Whole-Cell ... - ACS Publications

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

Simultaneous enzyme/whole-cell biotransformation of plant oils into C9 carboxylic acids Eun-Yeong Jeon, Joo-Hyun Seo, Woo-Ri Kang, Min-Ji Kim, Jung-Hoo Lee, Deok-Kun Oh, and Jin-Byung Park ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01884 • Publication Date (Web): 29 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 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.

ACS Catalysis 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 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

1

Simultaneous enzyme/whole-cell biotransformation of plant oils

2

into C9 carboxylic acids

3 4

Eun-Yeong Jeon1,†, Joo-Hyun Seo1,†, Woo-Ri Kang2,†, Min-Ji Kim1, Jung-Hoo Lee1, Deok-

5

Kun Oh2,*, and Jin-Byung Park1,*

6 7

1

8 9

Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea

2

10

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

11 12

†

These authors are equally contributed first authors.

13 14

*

15

Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea; tel:

16

+82-2-454-3118; fax: +82-2-444-5518; e-mail: [email protected]), Prof. Jin-Byung

17

Park (address: Department of Food Science and Engineering, Ewha Womans University,

18

Seoul 120-750, Republic of Korea; tel: +82-2-3277-6685/4509; fax: +82-2-3277-4213; e-

19

mail: [email protected]).

To whom correspondence should be addressed: Prof. Duk-Keon Oh (address: Department of

20 21

1

ACS Paragon Plus Environment

ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

22

Abstract

23

Oxyfunctionalization of plant oils such as olive oil and soybean oil into C9 carboxylic acids

24

(e.g., n-nonanoic acid and 9-hydroxynonanoic acid) was investigated. The biotransformation

25

was composed of hydrolysis of plant oils by the Thermomyces lanuginosus lipase (TLL) and

26

C9-C10 double-bond cleavage in unsaturated fatty acids by a serial reaction of a fatty acid

27

double bond-hydratase of Stenotrophomonas maltophilia, an alcohol dehydrogenase of

28

Micrococcus luteus, and a Baeyer-Villiger monooxygenase (BVMO) of Pseudomonas putida

29

KT2440 expressed in Escherichia coli. The newly cloned oleate hydratase allowed to

30

produce 10-hydroxyoctadecanoic acid and 10-hydroxyoctadec-12-enoic acid to a high rate

31

from oleic acid and linoleic acid, respectively, which are major fatty acid constituents of

32

many plant oils. Furthermore, overexpression of a long chain fatty acid transporter FadL in

33

the recombinant E. coli led to a significant increase of whole-cell biotransformation rates of

34

oleic acid and linoleic acid into the corresponding esters. The resulting esters (the BVMO

35

reaction products) were hydrolyzed in situ by the TLL, generating nonanoic acid, non-3-enoic

36

acid, and 9-hydroxynonanoic acid, which can be further oxidized to 1,9-nonanedioic acid.

37

This study demonstrated that industrially relevant C9 carboxylic acids could be produced

38

from olive oil or soybean oil by simultaneous enzyme/whole-cell biocatalysis.

39 40 41

Keywords: oleate hydratase, FadL, 9-hydroxynonanoic acid, olive oil, soybean oil,

42

simultaneous enzyme/whole-cell biocatalysis

43

2


Page 2 of 29

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

44

Introduction

45

Plant oils are one of the cheap and abundant renewable resources.1 For instance, 44.9 million

46

metric tons of soybean oil were produced worldwide in 2013–2014.2 Most were used as

47

cooking oil, while only a small portion was used in the chemical industry to manufacture

48

biodiesel and (bio)chemicals.

49

A number of emerging ideas to enhance the value of the plant oils and fatty acids have

50

been recently reported.3 A variety of fatty acids (e.g., oleic acid, linoleic acid) could be

51

converted into hydroxy fatty acids through hydration by the fatty acid double-bond

52

hydratases,4 oxygenation by the lipoxygenases,5 or hydroxylation by the monooxygenases,6

53

which can be further transformed into the corresponding keto fatty acids by the long chain

54

secondary alcohol dehydrogenases.3c, 7 For instance, linoleic acid, the major fatty acid

55

constituent of soybean oil, was converted into 10-oxo-octadec-12-enoic acid and 13-oxo-

56

octadec-9-enoic acid via 10-hydroxyoctadec-12-enoic acid and 13-hydroxyoctadec-9-enoic

57

acid, respectively,3c, 8 which were reported to have diverse biological and chemical

58

functions.4c, 9

59

The keto-fatty acids could be further oxidized to the ester fatty acids via oxidative C-

60

C bond cleavage reaction by the Baeyer-Villiger monooxygenases (BVMOs).3c, 10 For

61

instance, 10-keto-octadecanoic acid (4) was transformed into the ester fatty acid (5) by the

62

BVMOs from Pseudomonas putida KT2440 (i.e., EthA) and Rhodococcus jostii RHA1

63

(MO16) (Fig. 1). The ester fatty acid was then hydrolyzed by a lipase or esterase generating

64

n-nonanoic acid and 9-hydroxynonanoic acid, which can be further transformed into 1,9-

65

nonanedioic acid and 9-aminnonanoic acid.11

66 67

Most plant oils consist of glycerol and fatty acids, which are mainly composed of oleic acid and linoleic acid.12 Thereby, plant oils such as soybean oil and olive oil are an 3


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

68

excellent source of unsaturated fatty acids, which can be converted into diverse valuable

69

chemical products as described above. One of the hurdles, however, in biotransformation of

70

plant oils may include low hydration activity of the fatty acid double-bond hydratases with

71

linoleic acid and low fatty acid transport rate into the cascade enzymes inside whole-cells.5c,

72

13

73

which is very active to linoleic acid as well as oleic acid. Another goal was to engineer E. coli

74

cells to enhance fatty acid transport rate into intracellular cascade enzymes. We have also

75

investigated a simultaneous enzyme/whole-cell biocatalysis, which may catalyze

76

oxyfunctionalization of plant oils including soybean oil and olive oil into C9 carboxylic acids

77

(e.g., 9-hydroxynonanoic acid) in one-pot process (Fig. 1), which are currently manufactured

78

via ozonolysis in chemical industry.14

Thereby, this study has focused on cloning of a new fatty acid double-bond hydratase,

79 80

Results and Discussion

81

Whole-cell biotransformation of oleic acid into ester

82

One of the major fatty acid constituents of plant oils is oleic acid. Thereby, biotransformation

83

of oleic acid into the ester (5) (Fig. 1) was first investigated. Based on our previous studies11b,

84

15

85

fatty acid double-bond hydratase (OhyA) of Stenotrophomonas maltophilia, a long chain

86

secondary alcohol dehydrogenase (ADH) of Micrococcus luteus, and an engineered BVMO

87

(E6BVMO) of P. putida KT2440 (Fig. 1), was constructed and applied to the

88

biotransformation of oleic acid (Fig. 2A). Oleic acid was converted into the ester (5) via 10-

89

hydroxyoctadecanoic acid (3) and 10-ketooctadecanoic acid (4). However, the oleic acid

90

biotransformation rate and thus the ester production rate remained rather low (Fig. 2A).

, the recombinant E. coli BL21(DE3) pACYC-ADH-OhyA, pJOE-E6BVMO, expressing a

4


Page 4 of 29

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

91

With an aim to increase the oleic acid biotransformation rate, expression level of the

92

long chain fatty acid transporter, FadL16 in the outer membrane of the recombinant E. coli

93

BL21(DE3) pACYC-ADH-OhyA, pJOE-E6BVMO was enhanced by introducing the fadL

94

gene with the strong inducible T7 promoter (Fig. S1). The resulting recombinant E. coli

95

BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-E6BVMO led to ca. 6-fold higher oleic acid

96

biotransformation rate and the ester (5) production rate at the identical condition (Fig. 2B)

97

(Table 1). The specific ester (5) production rate of recombinant E. coli BL21(DE3) pACYC-

98

ADH-FadL-OhyA, pJOE-E6BVMO was also 4-fold greater as compared to that of the two

99

cell system, which consisted of E. coli BL21(DE3) pET-OhyA expressing the oleate

100

hydratase of S. maltophilia and E. coli BL21(DE3) pACYC-ADH, pJOE-BVMO11b. These

101

results indicated that transport of oleic acid and reaction intermediates into the cascade

102

enzymes inside cells is a key factor to determine the whole-cell oleic acid biotransformation

103

rate.

104

In order to enhance the final ester concentration and the ester production rate, the

105

oleic acid biotransformation was initiated at a high cell density of the recombinant E. coli

106

BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-BVMO (i.e., 25 g dry cell/L). The

107

biotransformation at the high cell density allowed the ester (5) to accumulate to a final

108

concentration of 42 mM with a volumetric productivity of ca. 5.3 mM/h (Fig. 2C). The

109

final product concentration and volumetric productivity were 9.6- and 5.9-fold greater than

110

the values in the experiment shown in Fig. 2B. This result indicated that the ester fatty acid

111

could be produced to a high concentration with a high volumetric productivity through the

112

biotransformation at high density of the whole-cells overexpressing FadL in the outer

113

membrane. 5


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

114 115

Enzyme/whole-cell biotransformation of olive oil into C9 carboxylic acids

116

One of the critical points in the biotransformation of olive oil into C9 carboxylic acids (6, 7)

117

may include selection of a suitable lipase, because the lipase is involved not only in the step

118

of olive oil hydrolysis but also in hydrolysis of the BVMO reaction product (5) (Fig. 1).

119

Among lipases, the lipases from Candida rugosa (CRL)17 and Thermomyces lanuginosus

120

(TLL)18 were extensively used for biocatalysis as well as soybean oil hydrolysis.19 The both

121

enzymes showed rather high activity to hydrolysis of olive oil as reported, whereas the TLL

122

was more active with the BVMO reaction product (5) (data not shown). Thereby, TLL was

123

applied to the further biotransformations.

124

The biotransformation of olive oil into n-nonanoic acid (6) and 9-hydroxynonanoic

125

acid (7) was carried out by adding olive oil and TLL into culture broth of the recombinant E.

126

coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-E6BVMO at the stationary growth phase

127

(Fig. 3). Olive oil was quickly hydrolyzed into glycerol and fatty acids (e.g., oleic acid and

128

palmitic acid) by the TLL. Glycerol was used up as a carbon source for the recombinant E.

129

coli, while oleic acid (2) was converted into C9 carboxylic acids (6, 7) via 10-

130

hydroxyoctadecanoic acid, 10-ketooctadecanoic acid and the ester (5). Palmitic acid remained

131

unreacted in the reaction medium. Overall, 9-hydorxynonanoic acid (7) was produced to 9.7

132

mM at t = 9 h in the culture broth from 5 g/L olive oil. After isolation of the reactants

133

including 9-hydorxynonanoic acid (7) via solvent extraction and concentration via

134

evaporation, the reactants were subjected to oxidation to 1,9-nonanedioic acid (i.e., azelaic

135

acid), as previously reported11b. The dicarboxylic acid product was purified from the reaction

136

medium via recrystallization from ethyl acetate-hexane, resulting in an isolated yield of ca 80% 6


Page 6 of 29

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

137 138

ACS Catalysis

with a purity of 94%. The specific ester (5) production rate of the recombinant E. coli BL21(DE3) pACYC-

139

ADH-FadL-OhyA, pJOE-E6BVMO from olive oil, which was calculated as sum of the ester

140

(5) concentration and the C9 acid (7) concentration during the biotransformation, reached

141

approximately 6.4 U/g dry cells (Fig. 3). This value is comparable to the specific ester

142

production rate of the whole-cells from oleic acid under similar conditions (Fig. 2B) (Table 1).

143

This result indicated that the target C9 products could be efficiently produced from olive oil

144

by the simultaneous enzyme/whole-cell biocatalysis.

145

One of the interesting points in the simultaneous enzyme/whole cell biotransformation

146

of olive oil (Fig. 3) would be the lipase-driven hydrolysis of the BVMO reaction product (5),

147

which was suggested to mostly exist in the lipid bilayer of E. coli cells.11b, 15 The fast

148

formation of 9-hydorxynonanoic acid (7) from the ester (5) (Fig. 3) indicated that the

149

hydrophobic long chain fatty acid derivatives partitioned in the cell membrane could be

150

attacked by the lipases present in the extracellular space. Probably, some of the ester fatty

151

acids might diffuse out of the cells and be subjected to hydrolysis by the lipases. Overall, it

152

was assumed that the industrially relevant chemicals 9-hydorxynonanoic acid (7) and n-

153

nonanoic acid (6) could be produced to a high rate from olive oil by the simultaneous

154

enzyme/whole cell biocatalysis containing the TLL and the recombinant E. coli

155

overexpressing fadL in the outer membrane in addition to the cascade enzymes as

156

biocatalysts.

157 158

Cloning of a novel fatty acid double-bond hydratase

159

Not only oleic acid but also linoleic acid is one of the major constituents of the renewable oils 7


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

160

(e.g., soybean oil12, microalgae oils20, and yeast oils21). However, most of fatty acid double-

161

bond hydratases reported showed a high activity with oleic acid, whereas rather low activity

162

with linoleic acid.4c, 5c, 13, 22 Thereby, cloning of a novel fatty acid hydratase, which is active

163

with linoleic acid as well as oleic acid, was investigated.

164

Intensive genome scanning of S. maltophilia, which was reported to have large

165

hydration activity of unsaturated fatty acids,4b has discovered another putative fatty acid

166

double-bond hydratase gene (NCBI accession number WP_017356052) in addition to the

167

previously reported ohyA (NCBI accession number WP_024958135).4b The putative fatty

168

acid hydratase (i.e., named as OhyA2) was cloned and subjected to activity assay after

169

expression in E. coli and purification via His-Trap affinity chromatography (Fig. S2). The

170

amino acid sequence of the putative hydratase was 37% identity with the OhyA of S.

171

maltophilia but more similar to the oleate hydratase of Stenotrophomonas nitritireducens

172

(identity: 72%)4d and of Elizabethkingia meningoseptica (identity: 58%)23 (Table 2). However,

173

the specific activities of the OhyA2 with oleic acid were 26- and 3-fold higher than that of

174

oleate hydratase of E. meningoseptica and S. nitritireducens, respectively. Remarkably, the

175

specific activities of the OhyA2 with oleic acid and linoleic acid were 1.5- and 1.9-fold

176

greater than that of OhyA from S. maltophilia, respectively (Table 2). This is the greatest

177

hydration activity with oleic acid and linoleic acid to our knowledge.

178 179

Whole-cell biotransformation of linoleic acid into ester

180

We have next examined the biotransformation of linoleic acid (9) into the ester (12) (Fig. 4)

181

by using the recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-BVMO and

182

the recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA2, pJOE-BVMO. Linoleic 8


Page 8 of 29

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

183

acid was converted into the ester (12) via 10-hydroxyoctadec-12-enoic acid and 10-

184

ketooctadec-12-enoic acid at a rate of 0.7 mM/h until t = 3 h (Fig. 5A). The ester production

185

rate was approximately 8-fold higher than that of the recombinant E. coli BL21(DE3)

186

pACYC-ADH-OhyA, pJOE-BVMO (Fig. S3) (Table 3). Afterwards, linoleic acid

187

concentration remained rather unchanged, indicating that hydration of linoleic acid into 10-

188

hydroxyoctadec-12-enoic acid was ceased. Probably, OhyA, which belongs to flavin enzymes,

189

was deactivated rather fast during the biotransformation, because the reaction was conducted

190

under aerobic condition4a, 24.

191

On the other hand, linoleic acid was completely converted into the ester (12) by the

192

recombinant E. coli pACYC-ADH-FadL-OhyA2, pJOE-BVMO (Fig. 5B). The specific ester

193

production rate at t < 3 h reached 6.7 U/g dry cells, which was ca. 70% greater than that of

194

the recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-BVMO (Table 3). The

195

complete biotransformation of linoleic acid at the high rate could be allowed by high activity

196

of the newly cloned fatty acid hydratase OhyA2 with linoleic acid.

197 198

Enzyme/whole-cell biotransformation of soybean oil into C9 carboxylic acids

199

The biotransformation of soybean oil into C9 carboxylic acids including 9-hydroxynonanoic

200

acid (7) was carried out by adding soybean oil and TLL into culture broth of the recombinant

201

E. coli BL21(DE3) pACYC-ADH-FadL-OhyA2, pJOE-BVMO at the stationary growth

202

phase. Soybean oil was quickly hydrolyzed into glycerol and fatty acids (e.g., oleic acid,

203

linoleic acid, and palmitic acid) by the TLL (Fig. 6). Glycerol was used up as carbon source

204

of the recombinant E. coli, while oleic acid (2) and linoleic acid (9) were converted into the

205

target product C9 carboxylic acids including 9-hydorxynonanoic acid (7) via 10-hydroxy 9


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

206

fatty acids (3, 10), 10-oxo-fatty acids (4, 11), and ester fatty acids (5, 12), respectively.

207

Overall, 9-hydorxynonanoic acid (7) was produced to 7.0 mM in the culture broth from 5 g/L

208

soybean oil. The specific ester production rate during the biotransformation reached ca. 5.7

209

U/g dry cells (Fig. 6), which is comparable to the whole-cell biotransformation rate of

210

linoleic acid (9) into the ester (12) under similar conditions (Fig. 5B) (Table 3). This result

211

indicated that not only olive oil but also soybean oil could be converted into C9 carboxylic

212

acids via the esters (5, 12) by the simultaneous enzyme/whole cell biocatalysis containing the

213

TLL and the recombinant E. coli overexpressing fadL in the outer membrane and the OhyA2

214

in addition to the ADH and the BVMO as biocatalysts.

215

The productivity of enzyme/whole-cell biotransformations could be influenced by many

216

factors including functional expression of the cascade enzymes in whole-cells10, 15, 25, activity

217

and stability of the cascade enzymes under process conditions3f, 15, substrate and product

218

toxicity3d, 11b, 26, and mass transport3e, 13. In this study, we showed that activity of fatty acid

219

double-bond hydratases and substrate transport into the cascade enzymes played a key role in

220

enzyme/whole-cell biotransformation of long chain fatty acids (e.g., oleic acid and linoleic

221

acid). Most of all, overexpression of the long chain fatty acid transporter FadL16 in the

222

recombinant E. coli cells was essential to obtain high productivity of the whole-cell

223

biotransformations (Tables 1 and 3).

224

Expression of FadL in E. coli was reported to be affected by a number of factors.27 The

225

fadL promoter of E. coli contains several binding sites for the transcription factors such as

226

FadR, ArcA and cAMP receptor protein (CRP) (http://www.ecocyc.org). FadR, a fatty acid-

227

responsive transcription factor is involved in repression of the fadL transcription in the

228

absence of long chain fatty acids.27a The fadL transcription is also repressed under anaerobic 10


Page 10 of 29

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

229

condition by ArcA and in the presence of glucose, because CRP-cAMP complex generally

230

acts as activator of fad regulon.27b, 27c The recombinant E. coli expressing the cascade

231

enzymes was grown in the glucose-containing Riesenberg medium without fatty acids.

232

Consequently, FadL expression level might remain low limiting transport of fatty acid

233

substrates into the cells during biotransformation at the stationary growth phase. This

234

assumption is also supported by the low hydration rates of oleic acid and linoleic acid (Tables

235

1 and 3). Thereby, it was concluded that increased expression of FadL in the recombinant

236

cells led to increase of fatty acid transport rates into the cascade enzymes, which in turn

237

allowed enhancement of the whole-cell fatty acid biotransformation rates.

238

Medium chain fatty acids (e.g., n-heptanoic acid, n-octanoic acid, and n-nonanoic acid)

239

were reported to be toxic to E. coli cells11b, 26, 28. The medium chain fatty acids may result in

240

the formation of transient or permanent pores through the interaction with the cellular

241

membranes when entering into the microbial cells in the undissociated form28a. The fatty

242

acids may also result in intracellular acidification by generating protons within the cells,

243

thereby cause the destabilization of DNA and proteins and the reduction in ATP production

244

due to the decrease of proton gradient. As a result, toxicity of medium chain fatty acids was

245

assumed as one of the factors limiting the productivity of whole-cell biotransformations.

246

Thereby, improvement of tolerance of E. coli to toxic effects of medium chain fatty acids

247

would be essential to achieve high productivity and high product concentration via whole-cell

248

biotransformation.

249

Most of renewable oils such as plant oils, microalgae oils20, and yeast oils21 possess

250

oleic acid and/or linoleic acid as the major fatty acid constituents. For instance, the fatty acids

251

of yeast oils, which were produced from marine biomass Laminaria japonica via

252

fermentation of Cryptococcus curvatus, consisted of palmitic acid (13.2%), stearic acid 11


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

253

(22.6%), oleic acid (50.7%), and linoleic acid (11.2%)21. Thereby, the biotransformation

254

process of olive oil and soybean oil developed in this study could be applied to

255

biotransformation of other renewable oils.

256

257

Conclusions

258

We have cloned a novel fatty acid double-bond hydratase (i.e., OhyA2), which is highly

259

active with both oleic acid and linoleic acid. This enzyme allowed efficient hydration of

260

unsaturated fatty acids, which were released from olive oil and soybean oil. This study has

261

also demonstrated that the plant oils could be oxyfunctionalized into the C9 carboxylic acids

262

(e.g., 9-hydroxynonanoic acid (7)) via simultaneous enzyme/whole-cell biocatalysis. The

263

productivity of whole-cell biotransformations were significantly improved by overexpression

264

of FadL in E. coli. The present study could be used to biotransformation of other plant oils

265

and renewable oils, which are mainly composed of unsaturated fatty acids (e.g., oleic acid

266

and linoleic acid).

267 268

Acknowledgements

269

This study was supported by the Marine Biomaterials Research Center grant from the Marine

270

Biotechnology Program funded by the Ministry of Oceans and Fisheries, Republic of Korea

271

(No. D11013214H480000100). J.-H. Seo was partially supported by RP-Grant 2016 of Ewha

272

Womans University.

273 274

Supporting information

275

Materials and methods, Table S1 and Figures S1~S3. 12


Page 12 of 29

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

276

References

277

1.

278 279

85, 925-930. 2.

280 281

Rodrigues, R. C.; Volpato, G.; Wada, K.; Ayub, M. A. Z. J. Am. Oil Chem. Soc. 2008,

United States Department of Agriculture. World agricultural supply and demand estimates, Washington, DC. 2015

3.

(a) Seo, J.-H.; Lee, S.-M.; Lee, J.; Park, J.-B. J. Biotechnol. 2015, 216, 158-166; (b)

282

Biermann, U.; Bornscheuer, U.; Meier, M. A.; Metzger, J. O.; Schafer, H. J. Angew.

283

Chem. Int. Ed. 2011, 50, 3854-71; (c) Song, J.-W.; Jeon, E.-Y.; Song, D.-H.; Jang, H.-Y.;

284

Bornscheuer, U. T.; Oh, D.-K.; Park, J.-B. Angew. Chem. Int. Ed. 2013, 52, 2534-2537;

285

(d) Jang, H.-Y.; Singha, K.; Kim, H.-H.; Kwon, Y.-U.; Park, J.-B. Green Chem. 2016,

286

18, 1089-1095; (e) Schrewe, M.; Julsing, M. K.; Lange, K.; Czarnotta, E.; Schmid, A.;

287

Buhler, B. Biotechnol. Bioeng. 2014, 111, 1820-1830; (f) Ladkau, N.; Assmann, M.;

288

Schrewe, M.; Julsing, M. K.; Schmid, A.; Buhler, B. Meta. Eng. 2016, 36, 1-9; (g)

289

Bornscheuer, U. T. Eur. J. Lipid Sci. Technol. 2014, 116, 1322-1331; (h) Otte, K. B.;

290

Kirtz, M.; Nestl, B. M.; Hauer, B. ChemSusChem 2013, 6, 2149-2156.

291

4.

(a) Engleder, M.; Pavkov-Keller, T.; Emmerstorfer, A.; Hromic, A.; Schrempf, S.;

292

Steinkellner, G.; Wriessnegger, T.; Leitner, E.; Strohmeier, G. A.; Kaluzna, I.; Mink, D.;

293

Schurmann, M.; Wallner, S.; Macheroux, P.; Gruber, K.; Pichler, H. ChemBioChem

294

2015, 16, 1730-1734; (b) Joo, Y.-C.; Seo, E.-S.; Kim, Y.-S.; Kim, K.-R.; Park, J.-B.; Oh,

295

D.-K. J. Biotechnol. 2012, 158, 17-23; (c) Hirata, A.; Kishino, S.; Park, S.-B.; Takeuchi,

296

M.; Kitamura, N.; Ogawa, J. J. Lipid Res. 2015, 56, 1340-1350; (d) Kang, W.-R.; Seo,

297

M.-J.; Shin, K.-C.; Park, J.-B.; Oh, D.-K. Biotechnol. Bioeng. 2016, in press.

298

http://dx.doi.org/10.1002/bit.26058; (e) Kim, B. J.; Shin, K. C.; Oh, D. K. J. Microbiol.

299

Biotechnol. 2014, 24, 359-62.

300

5.

(a) Kim, K.-R.; An, J.-U.; Lee, S.-H.; Oh, D.-K. PLoS One 2015, 10, e0137785; (b)

301

Kim, K.-R.; Seo, M.-H.; Park, J.-B.; Oh, D.-K. J. Mol. Catal. B: Enzym. 2014, 104, 56-

302

63; (c) Jeon, E.-Y.; Lee, J.-H.; Yang, K.-M.; Joo, Y.-C.; Oh, D.-K.; Park, J.-B. Process

303

Biochem. 2012, 47, 941-947.

304 305

6.

(a) Lu, W. H.; Ness, J. E.; Xie, W. C.; Zhang, X. Y.; Minshull, J.; Gross, R. A. J. Am. Chem. Soc. 2010, 132, 15451-15455; (b) Hlavica, P.; Lehnerer, M. Curr. Drug Metab. 13


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

306

2010, 11, 85-104; (c) Julsing, M. K.; Cornelissen, S.; Buhler, B.; Schmid, A. Curr. Opin.

307

Chem. Biol. 2008, 12, 177-186; (d) Schrewe, M.; Julsing, M. K.; Buhler, B.; Schmid, A.

308

Chem. Soc. Rev. 2013, 42, 6346-6377.

309

7.

Niehaus, W. G., Jr.; Frielle, T.; Kingsley, E. A., Jr. J. Bacteriol. 1978, 134, 177-183.

310

8.

Oh, H.-J.; Kim, S.-U.; Song, J.-W.; Lee, J.-H.; Kang, W.-R.; Jo, Y.-S.; Kim, K.-R.;

311 312

Bornscheuer, U. T.; Oh, D.-K.; Park, J.-B. Adv. Synth. Catal. 2015, 357, 408-416. 9.

Goto, T.; Kim, Y.-I.; Furuzono, T.; Takahashi, N.; Yamakuni, K.; Yang, H.-E.; Li, Y.;

313

Ohue, R.; Nomura, W.; Sugawara, T.; Yu, R.; Kitamura, N.; Park, S.-B.; Kishino, S.;

314

Ogawa, J.; Kawada, T. Biochem. Biophys. Res. Commun. 2015, 459, 597-603.

315

10.

316 317

Baek, A. H.; Jeon, E.-Y.; Lee, S.-M.; Park, J.-B. Biotechnol. Bioeng. 2015, 112, 889895.

11.

(a) Song, J.-W.; Lee, J.-H.; Bornscheuer, U. T.; Park, J.-B. Adv. Synth. Catal. 2014, 356,

318

1782-1788; (b) Koppireddi, S.; Seo, J.-H.; Jeon, E.-Y.; Chowdhury, P. S.; Jang, H.-Y.;

319

Park,

320

http://dx.doi.org/10.1002/adsc.201600216.

J.-B.;

Kwon,

Y.-U.

Adv.

Synth.

Catal.

2016,

in

press.

321

12.

Karmakar, G.; Ghosh, P. ACS Sustain. Chem. Eng. 2015, 3, 19-25.

322

13.

Jung, S. M.; Seo, J. H.; Lee, J. H.; Park, J. B.; Seo, J. H. Biotechnol. J. 2015, 10, 1887-

323 324

1893. 14.

325 326

(a) Schorken, U.; Kempers, P. Eur. J. Lipid Sci. Technol. 2009, 111, 627-645; (b) Köckritz, A.; Martin, A. Eur. J. Lipid Sci. Technol. 2011, 113, 83-91.

15.

327

Seo, J.-H.; Kim, H.-H.; Jeon, E.-Y.; Song, Y.-H.; Shin, C.-S.; Park, J.-B. Sci. Rep. 2016, 6, 28223.

328

16.

van den Berg, B. Curr. Opin. Struct. Biol. 2005, 15, 401-407.

329

17.

de Maria, P. D.; Sanchez-Montero, J. M.; Sinisterra, J. V.; Alcantara, A. R. Biotechnol.

330

Adv. 2006, 24, 180-196.

331

18.

332

19. (a) Ozmen, E. Y.; Yilmaz, M. Colloids Surf., B 2009, 69, 58-62; (b) Cavalcanti-Oliveira,

333

E. d. A.; da Silva, P. R.; Ramos, A. P.; Aranda, D. A.; Freire, D. M. Enzyme Res. 2011,

334

2011, 618692.

335 336

20.

Fernandez-Lafuente, R. J. Mol. Catal. B: Enzym. 2010, 62, 197-212.

Custodio, L.; Soares, F.; Pereira, H.; Barreira, L.; Vizetto-Duarte, C.; Rodrigues, M. J.; Rauter, A. P.; Albericio, F.; Varela, J. J. Appl. Phycol. 2014, 26, 151-161. 14


Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

337

ACS Catalysis

21.

338

Xu, X.; Kim, J. Y.; Cho, H. U.; Park, H. R.; Park, J. M. Chem. Eng. J. 2015, 264, 735743.

339

22.

Kim, K.-R.; Oh, D.-K. Biotechnol. Adv. 2013, 31, 1473-1485.

340

23.

Bevers, L. E.; Pinkse, M. W. H.; Verhaert, P.; Hagen, W. R. J. Bacteriol. 2009, 191,

341 342

5010-5012. 24.

343 344

Takeuchi, M.; Kishino, S.; Hirata, A.; Park, S. B.; Kitamura, N.; Ogawa, J. J. Biosci. Bioeng. 2015, 119, 636-641.

25.

(a) Song, J. W.; Woo, J. M.; Jung, G. Y.; Bornscheuer, U. T.; Park, J. B. Sci. Rep. 2016,

345

6, 10; (b) Jeon, E.-Y.; Baek, A.-H.; Bornscheuer, U. T.; Park, J.-B. Appl. Microbiol.

346

Biotechnol. 2015, 99, 6267-6275.

347

26.

348

Jang, H.-Y.; Jeon, E.-Y.; Baek, A. H.; Lee, S.-M.; Park, J.-B. Process Biochem. 2014, 49, 617-622.

349

27. (a) Feng, Y. J.; Cronan, J. E. Plos One 2012, 7; (b) Fujita, Y.; Matsuoka, H.; Hirooka, K.

350

Mol. Microbiol. 2007, 66, 829-839; (c) Pauli, G.; Ehring, R.; Overath, P. J. Biotechnol.

351

1974, 117, 1178-1183.

352

28.

(a) Desbois, A. P.; Smith, V. J. Appl. Microbiol. Biotechnol. 2010, 85, 1629-1642; (b)

353

Woo, J.-M.; Kim, J.-W.; Song, J.-W.; Blank, L. M.; Park, J.-B. PLoS ONE 2016,

354

accepted.

355

29.

356 357

94, 907-915. 30.

358 359

Joo, Y.-C.; Jeong, K.-W.; Yeom, S.-J.; Kim, Y.-S.; Kim, Y.; Oh, D.-K. Biochimie 2012,

Kim, B.-N.; Joo, Y.-C.; Kim, Y.-S.; Kim, K.-R.; Oh, D.-K. Appl. Microbiol. Biotechnol. 2012, 95, 929-937.

31.

O'Connell, K. J.; Motherway, M. O.; Hennessey, A. A.; Brodhun, F.; Ross, R. P.;

360

Feussner, I.; Stanton, C.; Fitzgerald, G. F.; van Sinderen, D. Bioengineered 2013, 4,

361

313-321.

362 363

15


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

364

Table 1. Oleic acid/olive oil biotransformation activity of the recombinant Escherichia coli-

365

based biocatalysts Biotransformation 1 a Biotransformation 2 a

E. coli BL21(DE3)

TLL and E. coli

pACYC-ADH-FadL-

BL21(DE3) pACYC-

OhyA, pJOE-

ADH-FadL-OhyA,

E6BVMO

pJOE-E6BVMO

Oleic acid

Oleic acid

Olive oil

1.0±0.1

6.3±0.3

6.4±0.3

0.2±0.01

1.2±0.1

1.1±0.1

1.3±0.1

3.9±0.1

10.3±0.8

E. coli BL21(DE3)

Biocatalyst(s)

pACYC-ADH-OhyA, pJOE-E6BVMO

Substrate

Biotransformation 3 a

Specific ester production rate (U/g dry cells)

b

Volumetric productivity (mM/h) b

Final ester concentration (mM) b

366

a

367

respectively.

368

b

369

the product concentration, which was determined by gas chromatography/liquid

370

chromatography (GC/MS), and biotransformation time, which was measured when > 90% of

371

the starting material was converted to the products. The ester concentration at the

372

Biotransformation 3 was calculated as sum of the ester (5) concentration and the C9 acid (7)

373

concentration.

Biotransformation 1, 2, and 3 indicates the experiment shown in Figs. 2A, 2B, and 3,

The specific ester production rate and the volumetric productivity were calculated based on

374 16


Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

375

17


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

376

Table 2. Comparison of OhyA2 to other fatty acid double-bond hydratases reported

Amino acid accession number

Strain

WP_017356052

Stenotrophomonas

(OhyA2)

maltophilia

WP_024958135

Stenotrophomonas

(OhyA)

maltophilia

KX162589

GQ_144652

NC_011999

ZP_07049769

WP_015438992 377

Page 18 of 29

Stenotrophomonas nitritireducens Elizabethkingia meningoseptica Macrococcus caseolyticus Lysinibacillus fusiformis Bifidobacterium brevis NCFB 2258

Identity (%)a

Specific activity (µmol/min/mg)

Reference

to oleic acid

to linoleic acid

100

5.36 ± 0.07

1.67 ± 0.19

This study

37

3.69 ± 0.02

0.91 ± 0.00

Joo et al.4b

72

1.70±0.16

1.51±0.09

59

0.21 ± 0.02

NR

38

3.30 ± 0.01

0.06 ± 0.00

37

1.58 ± 0.00

0.11 ± 0.00

36

NR

NR

NR: not reported

378

18


Kang et al.4d Engleder et al.4a Joo et al.29 Kim et al.30 O’Connell et al.31

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

379 380

ACS Catalysis

Table 3. Linoleic acid/soybean oil biotransformation activity of the recombinant Escherichia coli-based biocatalysts

Biocatalyst(s)

Substrate

Biotrans 1 a

Biotrans 2 a

Biotrans 3 a

Biotrans 4 a

E. coli

E. coli

E. coli

TLL and E. coli

BL21(DE3)

BL21(DE3)

BL21(DE3)

BL21(DE3)

pACYC-ADH-

pACYC-ADH-

pACYC-ADH-

pACYC-ADH-

OhyA, pJOE-

FadL-OhyA,

FadL-OhyA2,

FadL-OhyA2,

E6BVMO

pJOE-E6BVMO

pJOE-E6BVMO

pJOE-E6BVMO

Linoleic acid

Linoleic acid

Linoleic acid

Soybean oil

0.4

4.0±0.2

6.7±0.6

5.7±0.6

0.08

0.7±0.03

1.2±0.1

1.0±0.1

0.4

3.9±0.2

4.2±0.2

9.1±0.5

Specific ester production rate (U/g dry cells)

b

Volumetric productivity (mM/h) b

Final ester concentration (mM) b

381

a

382

respectively.

383

b

384

the product concentration, which was determined by gas chromatography/liquid

385

chromatography (GC/MS), and biotransformation time, which was measured when > 90% of

386

the starting material was converted to the products. The ester concentration at the Biotrans 4

387

was calculated as sum of the ester (12) concentration and the C9 acid (7) concentration.

Biotransformation 1, 2, 3, and 4 indicates the experiment shown in Figs. S3, 5A, 5B, and 6,

The specific ester production rate and the volumetric productivity were calculated based on

388 19


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

389

Figure legends

390

Fig. 1. Designed biotransformation pathway to produce C9 carboxylic acids from olive oil.

391

This pathway was designed on a basis of our previous studies.3c, 11a

392 393

Fig. 2. Time course of the biotransformation of oleic acid into the ester (5). Oleic acid was

394

added to a concentration of 5 mM into culture broth of the recombinant E. coli BL21(DE3)

395

pACYC-ADH-OhyA, pJOE-E6BVMO, expressing the fatty acid double-bond hydratase

396

(OhyA) of Stenotrophomonas maltophilia, the long chain secondary alcohol dehydrogenase

397

(ADH) of Micrococcus luteus, and the engineered Baeyer-Villiger monooxygenase

398

(E6BVMO) of Pseudomonas putida KT2440 at the stationary growth phase (cell density: 3 g

399

dry cells/L) (A). Oleic acid was added to a concentration of 5 mM into culture broth of the

400

recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-E6BVMO, expressing the

401

long chain fatty acid transporter (FadL) in addition to the OhyA of S. maltophilia, the ADH of

402

M. luteus, and the engineered BVMO of P. putida KT2440 at the stationary growth phase

403

(cell density: 3 g dry cells/L) (B). Oleic acid was added to a concentration of 60 mM into

404

culture broth of the recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-

405

E6BVMO after high cell density cultivation (cell concentration: 25 g dry cells/L) (C). The

406

error bars indicate standard deviations. The symbols indicate concentration of oleic acid (2)

407

(filled circle), 10-hydroxyoctadecanoic acid (3) (open triangle up), 12-ketooctadecanoic acid

408

(4) (open triangle down), and the ester (5) (filled square).

409 410

Fig. 3. Time course of the simultaneous enzyme/whole cell biotransformation of olive oil into

411

9-hydroxynonanoic acid (7). Olive oil and the lipase from Thermomyces lanuginosus (TLL) 20


Page 20 of 29

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

412

were added to a concentration of 5 g/L and 10 U/mL, respectively, into culture broth of the

413

recombinant E. coli BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-E6BVMO at the

414

stationary growth phase (cell density: 3 g dry cells/L). The error bars indicate standard

415

deviations. The symbols indicate concentration of olive oil (1) (open square), oleic acid (2)

416

(filled circle), 10-hydroxyoctadecanoic acid (3) (open triangle up), 12-ketooctadecanoic acid

417

(4) (open triangle down), the ester (5) (filled square), and 9-hydroxynonanoic acid (7) (filled

418

triangle up).

419 420

Fig. 4. Designed biotransformation pathway to produce C9 carboxylic acids from linoleic

421

acid. This pathway was designed on a basis of our previous studies.3c, 11a

422 423

Fig. 5. Time course of the biotransformation of linoleic acid into the ester (12). Linoleic acid

424

was added to a concentration of 5 mM into culture broth of the recombinant E. coli

425

BL21(DE3) pACYC-ADH-FadL-OhyA, pJOE-E6BVMO, expressing the OhyA of S.

426

maltophilia, the ADH of M. luteus, the engineered BVMO of P. putida KT2440, and the FadL

427

of E. coli at the stationary growth phase (cell density: 3 g dry cells/L) (A). Linoleic acid was

428

added to a concentration of 5 mM into culture broth of the recombinant E. coli BL21(DE3)

429

pACYC-ADH-FadL-OhyA2, pJOE-E6BVMO, expressing the newly cloned oleate hydratase

430

OhyA2 of S. maltophilia, the ADH of M. luteus, the engineered BVMO of P. putida KT2440,

431

and the FadL of E. coli at the stationary growth phase (cell density: 3 g dry cells/L) (B). The

432

error bars indicate standard deviations. The symbols indicate concentration of linoleic acid (9)

433

(open circle), 10-hydroxyoctadec-9-enoic acid (10) (open triangle up), 12-ketooctadec-9-

434

enoic acid (11) (open triangle down), and the ester (12) (filled square).

435 21


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

436

Fig. 6. Time course of the simultaneous enzyme/whole cell biotransformation of soybean oil

437

into 9-hydroxynonanoic acid (7). Soybean oil and TLL were added to a concentration of 5

438

g/L and 20 U/mL, respectively, into culture broth of the recombinant E. coli BL21(DE3)

439

pACYC-ADH-FadL-OhyA2, pJOE-E6BVMO, expressing the newly cloned oleate hydratase

440

OhyA2 of S. maltophilia, the ADH of M. luteus, the engineered BVMO of P. putida KT2440,

441

and the FadL of E. coli at the stationary growth phase (cell density: 3 g dry cells/L). The error

442

bars indicate standard deviations. The symbols indicate the concentration of soybean oil

443

(filled diamond), oleic acid (2) (filled circle), linoleic acid (9) (open circle), 10-

444

hydroxystearic acid (3) (open triangle up), 10-hydroxy-12-octadecenoic acid (10) (open

445

diamond), 10-ketostearic acid (4) (open triangle down), 10-keto-12-octadecenoic acid (11)

446

(filled triangle down), the ester from oleic acid (i.e., 9-nonanoyloxynonanoic acid) (5) (filled

447

square), the ester from linoleic acid (i.e., 9-(Z)-non-3-enoyloxynonanoic acid) (12) (open

448

square), and 9-hydroxynonanoic acid (7) (filled triangle up).

449

22


Page 22 of 29

Page 23 of 29

450

Fig. 1 Olive oil (1) L L T

e s a p i L

O 2 H

(

)

OH

(

O

A y h O

e s a t a r d y H

O 2 H

2

)

OH OH +

D A N

e s a n e g o r d y h e d l o h o c l A

H D A

O (

3

)

O OH O

(

O M V B

4

e s a n e g y x o o n o m r e g i l l i V r e y e a B

2

O , H P D A N

)

O OH

O O (

6

)

+

OH

L L T

5

e s a p i L

O 2 H

HO

OH

O

7

O

n o i t a d i x o c i t a m y z n e r o l a c i m e h C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

HO 451

OH O

452

23


O

8

ACS Catalysis

453

Fig. 2 A Concentration (mM)

5

4

3

2

1

0 0

2

4

6

8

Reaction time (h)

B

5

Concentration (mM)

4

3

2

1

0 0

2

4

6

8

6

8

Reaction time (hr)

C 60

50

Concentration (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

40

30

20

10

0 0

454

2

4

Reaction time (h)

455

24


Page 25 of 29

456 457

Fig. 3

16

12

4

3 8 2 4 1

0

0 0

458

2

4

6

Reaction time (h)

25


8

Fatty acid concentration (mM)

5

Olive oil concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

ACS Catalysis

459

Fig. 4 OH O

(

2 y h O

e s a t a r d y H

O 2 H

9

)

OH OH +

D A N

e s a n e g o r d y h e d l o h o c l A

H D A

O (

10

)

O OH O

(

O O

13

)

+

460

L L T

e s a p i L

O 2 H

(

OH

)

OH

O

12

O M V B

e s a n e g y x o o n o m r e g i l l i V r e y e a B

2

11

O , H P D A N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

HO

O

OH O

461

26


7

Page 27 of 29

462

Fig. 5

A

6

Concentration (mM)

5

4

3

2

1

0 0

2

4

6

8

Reaction time (h)

B

6

5

Concentration (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

4

3

2

1

0 0

463

2

4

6

8

Reaction time (h)

464

27


ACS Catalysis

Fig. 6

10

10 Soybean oil Oleic acid (2) Linoleic acid (9) 9-Nonanoyloxynonanoic acid (5) 9-(Z)-Non-3-enoyloxynonanoic acid (12) 9-Hydroxynonanoic acid (7)

8

8

6

6

4

4

2

2

0

0 0

466

2

4

6

Reaction time (h)

467

28


8

Fatty acid concentration (mM)

465

Soybean oil concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 29

Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

468 469

29


Whole-Cell ... - ACS Publications

Recommend Documents