Oxyfunctionalization of plant oils such as olive oil and soybean oil into C9 carboxylic acids (e.g., n-nonanoic acid and 9-hydroxynonanoic acid) was investigated. The biotransformation was composed of hydrolysis of plant oils by the Thermomyces lanuginosus lipase (TLL) and C9−C10 double-bond cleavage in unsaturated fatty acids by a serial reaction of a fatty acid double bond-hydratase of Stenotrophomonas maltophilia, an alcohol dehydrogenase of Micrococcus luteus, and a Baeyer−Villiger monooxygenase (BVMO) of Pseudomonas putida KT2440 expressed in Escherichia coli. The newly cloned oleate hydratase allowed one to produce 10-hydroxyoctadecanoic acid and 10hydroxyoctadec-12-enoic acid at a high rate from oleic acid and linoleic acid, respectively, which are major fatty acid constituents of many plant oils. Furthermore, overexpression of a long chain fatty acid transporter FadL in the recombinant E. coli led to a significant increase of whole-cell biotransformation rates of oleic acid and linoleic acid into the corresponding esters. The resulting esters (the BVMO reaction products) were hydrolyzed in situ by TLL, generating nonanoic acid, non-3-enoic acid, and 9-hydroxynonanoic acid, which can be further oxidized to 1,9-nonanedioic acid. This study demonstrated that industrially relevant C9 carboxylic acids could be produced from olive oil or soybean oil by simultaneous enzyme/whole-cell biocatalysis.

Hydroxy fatty acids are used as starting materials for the production of secondary metabolites and signalling molecules as well as in the manufacture of industrial fine chemicals. However, these compounds are usually difficult to produce from renewable biomass by chemical means. In this study, linoleate double bond hydratases of Lactobacillus acidophilus NBRC 13951 were cloned for the first time. These enzymes were highly specific for the hydration of the C‐9 or the C‐12 double bond of unsaturated fatty acids (e.g., linoleic acid). Thereby, the enzymes allowed the selective production of hydroxy fatty acids such as 13‐hydroxy‐cis‐9‐octadecenoic acid and 10‐hydroxy‐cis‐12‐octadecenoic acid from linoleic acid. In addition, the hydroxy fatty acids were further converted into industrially relevant carboxylic acids (e.g., 12‐hydroxy‐cis‐9‐dodecenoic acid, α,ω‐tridec‐9‐enedioic acid) and lactones (i.e., δ‐decalactone, γ‐dodecelactone) via whole‐cell biocatalysis using an enzyme cascade. This study thus contributes to the preparation of hydroxy fatty acids, unsaturated carboxylic acids, and lactones from renewable unsaturated fatty acids.magnified image

Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and -hydroxycarboxylic and ␣, -dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, ␣-linolenic acid, and ␥-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of ␥-linolenic acid, was the highest among all reported OhyAs. IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, ␣-linolenic acid, and ␥-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials. KEYWORDS Stenotrophomonas maltophilia, enzyme characterization, oleate hydrataseO leate hydratase (OhyA) converts unsaturated fatty acids to 10-hydroxy fatty acids, which are then converted to ␥-lactones by the yeasts Candida boidinii and Waltomyces lipofer (1, 2). ␥-Lactones are flavor compounds used in food and cosmetics (3). Multistep enzymatic reactions involving OhyA, alcohol dehydrogenase, Baeyer-Villiger monooxygenases, and esterase convert unsaturated fatty acids to -hydroxycarboxylic and ␣, -dicarboxylic acids (4), which are used in the preparation of resins, nylons, plastics, and lubricants (5). An effective OhyA is necessary for the efficient production of ␥-lactones, -hydroxycarboxylic acids, and ␣, -dicarboxylic acids.

Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis-9 polyunsaturated fatty acids (PUFAs) to 10-hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double-bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double-bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full-length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10-hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α-linolenic acid and/or γ-linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10-hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA-containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74-82. © 2016 Wiley Periodicals, Inc.