Whole-Cell Biotransformation of Plant Oils into

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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

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Simultaneous enzyme/whole-cell biotransformation of plant oils

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into C9 carboxylic acids

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

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

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

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

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†

These authors are equally contributed first authors.

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*

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

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

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

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

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mail: [email protected]).

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

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ACS Paragon Plus Environment

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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simultaneous enzyme/whole-cell biocatalysis

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Introduction

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

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

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

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biodiesel and (bio)chemicals.

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

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

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

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

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

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

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

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

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

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functions.4c, 9

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

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

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

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

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

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

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nonanedioic acid and 9-aminnonanoic acid.11

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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


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

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

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

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

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13

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

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

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

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

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

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via ozonolysis in chemical industry.14

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

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Results and Discussion

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Whole-cell biotransformation of oleic acid into ester

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

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

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

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

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

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

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

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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


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

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

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

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

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

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

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

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

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

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

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

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

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rate.

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

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

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

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

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

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

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

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

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

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membrane. 5


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

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

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

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

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

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

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

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

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applied to the further biotransformations.

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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by the simultaneous enzyme/whole-cell biocatalysis.

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

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

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

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

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

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

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

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

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

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

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

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biocatalysts.

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Cloning of a novel fatty acid double-bond hydratase

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


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

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

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

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

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

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

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

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

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

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

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

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maltophilia but more similar to the oleate hydratase of Stenotrophomonas nitritireducens

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

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

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

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

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

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

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Whole-cell biotransformation of linoleic acid into ester

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

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

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


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

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

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

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

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

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

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

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under aerobic condition4a, 24.

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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in addition to the ADH and the BVMO as biocatalysts.

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

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

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

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

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

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

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

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

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biotransformations (Tables 1 and 3).

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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biotransformation.

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

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

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

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


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

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

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biotransformation of other renewable oils.

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Conclusions

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

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

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

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

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

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productivity of whole-cell biotransformations were significantly improved by overexpression

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

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

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and linoleic acid).

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Acknowledgements

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

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

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

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Womans University.

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Supporting information

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


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26.

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354

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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,

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31.

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

360

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361

313-321.

362 363

15


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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


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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

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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 Biotransformation of Plant Oils into

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