beef, in which the highest

65

amounts of 1 were detected in the bacteria isolated from the rumen of bovine

66

animals (475 µg/g air-dry matter) followed by protozoa (229 µg/g).10 The

67

concentration of 1 in beef is correlated to the animal’s phospholipid content and

68

increases with its age. Up to 810 µg/g phospholipid were reported after SIDA.11

69

Additionally, the formation is effected by the feed. Pasture-fed bulls showed higher

70

concentrations compared to concentrate-fed animals.12 Nevertheless, the

71

concentrations of 1 in natural sources are generally very low. Even its chemical

72

synthesis is comparatively complex and therefore not economic or uses

73

environmental unfriendly chemicals.8,13 Albeit more convenient strategies have been

74

published recently, chemically synthesized 1 may not marketed as natural.14

75

Therefore, to the best of our knowledge, no natural 1 is available as a flavoring

76

substance on the world market, and no biotechnological route towards 1 has been

77

commercialized so far.

78

For the reduction of MBFAs to their corresponding branched aldehydes, enzymes

79

with carboxylic acid reduction ability are required.15 Carboxylic acid reductases

80

(CAR) are oxidoreductases, which catalyze the reduction of aromatic and short-chain

81

carboxylic acids.16 Recently, a CAR from Mycobacterium marinum was described

4 Environment ACS Paragon Plus

Page 4 of 27

Page 5 of 27

Journal of Agricultural and Food Chemistry

82

with the ability to reduce aliphatic fatty acids up to C18.17 Concerning MBFAs, the

83

reduction of 3-methylbutyrate and 3-methylpentanoate has been reported for a CAR

84

from Clostridium thermoaceticum.18 No data on the reduction of longer MBFAs have

85

been published so far.

86 87

MATERIALS AND METHODS

88

Chemicals. Adenosine-5'-triphosphate disodium salt (ATP), agar-agar (Kobe I), D-

89

glucose, n-hexane, L-leucine, 2-mercaptoethanol, methanol, β-nicotinamide adenine

90

dinucleotide phosphate tetrasodium salt (NADPH), potassium hydrogen sulfate,

91

sodium chloride, and sodium sulfate were purchased from Carl Roth GmbH + Co.

92

KG (Karlsruhe, Germany). tert-Butyl methyl ether (TBME), dichloromethane, diethyl

93

ether, lipase from Candida rugosa (2.9 U/mg), magnesium chloride hexahydrate,

94

page ruler unstained protein ladder (10–200 kDa), and yeast extract were bought

95

from Fisher Scientific GmbH (Schwerte, Germany). Lithium aluminum hydride

96

(LiAlH4) (for synthesis) was purchased from Merck KGaA (Darmstadt, Germany).

97

Malt extract, petroleum ether (40–60 °C), and Supelco 37 component FAME mix

98

were purchased from Sigma-Aldrich GmbH (Taufkirchen, Germany). 11-

99

Methyltridecanoic acid, 12-methyltridecanal (1), 12-methyltridecanoic acid (2) (Figure

100

1), 12-methyltetradecanal, 12-methyltetradecanoic acid, 13-methyltetradecanoic

101

acid, 13-methylpentadecanoic acid, and 14-methylpentadecanoic acid were provided

102

by Symrise AG (Holzminden, Germany). Boron trifluoride (20% in methanol) and

103

hydrochloric acid (32%) were obtained from VWR International GmbH (Darmstadt,

104

Germany).

105

Microorganisms. The basidiomycetous fungus Armillaria mellea DSMZ 2941

106

(honey fungus) was obtained from the German Collection of Microorganisms and

5 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

107

Cell Cultures (Brunswick, Germany). The fungi Conidiobolus denaeosporus CBS

108

137.57, Conidiobolus heterosporus CBS 543.63, C. heterosporus CBS 333.74, and

109

C. heterosporus CBS 138.57 (ATCC 12941) were obtained from CBS-KNAW Fungal

110

Biodiversity Centre (Utrecht, the Netherlands). Conidiobolus lobatus ATCC 18153

111

was purchased from the American Type Culture Collection (Manassas, VA).

112

Escherichia coli BL21(DE3) was purchased from Merck KGaA (Darmstadt,

113

Germany). The E. coli strain BL21(DE3)pET-PC2, which is able to co-express a

114

carboxylic acid reductase from Mycobacterium marinum and a phosphopantetheinyl

115

transferase (PPT) of Bacillus subtilis was provided by Pauli Kallio, University of

116

Turku, Finland and Patrick R. Jones, Imperial College London, England.

117

Cultivation. Stock cultures and precultures were maintained and grown as described

118

previously.19 Yeast extract (3 g/L) and malt extract (30 g/L) were used for stock

119

cultures and preculture media (YM medium). Growth periods on agar plates were: 5

120

days for C. heterosporus CBS 543.63 and CBS 333.74, 14 days for CBS 138.57, C.

121

denaeosporus, and C. lobatus, and 21 days for A. mellea. The standard cultivation

122

period of the precultures was 3 days, and 100 mL YM medium in 250 mL Erlenmeyer

123

flasks was used. Afterwards, the precultures were homogenized for 30 s at 9800 rpm

124

using an Ultra Turrax homogenizer (IKA, Staufen, Germany). For the main cultures,

125

20 mL of the homogenized suspension was used to inoculate 200 mL YM medium in

126

500 mL Erlenmeyer flasks. The cultivations were carried out in an Ecotron incubation

127

shaker (25 mm shaking diameter, 150 rpm, 24 °C) (Infors GmbH, Einsbach,

128

Germany) for 5 d (if not stated otherwise). Supplementation with L-valine was done

129

by adding 0.2 g/L or 2.0 g/L to the main culture medium prior to autoclaving. For

130

harvesting, the fungal mycelium was separated from the culture broth by vacuum

6 Environment ACS Paragon Plus

Page 6 of 27

Page 7 of 27

Journal of Agricultural and Food Chemistry

131

filtration with a Buchner funnel (110 mm) and DP 595 cellulose filter paper (Albet

132

LabScience, Dassel, Germany).

133

Lipid Extraction. The harvested biomass was treated with 200 mL of 4 M

134

hydrochloric acid with boiling for 30 min. After addition of 100 mL hot water, filtration

135

of the hydrolysis solution and neutralization, the residue was dried in a drying cabinet

136

at 103 °C for 3 h. Subsequently, Soxhlet extraction was performed with 180 mL

137

petroleum ether for 4 h. The solvent was evaporated by means of a rotary

138

evaporator (40 °C, ≤150 mbar), and the lipid extract was dried in the drying cabinet

139

at 103 °C to constant weight. After addition of 2 mL n-hexane, the lipid extract was

140

stored at -20 °C until saponification and esterification.

141

Preparation of fatty acid methyl esters (FAMEs). 150 mg fungal lipid extract was

142

saponified in a derivatization vial with a Teflon septum by adding 4 mL 0.5 M

143

methanolic sodium hydroxide solution and heating to 80 °C for 10 min. After cooling

144

to room temperature, 3.5 mL of boron trifluoride (20% in methanol) was added. The

145

mixture was firmly shaken and incubated for 5 min (80 °C). After cooling, 5 mL n-

146

hexane was added, the mixture was heated for 1 min (80 °C), and 5 mL saturated

147

sodium chloride solution was added. The organic phase was separated and dried

148

over anhydrous sodium sulfate. Reference standards were directly esterified by

149

addition of 1 mg fatty acid to 1 mL boron trifluoride solution. The generated FAMEs

150

were analyzed by means of gas chromatography (GC).

151

Protein expression. Overnight cultures were grown according to Akhtar et al. in Luria-

152

Bertani (LB) medium.17 100 µL of these cultures was used for inoculation of 200 mL

153

Overnight Express Instant TB Medium (Merck KGaA). Cells were incubated for 20 h

154

in an incubation shaker (30 °C, 250 rpm, 25 mm shaking diameter), then separated

7 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

155

by centrifugation (4 °C, 4713 x g) and stored at -20 °C. After thawing on ice, the cell

156

pellets were employed for enzyme purification or biotransformation.

157

Enzyme purification. The His-tagged protein was purified according to the Protino Ni-

158

TED protocol (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) using 2 mg/mL

159

lysozyme.17 The enzyme containing fractions were combined and 2-mercaptoethanol

160

(3%, v/v) was added. 10 mL aliquots were washed seven times with phosphate

161

buffer (50 mM, pH 7.5) using 30 K Macrosep Advance Centrifugal Devices (Pall Life

162

Sciences, Port Washington, NY) by means of centrifugation (4 °C, 4713 x g).

163

Determination of protein concentration. The protein concentration was measured by

164

means of a Roti Nanoquant-Kit K880 (Carl Roth GmbH + Co. KG) according to the

165

manufacturer’s protocol. The purified CAR enzyme solution was diluted with 50 mM

166

phosphate buffer (pH 7.5) to a final protein concentration of 100 µg/mL and used for

167

in vitro bioconversion experiments.

168

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and

169

Coomassie staining. Discontinuous SDS-PAGE was performed according to

170

Laemmli.20 The gels were stained overnight at room temperature with Coomassie

171

brilliant blue G250 solution.21

172

Biotransformation – whole cell catalysis. Twenty-five mg cells were suspended in 1

173

mL phosphate buffer (100 mM, pH 7.5) in a 10 mL reaction vial and 0.5 mM 2 (in n-

174

hexane) and 100 mM D-glucose were added. If not stated otherwise, the vials were

175

incubated for 24 h in an incubation shaker at 30 °C and 250 rpm. Prior to solvent

176

extraction with 1 mL dichloromethane, the pH was adjusted to pH 2 with hydrochloric

177

acid (18%, w/w). As controls, analogous transformations with BL21(DE3) E. coli

178

cultures and an empty plasmid were performed with 0.25 mM 1 and 0.5 mM 2 as

179

substrates. For additional negative controls, 25 mg E. coli BL21(DE3)pET-PC2 cell

8 Environment ACS Paragon Plus

Page 8 of 27

Page 9 of 27

Journal of Agricultural and Food Chemistry

180

pellets were heated for 30 min at 95 °C in a water bath prior to the addition of the

181

substrate and D-glucose.

182

In vitro conversion with purified enzymes. For the conversion of 2, ATP (1 mM),

183

NADPH (1 mM), MgCl2 (10 mM), and 2 (0.5 mM) were added to 0.9 mL CAR-

184

solution (100 µg/mL in 50 mM phosphate buffer, pH 7.5) in a total reaction volume of

185

1011 µL. If not stated otherwise, the reaction mixture was incubated for 24 h at 30 °C

186

and subsequently extracted as described above. Alternatively, 150 mg lipid extract of

187

C. heterosporus CBS 333.74 was suspended in phosphate buffer containing ATP (1

188

mM), NADPH (1 mM), MgCl2 (10 mM), 2 (0.5 mM), 0.9 mL CAR-solution (100 µg/mL

189

in 50 mM phosphate buffer, pH 7.5), and 200 µL lipase solution (Candida rugosa; 12

190

mg/mL, 7 U in phosphate buffer) was added. The final volume of the reaction mixture

191

was 1210 µL. The mixture was incubated for 2 h at 30 °C and subsequently

192

extracted as described above. For negative controls, the CAR solutions were heated

193

for 30 min at 95 °C prior to the reaction. In additional controls, the enzyme solution

194

was substituted by the same volume of 50 mM phosphate buffer.

195

Gas chromatography. GC analyses were performed according to Kleofas et al.22 For

196

compound identification, GC-mass spectrometry (MS) analyses on two columns of

197

different polarity and calculation of the corresponding retention indices (RIs) were

198

performed. For quantitation, response factors were determined by means of a GC

199

equipped with a flame ionization detector (FID). In general, 1 µL sample was injected

200

with a split of 10:1 for GC-MS and 2:1 for GC-FID. The RIs of the methyl esters of

201

the MBFAs were as follows: methyl 11-methyltridecanoate 1977 (HP-Innowax) and

202

1761 (DB-5), methyl 12-methyltridecanoate 1962 and 1754, methyl 12-

203

methyltetradecanoate 2080 and 1865, methyl 13-methyltetradecanoate 2065 and

9 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

204

1857, methyl 13-methylpentadecanoate 2210 and 1969, and methyl 14-

205

methylpentadecanoate 2196 and 1961.

206

Synthesis of 12-methyltridecanol. For the synthesis of 12-methyltridecanol (3)

207

(Figure 1) 2.00 g 2 dissolved in 10 mL diethyl ether was added dropwise to 665 mg

208

of LiAlH4 in 20 mL diethyl ether, and the mixture was stirred overnight. The reaction

209

was stopped by addition of ice-water and the crude product was extracted with 50

210

mL diethyl ether. After drying with sodium sulfate and removal of the solvent by

211

distillation, the crude product was purified by column-chromatography (MTBE:n-

212

hexane 1:3, v/v).

213

1.68 g (90%) of a clear oil was isolated. EI-MS: 69 (100), 55 (100), 43 (90), 41 (88),

214

83 (78), 97 (54), 111 (26), 140 (10), 168 (7), 125 (6); RI: 2118 (HP-Innowax), 1647

215

(DB-5)

216

Statistics. Analyses were performed at least in duplicate, and results are reported as

217

means ± half range or standard deviations, respectively.

218 219

RESULTS AND DISCUSSION

220

Production of methyl-branched fatty acids. After screening of the literature for

221

MBFAs-producing fungi, six different fungi were chosen for further investigations.

222

Five fungi were Conidiobolus species, three of them C. heterosporus strains from

223

different origins. All of them grew well in submerged cultures, and yeast extract / malt

224

extract medium was found to be an appropriate standard medium. After harvesting

225

and lipid extraction, the fatty acid profiles of the fungi were analyzed. BCFAs were

226

detected in all lipid samples, but in varying quantities (Table 1). The C. heterosporus

227

strains showed the highest share of iso- and anteiso-FAs. More than 55% of MBFAs

228

were detected in the lipid extracts of C. heterosporus CBS 543.63. In the literature,

10 Environment ACS Paragon Plus

Page 10 of 27

Page 11 of 27

Journal of Agricultural and Food Chemistry

229

~40% 2 have been reported for C. heterosporus ATCC 12941 (CBS 138.57) and for

230

Conidiobolus lobatus.4,5 In our studies, up to 40% 2 was observed with C.

231

heterosporus CBS 543.63 and CBS 333.74, while C. heterosporus CBS 138.57 and

232

C. lobatus only formed ~ 20% 2. The observed differences might be related to

233

different media used or culture parameters applied.

234

According to Řezanka and Mareš the basidiomycetous fungus Armillaria mellea

235

(honey fungus) contains ~10% of BCFA, including small amounts of the odd-

236

numbered iso-FA 13-methyltetradecanoic acid (1.8%).7 Odd-numbered iso-FAs have

237

not been reported for other fungal species. Therefore, the fatty acid profile of A.

238

mellea was analyzed as well. However, all identified iso-FAs were even-carbon-

239

numbered while the anteiso-FAs were odd-carbon-numbered (Table 1). For the

240

Conidiobolus species, this was in accordance with Tyrrell and Weatherston.5

241

C. heterosporus CBS 333.74 was chosen for all further experiments due to its overall

242

better ability to be cultivated in liquid media compared to other C. heterosporus

243

strains. Its growth in submerged cultures was investigated over a period of two

244

weeks. Both, the biomass and the lipid yield increased steadily over time (Figure 2).

245

The lipid content of the dry biomass was 25–30%. The amounts of saturated fatty

246

acids decreased until day 14 by about 10%, whereas the share of unsaturated fatty

247

acids increased slightly (data not shown). On day 14, the highest levels of 2 were

248

observed.

249

For certain bacteria and some Conidiobolus species it is known that supplementation

250

of the culture medium with the amino acid L-valine may increase the formation of

251

even numbered iso-FAs, i.e. 2 or 14-methylpentadecanoic acid.5,23,24 Based on these

252

observations, the culture media of C. heterosporus CBS 333.74 were supplemented

253

with 0.2 g/L and 2.0 g/L L-valine, respectively. For 2, an increase of 10% (0.2 g/L)

11 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

254

and 38% (2.0 g/L) was observed. For 14-methylpentadecanoic acid, the increase

255

was less pronounced with 4% and 10%.

256

Biotransformation to methyl branched aldehydes. Initially, several enzymes were

257

considered for the reduction of MBFAs to their corresponding aldehydes. Various

258

enzymes have been described to reduce aromatic carboxylic acids. The well

259

investigated CAR from Nocardia species are known especially to reduce acids with

260

aromatic residues, but successful reduction of aliphatic acids has not been reported

261

for them yet.25,26 However, for the CAR from M. marinum, the reduction of aliphatic

262

fatty acids, with a range from C8–C18, was published recently.17 Because of the

263

structural similarities, the CAR from M. marinum was chosen for the

264

biotransformation of MBFAs. The CAR was heterologously expressed and purified to

265

electrophoretic homogeneity. The molecular weight of the CAR was determined to

266

be 127 kDa (Figure 3) which was in agreement with the results of Akhtar et al.17

267

For whole cell biotransformation, 2 was incubated for 24 h with different amounts of

268

E. coli cell pellets expressing CAR. The formation of up to 28.0 ± 1.8 mol% 3 was

269

observed, but no 1 was detected. To further elucidate the formation of 1, shorter

270

biotransformation periods were checked. Here, 1 was identified, with the highest

271

amounts (3.1 ± 0.6 mol%) after 2 h (Figure 4A). The concentration of 3 steadily

272

increased over the first 6 h (20 ± 3.4 mol%). In contrast, in none of the negative

273

controls (without CAR, with heat inactivated CAR, with BL21(DE3) and empty

274

plasmid) a reduction of 2 to 1 or 3 was detected. On the other hand, the E. coli strain

275

BL21(DE3) with empty plasmid converted 1 to 3 (data not shown).

276

To prevent the accumulation of 3 and thus to increase the amounts of 1, the in vitro

277

conversion of 2 was further investigated with the purified CAR enzyme. Therefore,

278

the recombinant polyhistidine-tagged CAR was purified by means of immobilized

12 Environment ACS Paragon Plus

Page 12 of 27

Page 13 of 27

Journal of Agricultural and Food Chemistry

279

metal ion affinity chromatography, and 2 was incubated with the isolated CAR in the

280

presence of the required cofactors for 2–8 h. After 2 h, 22.1 ± 0.53 mol% 1 were

281

detected, and the highest conversion yield was found with 28 ± 7.8 mol% after 8 h

282

(Figure 4B). In all samples, only trace amounts of 3 were determined. This led us to

283

the conclusion, that the CAR is essential for the initial reduction of 2 to 1, whereas

284

native E. coli enzymes (most probably alcohol dehydrogenases) are responsible for

285

the reduction of the formed aldehyde to its corresponding alcohol.

286

Next, the fatty acids produced by C. heterosporus CBS 333.74 were directly

287

employed. In addition to the CAR, a lipase from Candida rugosa was added to the

288

reaction mixture to release esterified fatty acids. Besides 12-methyltridecanal and

289

12-methyltetradecanal, several linear aldehydes, like dodecanal, tridecanal,

290

tetradecanal, pentadecanal, and hexadecanal were generated (Figure 5).

291

In conclusion, the enzymatic reduction of methyl-branched fatty acids to their

292

corresponding aldehydes has been shown in this study for the first time. 2 was

293

converted enzymatically to the highly sought after flavor compound 12-

294

methyltridecanal. Even though the initial concentrations obtained with the

295

investigated one-pot reaction were rather low, the general feasibility was clearly

296

shown. Future studies will concentrate on aspects of increasing the yields of 1 by

297

direct enzymatic conversion of fungal lipids.

298 299

ABBREVIATIONS

300

AEDA, aroma extract dilution analysis; BCFA, branched-chain fatty acid; MBFA,

301

methyl-branched fatty acid; OAV, odor activity value; PPT, phosphopantetheinyl

302

transferase; SIDA, stable isotope dilution analysis; YM, yeast extract-malt extract.

303

13 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

304

ACKNOWLEDGMENT

305

We are very grateful to Pauli Kallio, University of Turku, and Patrik R. Jones,

306

Imperial College London, who generously provided the BL21(DE3)pET-PC2 E. coli

307

strain.

308 309

ASSOCIATED CONTENT

310

Supporting Information

311

GC-MS chromatogram of a C. denaeosporus lipid extract after transesterification to

312

the corresponding fatty acid methyl esters and mass spectrum of methyl 12-

313

methyltridecanoate identified in the C. denaeosporus lipid extract. This material is

314

available free of charge via the Internet at http://pubs.acs.org.

315 316

Funding

317

The authors MAF and HZ are grateful for financial support by the excellence initiative

318

of the Hessian Ministry of Science and Art which encompasses a generous grant for

319

the LOEWE Center for Insect Biotechnology & Bioresources.

320 321

Notes

322

The authors declare no competing financial interest. Torsten Geissler, Egon Gross,

323

Jens-Michael Hilmer, and Jakob Ley are members of Symrise AG holding intellectual

324

property on 12-methyltridecanal.

325 326 327 328

REFERENCES (1)

Kaneda, T. Iso- and anteiso-fatty acids in bacteria: Biosynthesis, function,

and taxonomic significance. Microbiol. Rev. 1991, 55, 288–302.

14 Environment ACS Paragon Plus

Page 14 of 27

Page 15 of 27

329

Journal of Agricultural and Food Chemistry

(2)

Vlaeminck, B.; Fievez, V.; Cabrita, A. R. J.; Fonseca, A. J. M.; Dewhurst, R.

330

J. Factors affecting odd- and branched-chain fatty acids in milk: A review. Anim.

331

Feed Sci. Technol. 2006, 131, 389–417.

332

(3)

Tyrrell, D. The fatty acid composition of some Entomophthoraceae. II. The

333

occurrence of branched-chain fatty acids in Conidiobolus denaesporus Drechsl.

334

Lipids 1968, 3, 368–372.

335 336 337

(4)

Tyrrell, D. The fatty acid composition of some Entomophthoraceae. III. Can.

J. Microbiol. 1971, 17, 1115–1118. (5)

Tyrrell, D.; Weatherston, J. The fatty acid composition of some

338

Entomophthoraceae. IV. The occurrence of branched-chain fatty acids in

339

Conidiobolus species. Can. J. Microbiol. 1976, 22, 1058–1060.

340

(6)

Sinanoglou, V. J.; Zoumpoulakis, P.; Heropoulos, G.; Proestos, C.; Ćirić, A.;

341

Petrovic, J.; Glamoclija, J.; Sokovic, M. Lipid and fatty acid profile of the edible

342

fungus Laetiporus sulphurous. Antifungal and antibacterial properties. J. Food Sci.

343

Technol. (New Delhi, India) 2014, 52, 3264–3272.

344 345 346 347 348

(7)

Řezanka, T.; Mareš, P. Unusual and very long-chain fatty acids produced by

Basidiomycetes. J. Chromatogr. A 1987, 409, 390–395. (8)

Guth, H.; Grosch, W. 12-Methyltridecanal, a species-specific odorant of

stewed beef. Lebensm.-Wiss. Technol. 1993, 26, 171–177. (9)

Guth, H.; Grosch, W. Identification of the character impact odorants of

349

stewed beef juice by instrumental analyses and sensory studies. J. Agric. Food

350

Chem. 1994, 42, 2862–2866.

351

(10) Kerscher, R.; Nürnberg, K.; Voigt, J.; Schieberle, P.; Grosch, W. Occurrence

352

of 12-methyltridecanal in microorganisms and physiological samples isolated from

353

beef. J. Agric. Food Chem. 2000, 48, 2387–2390.

15 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

354 355 356

(11) Guth, H.; Grosch, W. Dependence of the 12-methyltridecanal concentration in beef on the age of the animal. Z. Lebensm.-Unters. Forsch. 1995, 201, 25–26. (12) Dannenberger, D.; Lorenz, S.; Nuernberg, G.; Scollan, N.; Ender, K.;

357

Nuernberg, K. Analysis of fatty aldehyde composition, including 12-methyltridecanal,

358

in plasmalogens from Longissimus muscle of concentrate- and pasture-fed bulls. J.

359

Agric. Food Chem. 2006, 54, 182–188.

360

(13) Werkhoff, P.; Bruening, J.; Emberger, R.; Guentert, M.; Hopp, R. Flavor

361

chemistry of meat volatiles: New results on flavor components from beef, pork, and

362

chicken. In Recent Dev. Flavor Fragrance Chem., Proc. Int. Haarmann Reimer

363

Symp.; VCH:Weinheim, 1993; pp 183–213.

364

(14) Yuasa, Y.; Tsuruta, H. Convenient syntheses of iso-methyl-branched long-

365

chain aliphatic aldehydes, known to contribute significantly to meat flavour. Flavour

366

Fragrance J. 2004, 19, 199–204.

367 368 369

(15) Napora-Wijata, K.; Strohmeier, G. A.; Winkler, M. Biocatalytic reduction of carboxylic acids. Biotechnol. J. 2014, 9, 822–843. (16) Lamm, A. S.; Venkitasubramanian, P.; Rosazza, J. P. N. Carboxylic acid

370

reductase. In Science of synthesis: Biocatalysis in organic synthesis; Faber, K.,

371

Fessner, W.-D., Turner, N. J., Eds.; Georg Thieme Verlag: Stuttgart, 2015; Vol. 2, pp

372

459–478.

373

(17) Akhtar, M. K.; Turner, N. J.; Jones, P. R. Carboxylic acid reductase is a

374

versatile enzyme for the conversion of fatty acids into fuels and chemical

375

commodities. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 87–92.

376

(18) Huber, C.; Skopan, H.; Feicht, R.; White, H.; Simon, H. Pterin cofactor,

377

substrate specificity, and observations on the kinetics of the reversible tungsten-

378

containing aldehyde oxidoreductase from Clostridium thermoaceticum. Arch.

16 Environment ACS Paragon Plus

Page 16 of 27

Page 17 of 27

379 380

Journal of Agricultural and Food Chemistry

Microbiol. 1995, 164, 110–118. (19) Fraatz, M. A.; Naeve, S.; Hausherr, V.; Zorn, H.; Blank, L. M. A minimal

381

growth medium for the basidiomycete Pleurotus sapidus for metabolic flux analysis.

382

Fungal Biol. Biotechnol. 2014, 1.

383 384 385

(20) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. (21) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins

386

in polyacrylamide gels including isoelectric focusing gels with clear background at

387

nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

388

Electrophoresis 1988, 9, 255–262.

389

(22) Kleofas, V.; Popa, F.; Fraatz, M. A.; Rühl, M.; Kost, G.; Zorn, H. Aroma

390

profile of the anise-like odour mushroom Cortinarius odorifer. Flavour Fragrance J.

391

2015, 30, 381–386.

392

(23) Kaneda, T. Biosynthesis of branched chain fatty acids. II. Microbial synthesis

393

of branched long chain fatty acids from certain short chain fatty acid substrates. J.

394

Biol. Chem. 1963, 238, 1229–1235.

395

(24) Kaneda, T. Biosynthesis of branched-chain fatty acids: IV. Factors affecting

396

relative abundance of fatty acids produced by Bacillus subtilis. Can. J. Microbiol.

397

1966, 12, 501–514.

398 399 400

(25) Chen, Y.; Rosazza, J. P. N. Microbial transformation of ibuprofen by a Nocardia species. Appl. Environ. Microbiol. 1994, 60, 1292–1296. (26) He, A.; Li, T.; Daniels, L.; Fotheringham, I.; Rosazza, J. P. N. Nocardia sp.

401

carboxylic acid reductase: Cloning, expression, and characterization of a new

402

aldehyde oxidoreductase family. Appl. Environ. Microbiol. 2004, 70, 1874–1881.

403

17 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

404

18 Environment ACS Paragon Plus

Page 18 of 27

Page 19 of 27

Journal of Agricultural and Food Chemistry

405

FIGURE CAPTIONS

406

Figure 1. Structures of 12-methyltridecanal (1), 12-methyltridecanoic acid (2), and

407

12-methyltridecanol (3).

408 409

Figure 2. Dry biomass, lipids, and relative lipid content of C. heterosporus CBS

410

333.74 grown in submerged cultures over a cultivation period of 14 d.

411 412

Figure 3. SDS-PAGE of the CAR enzyme preparation after heterologous expression

413

and purification according to the procedure described by Akhtar et al.;17 left lane

414

shows a molecular mass marker (kDa), right lane shows the isolated CAR.

415 416

Figure 4. Kinetics of the whole cell catalyzed reduction of 2 with CAR from M.

417

marinum co-expressed with PPT in E. coli for 1–6 h (A) and by the recombinant

418

CAR, purified by means of immobilized metal ion affinity chromatography, for 2–8 h

419

(B).

420 421

Figure 5. Section of a GC-MS chromatogram (total icon current, scan mode, m/z 33–

422

300) recorded after injection of an organic solvent extract of a reaction mixture of the

423

enzymatic hydrolysis of a fungal lipid and reduction of the thereby generated free

424

fatty acids by CAR.

425

19 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

Page 20 of 27

TABLES Table 1. Relative Fatty Acid (FA) Compositions (%) of A. mellea, C. denaeosporus, C. heterosporus CBS 543.63, CBS 333.74, CBS 138.57, and C. lobatus (CLO) after Lipid Extraction and Transesterification to the Corresponding Fatty Acid Methyl Esters.

fatty acid

C.

C. heterosporus

C. heterosporus

C. heterosporus

denaeosporus

CBS 543.63

CBS 333.74

CBS 138.57

C. lobatus

A. mellea

a14:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

i14:0

5.0

8.6

40.1

34.5

20.1

18.8

a15:0

1.4

11.1

13.6

12.0

12.7

7.8

i15:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

a16:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

i16:0

0.7

0.4

4.2

7.0

2.1

2.1

∑ unassigned FAs

28.3

28.6

19.6

21.4

7.7

7.8

∑ saturated FAs*

31.2

48.3

21.1

20.7

47.6

44.0

∑ unsaturated

33.4

3.0

1.4

4.5

9.8

19.5

20 ACS Paragon Plus Environment

Page 21 of 27

Journal of Agricultural and Food Chemistry

FAs

a14:0 11-methyltridanoic acid, i14:0 12-methyltridanoic acid, a:15:0 12-methyltetradecanoic acid, i15:0 13-methyltetradecanoic acid, a16:0 13-methylpentadecanoic acid, i16:0 14-methylpentadecanoic acid, n.d. not detected, * excluding listed methyl-branched fatty acids

21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

FIGURES

Figure 1

22 ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27

Journal of Agricultural and Food Chemistry

12

30

10

25

8

20

6

15

4

10

2

5

0

0 2

4

6

8

10

cultivation time [d] dry biomass

lipids

lipids / dry biomass

23 ACS Paragon Plus Environment

12

14

lipids / dry biomass [%]

dry biomass & lipids [g L-1]

Figure 2

Journal of Agricultural and Food Chemistry

Figure 3

24 ACS Paragon Plus Environment

Page 24 of 27

Page 25 of 27

Journal of Agricultural and Food Chemistry

Figure 4

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 27

tetradecanal

tridecanal

150000

dodecanal

abundance

200000

100000

50000

hexadecanal

250000

pentadecanal

300000

12-methyltetradecanal

12-methyltridecanal

Figure 5

0 30

31

32

33

34

35

36

37

38

39

40

41

[min]

26 ACS Paragon Plus Environment

42

43

44

45

46

Page 27 of 27

Journal of Agricultural and Food Chemistry

For Table of Contents Only

27 ACS Paragon Plus Environment