Production of Novel Tetrahydroxyfuranyl Fatty Acids from α-Linolenic Acid by Clavibacter sp. Strain ALA2

Abstract

Previously, it was reported that a newly isolated microbial culture, Clavibacter sp. strain ALA2, produced trihydroxy unsaturated fatty acids, diepxoy bicyclic fatty acids, and tetrahydroxyfuranyl fatty acids (THFAs) from linoleic acid (C. T. Hou, J. Am. Oil Chem. Soc. 73:1359-1362, 1996; C. T. Hou and R. J. Forman III, J. Ind. Microbiol. Biotechnol. 24:275-276, 2000; C. T. Hou, H. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 75:1483-1487, 1998; C. T. Hou, H. W. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 78:1167-1169, 2001). In this study, we found that Clavibacter sp. strain ALA2 produced novel THFAs, including 13,16-dihydroxy-12-THFA, 15-epoxy-9(Z)-octadecenoic acid (13,16-dihydroxy-THFA), and 7,13,16-trihydroxy-12, 15-epoxy-9(Z)-octadecenoic acid (7,13,16-trihydroxy-THFA), from α-linolenic acid (9,12,15-octadecatrienoic acid). The chemical structures of these products were determined by gas chromatography-mass spectrometry and proton and ¹³C nuclear magnetic resonance analyses. The optimum incubation temperature was 30°C for production of both hydroxy-THFAs. 13,16-Dihydroxy-THFA was detected after 2 days of incubation, and the concentration reached 45 mg/50 ml after 7 days of incubation; 7,13,16-trihydroxy-THFA was not detected after 2 days of incubation, but the concentration reached 9 mg/50 ml after 7 days of incubation. The total yield of both 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA was 67% (wt/wt) after 7 days of incubation at 30°C and 200 rpm. In previous studies, it was reported that Clavibacter sp. strain ALA2 oxidized the C-7, C-12, C-13, C-16, and C-17 positions of linoleic acid (n-6) into hydroxy groups. In this case, the bond between the C-16 and C-17 carbon atoms is saturated. In α-linolenic acid (n-3), however, the bond between the C-16 and C-17 carbon atoms is unsaturated. It seems that enzymes of strain ALA2 oxidized the C-12-C-13 and C-16-C-17 double bonds into dihydroxy groups first and then converted them to hydroxy-THFAs.

Hydroxy fatty acids are important industrial materials. They are used in plasticizers, surfactants, lubricants, and additives and in the manufacture of paints because the hydroxy group gives materials special properties, such as higher viscosity and reactivity to fatty acids (13). Furthermore, some hydroxy fatty acids are known to have interesting biological activities. For example, Kato et al. (20) and other researchers (3, 16, 25) reported that hydroxy fatty acids have antifungal activity. A few hydroxy fatty acids also exhibit cytotoxic activity against cancer cells (21, 26) and prostaglandin E-like activity (29).

Recently, production of hydroxy fatty acids through bioconversion by microorganisms has been a major focus of research (11). A number of microbial systems that convert oleic acid to monohydroxy and dihydroxy fatty acids have been found (1, 2, 6, 10, 14, 15, 27). Bioconversion of polyunsaturated fatty acids, such as linoleic acid and α- and γ-linolenic acids, has also been studied (10, 22, 28, 30). Recently, it was found that Clavibacter sp. strain ALA2, which is a gram-positive nonmotile rod isolated from a dry soil, converts linoleic acid to trihydroxy fatty acid (12). Strain ALA2 performs unique reactions and places hydroxyl groups at the C-12, C-13, and C-17 positions of linoleic acid. In addition, it also hydroxylates the C-7 and C-16 positions to a small extent (8, 17, 18). In previous studies, we and other workers identified eight new hydroxy fatty acids, including 12,13,17-trihydroxy-9(Z)-octadecenoic acid, 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid, and 12,17;13,17-diepoxy-9(Z)-octadecenoic acid (DEOA), produced from linoleic acid by Clavibacter sp. strain ALA2 (8, 12, 17). The unique structures of these novel cyclic fatty acids (tetrahydrofuranyl fatty acids [THFAs] and DEOA) mean that the compounds have the potential to be used in the biomedical as well as specialty chemical industries.

In this study, we investigated bioconversion of α-linolenic acid (n-3) by Clavibacter sp. strain ALA2. Novel THFAs were isolated, and their chemical structures were identified by gas chromatography (GC)-mass spectrometry (MS) and proton and ¹³C nuclear magnetic resonance (NMR) analyses as 13,16-dihydroxy-12,15-epoxy-9(Z)-octadecenoic acid (13,16-dihydroxy-THFA) and 7,13,16-trihydroxy-12,15-epoxy-9(Z)-octadecenoic acid (7,13,16-trihydroxy-THFA). Optimum conditions for production of THFAs were also investigated.

MATERIALS AND METHODS

Microorganism.

Clavibacter sp. strain ALA2 (= NRRL B-21660), which was isolated from a soil sample collected from McColla, Ala. (12), was aerobically cultivated at 30°C with shaking in 50 ml of culture medium at 200 rpm. The medium used contained (per liter) 5 g of dextrose, 15 g of yeast extract, 10 g of tryptone, 5 g of K₂HPO₄, 0.5 g of MgSO₄ · 7H₂O, 0.01 g of FeSO₄ · 7H₂O, 0.014 g of ZnSO₄, 0.008 g of MnSO₄ · H₂O, and 0.01 g of nicotinic acid. The pH of the medium was adjusted to 6.8 with dilute phosphoric acid.

Chemicals.

α-Linolenic acid (9,12,15-octadecatrienoic acid; purity, >99% [as determined by GC]) was purchased from Nu Chek Prep Inc. (Elysian, Minn.). A Silica Gel 60 F₂₅₄ thin-layer chromatography (TLC) plate was purchased from EM Science (Cherry Hill, N.J.). All chemicals were obtained from commercial sources.

Bioconversion.

Bioconversion was carried out by adding 70 to 250 μl of α-linolenic acid to a 1-day-old culture (50 ml) in a 125-ml Erlenmeyer flask and incubating it with shaking at 200 rpm and 30°C for 2 to 7days. At the end of the reaction, the culture broth was acidified to pH 2 with 6 N hydrochloric acid. The crude lipid fraction was extracted from the culture broth with 2 volumes of ethyl acetate, followed by diethyl ether. The solvent was removed from the combined extracts with a rotary evaporator.

Product analyses.

Crude extracts obtained from the culture of Clavibacter sp. strain ALA2 incubated with α-linolenic acid were methylated with diazomethane. The methyl ester derivative was analyzed with a Hewlett-Packard model 5890 GC equipped with a flame ionization detector, a Spelco (Bellafonte, Pa.) SPB-1 capillary column (15 m by 0.32 mm [inside diameter]; thickness, 0.25 μm), and a Hewlett-Packard model 6890 integrator. The column temperature was raised from 170 to 210°C at a rate of 3°C/min and then kept at 210°C. The injector and detector temperatures were 240 and 250°C, respectively. For quantitative analysis, palmitic acid was added as an internal standard prior to solvent extraction. A linear relationship was established for the peak area ratios of products to methyl palmitate.

Isolation and identification of the product with a GC RT of 17.8 min.

The crude extract was treated with diazomethane and was then subjected to preparative silica gel TLC to isolate the methyl ester of a product with a GC retention time (RT) of 17.8 min. Ethyl acetate was used as the development solvent. A band on the TLC plate (R_f, 0.69) detected with UV light was scraped off with a razor blade, and the material was eluted with ethyl acetate. The product band on the TLC plate was also confirmed by cutting off a small side of the TLC plate, and the corresponding product band was developed in iodine vapor. The recovered product was purified further by a second TLC performed with a different development solvent, n-hexane-ethyl acetate (1:4, vol/vol) (R_f, 0.64). The purity of the purified product was 85.5%, as determined by GC analysis. The chemical structure of the product with a GC RT of 17.8 min was determined by GC-MS and NMR. For GC-MS, silylation of the methylated product was accomplished by using a mixture of trimethylsilyl (TMSi) ether and pyridine (1:4, vol/vol) purchased from Supelco (Bellefonte, Pa.) for 30 min at room temperature. GC-MS analysis was performed with a Hewlett-Packard model 5890 GC interfaced with a model 5971 mass selective detector operating at 70 eV. The capillary column used was a Hewlett-Packard HP-5-MS cross-linked 5% phenylmethyl silicone column (30 m by 0.25 mm [inside diameter]; film thickness, 0.25 μm). The carrier gas was helium, and its flow rate was 0.65 ml/min. The temperature of the GC column was programmed to increase from 65 to 260°C at a rate of 20°C/min and then kept at 260°C for 20 min. NMR spectra were obtained with a model ARX-400 spectrometer (Bruker, Billerica, Mass.) equipped with a 5-mm ¹³C/¹H dual probe (¹³C NMR, 100 MHz; ¹H NMR, 400 MHz). NMR spectra were recorded with CDCl₃ as the internal standard and solvent. Isolated methyl esterified product was hydrogenated by stirring it in 2 ml of methanol under a hydrogen atmosphere with 20 mg of 5% palladium on calcium carbonate catalyst (Aldrich, Milwaukee, Wis.)

Isolation and identification of the product with a GC RT of 25.6 min.

To isolate the product with a GC RT of 25.6 min, the crude extract was applied to a preparative silica gel TLC plate and was developed with methylene chloride-methanol (9:1, vol/vol). The product band on the TLC plate (R_f, 0.50) was recovered by using the method described above. The material was eluted from the silica gel with methanol. It was then methylated with diazomethane and was purified again by silica gel TLC with methylene chloride-methanol (95:5, vol/vol) as the development solvent (R_f, 0.35). The methyl ester of the product with a GC RT of 25.6 min was eluted with ethyl acetate from silica gel scraped from the TLC plate. The purity of the purified product was 89.0%, as determined by GC analysis. The chemical structure of the product was identified by a procedure similar to the procedure described above.

RESULTS

Bioconversion of α-linolenic acid by Clavibacter sp. strain ALA2.

The crude extract obtained from incubation of α-linolenic acid and Clavibacter sp. strain ALA2 in culture media for 7 days was methylated with diazomethane. GC analyses of the methyl esters of the crude extract revealed several product peaks. The GC RT of the main product was 17.8 min, and there was a minor product at 25.6 min (Fig. 1). Since the RTs of α-linolenic acid, ricinoleic acid [12-hydroxy-9(Z)-octadecenoic acid], and 9,10-dihydroxystearic acid were 8.1, 12.2, and 16.5 min, respectively, we predicted that these new products have two or more hydroxy groups in their fatty acid molecules. As a control, when α-linolenic acid was incubated with autoclaved Clavibacter sp. strain ALA2 for 7 days, neither the product with an RT of 17.8 min nor the product with an RT of 25.6 min was detected. In another control Clavibacter sp. strain ALA2 was incubated in culture media without α-linolenic acid for 7 days, and again, neither product was observed. Therefore, these two compounds were formed from α-linolenic acid by Clavibacter sp. strain ALA2 through bioconversion. Previously, it was also reported that no nonenzymatic dehydrative cyclization of hydroxy fatty acids was observed under the GC conditions which we used (8, 9, 19).

FIG. 1.

GC chromatograms of product methyl esters obtained from α-linolenic acid bioconversion by Clavibacter sp. strain ALA2. (A) Bioconversion of α-linolenic acid by Clavibacter sp. strain ALA2. Peak I, product with a GC RT of 17.8 min; peak II, product with a GC RT of 25.6 min. (B) Control incubation mixture containing α-linolenic acid and autoclaved 1-day-old Clavibacter sp. strain ALA2 culture.

Structure analyses of the product with a GC RT of 17.8 min.

The structure was identified by GC-MS and NMR analyses. The electron impact mass spectrum obtained from the methyl ester-O-TMSi (OTMSi) ether of the purified product (Fig. 2) had a molecular ion at m/z 486 [M]⁺ (relative intensity, 0.2). Fragment ions were interpreted as follows: m/z 396, [M-TMSiOH]⁺ (relative intensity, 2); m/z 381, [M-CH₃-TMSiOH]⁺ (relative intensity, 1); m/z 355, [M-CH₃CH₂CHO-TMSi]⁺ (relative intensity, 2); m/z 289, [M-CH₂CH=CH(CH₂)₇COOCH₃]⁺ (relative intensity, 10); m/z 265, [355-TMSiOH]⁺ (relative intensity, 8); m/z 199, [289-TMSiOH]⁺ (relative intensity, 50); m/z 131, [CH₂CH₂CHO-TMSi]⁺ (relative intensity, 80); and m/z 73, [TMSi]⁺ (relative intensity, 100). There were also many other ions (e.g., m/z 429, m/z 337, m/z 233, m/z 181, m/z 157, and m/z 145). The methyl ester was further hydrogenated by oxidation with pyridinium chlorochromate and applied to a GC-MS. The mass spectrum of the hydrogenated product showed that there was a gain of two hydrogens, which corresponded to one double bond, as follows: m/z 473, [M-CH₃]⁺ (relative intensity, 0.1); m/z 398, [M-TMSiOH]⁺ (relative intensity, 0.2); m/z 383, [473-TMSiOH]⁺ (relative intensity, 0.7); m/z 357, [M-CH₃CH₂CHOTMSi]⁺ (relative intensity, 0.9); and m/z 267, [357-TMSiOH]⁺ (relative intensity, 40) (the base ion m/z 73 had a relative intensity of 100). As expected, a fragment ion at m/z 131, [CH₂CH₂CHO-TMSi]⁺ (relative intensity, 100), which corresponded to a hydroxy at the C-16 position, was observed even after hydrogenation and the relative intensity was enhanced, while the fragment ions at m/z 289 and and m/z 199 observed after cleavage between tetrahydrofuranyl and the double bond became small after hydrogenation.

FIG. 2.

MS analysis of methyl ester-OTMSi ether of the product with a GC RT of 17.8 min obtained from α-linolenic acid bioconversion by Clavibacter sp. strain ALA2.

The structure of the product with a GC RT of 17.8 min was further confirmed by ¹³C and ¹H NMR analyses (Table 1). The ¹³C NMR signals at 71.0 ppm (C-13) and 74.6 ppm (C-16) corresponded to C-13 and C-16 hydroxy groups in the molecule. The positions of the hydroxy groups were also supported by ¹H NMR signals at 4.00 ppm (H-13) and 3.38 ppm (H-16). Resonance signals of the olefinic proton were observed at 5.47 ppm. ¹³C NMR signals at 131.8 ppm (C-9) and 125.1 ppm (C-10) also indicated the presence of a double bond between C-9 and C-10. Since the signals of methylene carbon next to the olefinic carbon were C-8 (27.1 ppm) and C-11 (26.8 ppm) signals, the double bond is in the cis configuration (7). ¹³C NMR signals at C-12 and C-15 were observed at 83.5 and 78.9 ppm, respectively, and corresponded to —CH. Based on these data, the structure of the product with a GC RT of 17.8 min is 13,16-dihydroxy-12,15-epoxy-9(Z)-octadecenoic acid (13,16-dihydroxy-THFA) (Fig. 2).

TABLE 1.

Proton and ¹³C NMR signals and molecular assignments for the methyl ester product with a GC RT of 17.8 min

Carbon	Resonance signals (ppm)
	Proton NMR	13C NMR
1		174.4
2	2.32 t (J = 7.6 Hz)	33.8
3	1.60 m	24.7
4	1.33 m	28.8
5	1.33 m	28.9
6	1.33 m	28.9
7	1.33 m	29.4
8	2.09 m	27.1
9	5.47 m	131.8
10	5.47 m	125.1
11	2.40 m	26.8
12	3.63 m	83.5
13	4.00 m	71.0
14	1.84 m (J = 14 Hz)	37.9
	2.40
15	4.00 m	78.9
16	3.38 m	74.6
17	1.60 m	26.7
18	0.99 t (J = 7.6 Hz)	10.0
OCH3	3.66 s	51.1

Structure analyses of the product with a GC RT of 25.6 min.

The chemical structure of the product with a GC RT of 25.6 min was also identified by GC-MS and NMR analyses. The mass spectrum of the methyl ester-OTMSi ether of the purified product was interpreted as follows: m/z 484, [M-TMSiOH]⁺ (relative intensity, 0.1); m/z 469, [M-CH₃-TMSiOH]⁺ (relative intensity, 0.1); m/z 394, [484-TMSiOH]⁺ (relative intensity, 0.2); m/z 379, [469-TMSiOH]⁺ (relative intensity, 0.3); m/z 353, [M-CH₃CH₂CHOTMS-TMSiOH]⁺ (relative intensity, 0.4); m/z 289, [M-CH₂CH=CH(CH₂)₇COOCH₃]⁺ (relative intensity, 2); m/z 263, [353-TMSiOH]⁺ (relative intensity, 0.9); m/z 231, [CHOTMSi(CH₂)₅COOCH₃]⁺ (relative intensity, 98); m/z 199, [289-TMSiOH]⁺ (relative intensity, 18); m/z 131, [CH₃CH₂CHO-TMSi]⁺ (relative intensity, 32); and m/z 73, [TMSi]⁺ (relative intensity, 100). In addition, there were many other ions (e.g., m/z 310, m/z 157, and m/z 145) (Fig. 3). A fragment ion at m/z 231 corresponded to a hydroxy residue at the C-7 position and was characteristic of the product with a GC RT of 25.6 min.

FIG. 3.

MS analysis of methyl ester-OTMSi ether of the product with a GC RT of 25.6 min obtained from α-linolenic acid bioconversion by Clavibacter sp. strain ALA2.

The hydroxy residue at the C-7 position was confirmed by a ¹³C NMR chemical shift at 70.9 ppm and ¹H NMR signals at 3.60 ppm (Table 2). The ¹³C NMR chemical shifts at the C-6 and C-8 positions were also shifted to higher fields at 36.9 ppm (C-6) and 34.8 ppm (C-8) compared to shifts of 13,16-dihydroxy-THFA. Other ¹³C NMR chemical shifts and ¹H NMR resonance patterns were very similar to those of 13,16-dihydroxy-THFA. Based on these data, the structure of the product with a GC RT of 25.6 min is 7,13,16-trihydroxy-12,15-epoxy-9(Z)-octadecenoic acid (7,13,16-trihydroxy-THFA).

TABLE 2.

Proton and ¹³C NMR signals and molecular assignments for methyl ester product with a GC RT of 25.6 min

Carbon	Resonance signals (ppm)
	Proton NMR	13C NMR
1		174.5
2	2.82 t (J = 7.5 Hz)	33.7
3	1.57 m	24.6
4	1.34 m	28.9
5	1.28 m	26.4
6	1.35 m	36.9
	1.45
7	3.60 m	70.9
8	2.37 m	34.8
	2.52
9	5.52 m	127.3
10	5.52 m	127.8
11	2.21 m	26.9
	2.31
12	3.70 m	83.1
13	4.03 m	70.6
14	1.81 m	37.5
15	3.98 m	79.6
16	3.37 m	74.6
17	1.62 m	25.1
18	0.98 t	9.9
OCH3	3.66 s	51.1

Time course.

Conversions were carried out at 30°C for various times after addition of α-linolenic acid to a 1-day-old culture of Clavibacter sp. strain ALA2. The amount of 13,16-dihydroxy-THFA in the culture medium increased gradually with incubation time and reached 45 mg/50 ml after 7 days of incubation (Fig. 4). On the other hand, 7,13,16-trihydroxy-THFA was not detected after 2 days of incubation and then was produced slowly and reached a concentration of 9 mg/50 ml after 7 days of incubation (Fig. 4). Since the production of 7,13,16-trihydroxy-THFA was slower than the production of 13,16-dihydroxy-THFA, hydroxylation at the C-7 position of α-linolenic acid might occur after hydroxylation at the C-12, C-13, and C-16 positions, similar to the biosynthetic pathways of linoleic acid (9, 19).

FIG. 4.

Time course of production of hydroxy-THFAs from α-linolenic acid by Clavibacter sp. strain ALA2.

Effect of incubation temperature.

To investigate the effect of incubation temperature on the production of 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA, Clavibacter sp. strain ALA2 cultures were incubated for 7 days at temperatures between 20 and 40°C after addition of α-linolenic acid (Fig. 5). The production of 13,16-dihydroxy-THFA increased with increasing temperature until 30°C and decreased markedly at temperatures above 30°C. After incubation at 40°C, no 13,16-dihydroxy-THFA was detected. The effect of incubation temperature on production of 7,13,16-trihydroxy-THFA was similar to the effect on 13,16-dihydroxy-THFA production. The optimal incubation temperature was 30°C for both products.

FIG. 5.

Effect of incubation for 7 days at different temperatures on the production of hydroxy-THFAs from α-linolenic acid by Clavibacter sp. strain ALA2. Solid bars, 13,16-dihydroxy-THFA; open bars, 7,13,16-trihydroyxy-THFA.

Effect of α-linolenic acid concentration.

Bioconversion was carried out for 7 days with Clavibacter sp. strain ALA2 in culture media containing α-linolenic acid at different concentrations (Table 3). The concentration of 13,16-dihydroxy-THFA was highest with 125 μl of α-linolenic acid per 50 ml. In the presence of 188 μl of α-linolenic acid per 50 ml, the amount of 13,16-dihydroxy-THFA produced was 15 mg/50 ml, and the concentration of α-linolenic acid remained 73 mg/50 ml after 7 days of incubation. In contrast, the concentration of 7,13,16-trihydroxy-THFA produced was highest (21 mg/50 ml) when 75 μl of substrate per 50 ml was added and decreased with increasing substrate concentration in the culture medium. The total yield of 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA in the presence of 75 μl of α-linolenic acid per 50 ml of culture medium was 67% (wt/wt) after 7 days of incubation.

TABLE 3.

Effect of α-linolenic acid content in the culture medium on bioconversion to hydroxy-THFAs by Clavibacter sp. strain ALA2

Substrate α-linolenic acid concn (mg/50 ml)	Concn of:
	13,16-dihydroxy-THFA (mg/50 ml)a	7,13,16-trihydroxy-THFA (mg/50 ml)b
60 (75)	19 (32)	21 (35)
100 (125)	45 (45)	9 (9)
150 (188)	15 (10)	3 (2)
200 (250)	14 (7)	0 (0)

^aThe values in parentheses are volumes (in microliters).

^bThe values in parentheses are yields, expressed as percentages and calculated as follows: yield = (amount of product/amount of substrate) × 100.

DISCUSSION

It is known that some microorganisms convert α-linolenic acid to hydroxy fatty acids. Brodowsky et al. reported the bioconversion of 8-hydroxy-octadecatrienoic acid, 17-hydroxy-octadecatrienoic acid, and 7,8-dihydroxy-octadecatrienoic acid from α-linolenic acid by the fungus Gaeumannomyces graminis (4, 5). Bioconversion of 10-hydroxy-12(Z),15(Z)-octadecadienoic acid by Nocardia cholesterolicum and Flavobacterium sp. strain DS5 has also been reported (10, 22). However, these hydroxy fatty acids were monohydroxy and dihydroxy fatty acids with straight carbon chain structures. In this study, Clavibacter sp. strain ALA2 oxidized α-linolenic acid. The products, 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA, each had a tetrahydrofuranyl ring in the molecule. 7,13,16-Trihydroxy-THFA is a new chemical entity with a trihydroxy fatty acid having a cyclic structure. Clavibacter sp. strain ALA2 can oxidize the C-7, C-12, C-13, C-16, and C-17 positions of linoleic acid (8, 12, 17). Clavibacter sp. strain ALA2 also placed hydroxyl groups at the C-7, C-13, and C-16 positions of α-linolenic acid, an omega-3 polyunsaturated fatty acid. Therefore, it appears that the C-16 hydroxylation activity of Clavibacter sp. strain ALA2 is effective not only for double bonds but also for single bonds.

When linoleic acid was used as the substrate for bioconversion by Clavibacter sp. strain ALA2, both 12-hydroxy-THFA and 7,12-dihydroxy-THFA were produced (17). These compounds differ from 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA in the positions of their tetrahydrofuranyl ring and hydroxy groups. In addition, diepoxy bicyclic fatty acid products, such as DEOA, 7-hydroxy-DEOA, and 16-hydroxy-DEOA, were not observed when α-linolenic acid was the substrate. These results indicate that the chemical structure of products produced by Clabvibacter sp. strain ALA2 depended remarkably on the type of polyunsaturated fatty acid substrate.

In the time course studies, the amount of 13,16-dihydroxy-THFA increased with time and reached 45 mg/50 ml after 7 days of incubation. In the case of linoleic acid, most of the substrate was converted to 12,13,17-trihydroxy-9(Z)-octadecenoic acid, THFA, and DEOA after 2 days of incubation with strain ALA2 (19). Therefore, the hydroxylation of α-linolenic acid by strain ALA2 proceeded much more slowly than the hydroxylation when linoleic acid was the substrate.

Polyunsaturated fatty acids are metabolized to several unique products with various biological functions. These metabolites include prostaglandins, prostacyclines, thromboxanes, leukotrienes, lipoxins, and hydroxy, hydroperoxy, and epoxy fatty acids. Recently, it was reported that low doses of 9,(12)-oxy-10,13-dihydroxy-stearic acid and 10,(13)-oxy-9,12-dihydroxy stearic acid isolated from corn stimulated breast cancer cell proliferation in vitro and disrupted the estrous cycle in female rats (24). These dihydroxy-THFAs inhibited breast cancer and prostate cancer cell proliferation at high doses (23). However, little is known about biological functions and chemical properties of hydroxy-THFAs. Therefore, it should be interesting to determine the physiological functions and industrial applications of the novel hydroxy-THFAs.