Production of 7,10-dihydroxy-8(E)-octadecenoic acid using cell-free supernatant of Pseudomonas aeruginosa.

Cell-free synthesis has been adopted in the bioconversion process due to its known advantages, such as fast production rate, high product content, and no substrate/product inhibition effect. In this study, the cell-free supernatant of Pseudomonas aeruginosa was used to improve the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. DOD production using cell-free supernatant demonstrated reduction in bioconversion duration and higher product concentration than conventional method using whole cell culture. The maximum DOD concentration (6.41 g/L) was obtained after 36 h of biotransformation using 1 % v/v oleic acid as a substrate with a productivity of 0.178 g/L/h and a yield of 74.8 %. DOD concentration, productivity, and yield using cell-free supernatant were 2.12, 7.12, and 2.22 times higher, respectively, than using the conventional whole cell culture method. Of the carbon and nitrogen sources used in pre-culture, galactose and sodium glutamate along with diammonium phosphate were found to be the most effective for DOD production. An incubation temperature of 27 °C and pH 8.0 were found to be most favorable for DOD production. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis demonstrated the presence of enzymes related to DOD production in the cell-free supernatant, which was substantiated by performing DOD production experiment using the supernatant enzymes extracted from protein gel bands with oleic acid as a substrate. To the best of our knowledge, this is the first report on DOD production using a cell-free supernatant and verifying the existence of the relevant enzymes in the cell-free supernatant. Compared to whole cell process, cell-free DOD production holds several advantages, including higher DOD productivity which could be beneficial for large-scale production.

Monoacylglycerol of 7,10-Dihydroxy-8(E)-octadecenoic Acid Enhances Antibacterial Activities against Food-Borne Bacteria.

Conversion of free fatty acids into monoacylglycerol gives rise to new structural properties, particularly amphipathic property. Therefore, monoacylglycerols are widely used in pharmaceutical and food industries and are also reported to facilitate better absorption into the human body. A functional fatty acid when transformed into a monoacylglycerol will possibly conserve both the original functionality and amphipathic property. The compound 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was generated from oleic acid by Pseudomonas aeruginosa PR3 and was known to contain antimicrobial activities against a broad range of food-borne and plant pathogenic bacteria. Here, we attempted to convert DOD into its monoacylglycerol form using lipase for producing an amphipathic antibacterial agent. Consequently, the monoacylglycerol of DOD (DOD-MAG) was successfully produced by coincubating DOD, glycerol, and lipase at 30 °C. The maximum conversion yield reached 70% after 12 h of incubation. Antibacterial activity of DOD-MAG was enhanced by 8 times from the original activity of DOD against food-borne bacteria.

Microbial Conversion of Vegetable Oil to Hydroxy Fatty Acid and Its Application to Bio-Based Polyurethane Synthesis.

New polyurethanes were synthesized based on dihydroxy fatty acid obtained by the microbial conversion of olive oil. Monounsaturated 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced from olive oil by Pseudomonas aeruginosa PR3 and reacted with hexamethylene diisocyanate (HMDI) at different ratios to form polyurethanes. Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry confirmed the synthesis of DOD. The thermal and tensile properties of the polyurethanes were investigated by differential scanning calorimetry, thermogravimetric analysis, and a universal testing machine. At an isocyanate/hydroxyl ratio of 1.4, the polyurethane exhibited an elongation at break of 59.2% and a high tensile strength of 37.9 MPa. DOD was also mixed with polycaprolactone diol or polyethylene glycol at different weight ratios and then reacted with HMDI to produce new polyurethanes of various properties. These polyurethanes displayed higher elongation at break and good thermal stability. This is the first report on the synthesis of polyurethanes based on DOD produced by the microbial conversion of vegetable oil.

7,10-Epoxyoctadeca-7,9-dienoic Acid: A Small Molecule Adjuvant That Potentiates ß-Lactam Antibiotics Against Multidrug-Resistant Staphylococcus aureus.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infections with multi-drug resistance needs effective and alternative control strategies. In this study we investigated the adjuvant effect of a novel furan fatty acid, 7,10-epoxyoctadeca-7,9-dienoic acid (7,10-EODA) against multidrug-resistant S. aureus (MDRSA) strain 01ST001 by disc diffusion, checker board and time kill assays. Further the membrane targeting action of 7,10-EODA was investigated by spectroscopic and confocal microscopic studies. 7,10-EODA exerted synergistic activity along with ß-lactam antibiotics against all clinical MRSA strains, with a mean fractional inhibitory concentration index below 0.5. In time-kill kinetic study, combination of 7,10-EODA with oxacillin, ampicillin, and penicillin resulted in 3.8-4.2 log10 reduction in the viable counts of MDRSA 01ST001. Further, 7,10-EODA dose dependently altered the membrane integrity (p < 0.001) and increased the binding of fluorescent analog of penicillin, Bocillin-FL to the MDRSA cells. The membrane action of 7,10-EODA further facilitated the uptake of several other antibiotics in MDRSA. The results of the present study suggested that 7,10-EODA could be a novel antibiotic adjuvant, especially useful in repurposing ß-lactam antibiotics against multidrug-resistant MRSA.

Antibacterial activity of a 7,10-dihydroxy-8(E)-octadecenoic acid against plant pathogenic bacteria.

7,10-Dihydroxy-8(E)-octadecenoic acid (DOD), one of hydroxy fatty acids, was successfully produced from oleic acid and natural vegetable oils containing oleic acid by a bacterial strain Pseudomonas aeruginosa (PR3). However, biological properties of DOD remained unknown so far. In this study, as a trial to determine the biological properties of DOD molecule, antibacterial activities of DOD against plant pathogenic bacteria were determined qualitatively and quantitatively. DOD presented strong antibacterial activities against all the bacterial strains tested with MIC value being in the range of 125-1000µg/ml and there was no sensitivity preference detected between Gram-positive and Gram-negative strains.

Optimization of culture conditions for production of a novel cold-active lipase from Pichia lynferdii NRRL Y-7723.

Lipases with abnormal properties such as thermostability, alkalinity, acidity, and cold activity receive industrial attention because of their usability under restricted reaction conditions. Most microbial cold-active lipases originate from psychrotrophic and psychrophilic microorganisms found in Antarctic regions, which has led to difficulties in the practical production of cold-active lipase. Recently, a mesophilic yeast, Pichia lynferdii NRRL Y-7723, was reported to produce a novel cold-active lipase. This study focused on optimization of environmental factors, while giving particular attention to the relationships between given factors and incubation time, to maximize the production of a novel cold-active lipase from P. lynferdii NRRL Y-7723. Maximum lipase production was highly dependent on the incubation time at a given environmental factor. Lipase production varied with incubation time at a given temperature, and 20 °C was selected as the optimum temperature for lipase production. Fructose was selected as the best carbon source, and maximum lipase production was obtained when it was present at 0.7% (w/v). Yeast extract was an efficient organic nitrogen source, with maximum lipase production occurring at 0.9% (w/v). Specifically, at the optimum yeast extract level the lipase production was >10 times higher than the productivity under standard conditions. All natural oils tested showed lipase production, but their maximum productivities varied according to incubation time and oil species.

Production of a novel 9,12-dihydroxy-10(E)-eicosenoic acid from eicosenoic acid by Pseudomonas aeruginosa PR3.

Microbial conversions of unsaturated fatty acids often generate polyhydroxy fatty acids, giving them new properties such as higher viscosity and reactivity. A bacterial strain Pseudomonas aeruginosa (PR3) has been intensively studied to produce mono-, di-, and trihydroxy fatty acids from different 9-cis-monoenoic fatty acids such as oleic acid, ricinoleic acid, and palmitoleic acid. However, from the results and the postulated similar metabolic pathways involved in these transformations, it was assumed that the enzyme system involved in transformation of the monoenoic fatty acid by strain PR3 could utilize fatty acids with different chain lengths and locations of the double bond. In this study was used as a substrate for bioconversion by strain PR3 eicosenoic acid (C20:1, ω-9) containing a singular cis double bond at different positions from the carboxyl end as oleic acid, and it was confirmed that PR3 could produce a novel 9,12-dihydroxy-10(E)-eicosenoic acid (DED) with 6.2% yield from eicosenoic acid. The structure of DED was confirmed using GC-MS, FTIR, and NMR analyses. DED production was maximized at 72 h after the substrate was added to the 24 h culture. Some other nutritional factors were also studied for optimal production of DED.

Production of 7,10-dihydroxy-8(E)-octadecenoic acid from olive oil by Pseudomonas aeruginosa PR3.

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.

Production of a novel cold-active lipase from Pichia lynferdii Y-7723.

Lipase (triacylglycerol acylhydrolases, E.C. 3.1.1.3) is one of the most important enzymes applied to a broad range of industrial application fields. Especially, lipases with abnormal functionality such as thermostability and alkaline, acidic, and cold activities gain special attention because of their applicability in the restricted reaction conditions. In this study, 16 yeast strains prescreened for lipase induction were investigated for their actual lipase production, and we found a novel cold-active lipase produced from Pichia lynferdii Y-7723. The activity of lipase Y-7723 was retained by 74 and 70% at 20 and 10 degrees C, respectively, as compared to the maximum value at 35 degrees C. On the basis of an optimization study, the optimal lipase productivity was obtained at 96 h of incubation with 3% oil substrate in a medium composed of sucrose as a carbon source at pH 7.0. Among carbon sources tested, sucrose showed almost twice as high of a lipase production (184%) as the control, while the cell growth was similar (105%). Yeast extract and ammonium salts were effective as individual nitrogen sources for lipase production. This study demonstrated that the cold activity of lipase Y-7723 at 10 degrees C was highest among the cold-active lipases reported so far.

Optimal production of 7,10-dihydroxy-8(E)-hexadecenoic acid from palmitoleic acid by Pseudomonas aeruginosa PR3.

The hydroxylation of unsaturated fatty acids by bacterial strains is one type of value-adding bioconversion processes. This process generates new hydroxy fatty acids (HFA) carrying special properties such as higher viscosity and reactivity compared with normal fatty acids. Among microbial strains tested for HFA production, Pseudomonas aeruginosa PR3 is well known to utilize various unsaturated fatty acids to produce mono-, di- and tri-hydroxy fatty acids. Previously we reported that strain PR3 could produce a novel value-added hydroxy fatty acid 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) from palmitoleic acid (Bae et al. (2007) Appl. Microbiol. Biotechnol. 75, 435-440). In this study, we focused on the development of the optimal nutritional and environmental conditions for DHD production from palmitoleic acid by PR3. Optimal carbon and nitrogen sources for DHD production were fructose and yeast extract, respectively. Optimal initial medium pH and incubation temperature were pH 8.0 and 30 degrees C and magnesium ion was essentially required for DHD production. Substrate concentration and time of substrate addition were also optimized. Under optimized conditions, maximal DHD production was 1600mg/l representing 26.7% conversion yield.

Effect of surfactant on the production of oxygenated unsaturated fatty acids by Bacillus megaterium ALA2.

Bacillus megaterium ALA2 NRRL B-21660 has been well studied for the production of many oxygenated unsaturated fatty acids from linoleic acid. Its major product, 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA), inhibited the growth of many plant pathogenic fungi. However, we have been unable, until now, to demonstrate 12,13,17-THOA production in a fermentor. Here, we have investigated the effect of surfactants on 12,13,17-THOA production. Surfactant types (SO-25, Tween-80 and Triton X-100) at various concentrations were evaluated for their effects on cell growth and production of 12,13,17-THOA. Triton X-100 decreased cell growth and 12,13,17-THOA production while 1% Tween-80 increased 12,13,17-THOA production more than twofold over control. In a pH and dissolved oxygen (DO) controlled fermentor and with 1.0% Tween-80, both 12,13,17-THOA production and productivity increased to 553 mg/L and 24.0mg/L/hour, respectively. We further conducted THOA production in a fermentor without the control of pH and DO, but with aeration through medium surface rather than by Spurger, and with various amounts of Tween-80 to compare with the results obtained in flask runs. Both additions with 0.1% and 0.5% Tween-80 and the control runs produced poor amounts of product 12,13,17-THOA. The maximum 12,13,17-THOA production was observed at 1.0% Tween-80 at a yield of 650 mg/L. Thus fermentor production of 12,13,17-THOA was successfully demonstrated.

Production of oxygenated fatty acids from vegetable oils by Flavobacterium sp. strain DS5.

Flavobacterium sp. strain DS5 (NRRL B-14859) was used to convert two vegetable oils, olive oil and soybean oil, directly to oxygenated fatty acids such as 10-ketostearic acid (10-KSA) and 10-hydroxystearic acid (10-HSA). Lipase addition to the culture was required because strain DS5 did not induce lipase activity to release free fatty acids from vegetable oils. 10-KSA production was higher from olive oil than from soybean oil because olive oil contains more oleic acid, the precursor of 10-KSA. The optimum amounts of olive oil and lipase addition for 10-KSA production were determined as 0.3ml and 1mg (specific activity=700units/mg) per 50ml culture medium, respectively. At these conditions, 2.8g/L of 10-KSA and 0.40g/L of 10-HSA were obtained from olive oil as a substrate.

Biotechnology for fats and oils: new oxygenated fatty acids.

Among the three groups of natural products (starch, protein and fat), fat and oil are the most under-investigated. The US has a large amount of surplus soybean oil annually, and using vegetable oils or their component fatty acids as starting material provides a new opportunity for bioindustry. Vegetable oils are relatively inexpensive and can be used to manufacture value-added products such as oxygenated oils and fatty acids. Oxygenated fatty acids are common in nature and are important industrial materials. They exist both in mammals and plants. Microorganisms oxidize fatty acids either at the terminal carbon or inside of the acyl chain to produce hydroxyl or keto fatty acids. In our continuing effort to produce value-added products from vegetable oils, we discovered more than one dozen novel oxygenated fatty acids through biotransformation. Microbial hydratase is a carbon 10 positional specific enzyme. Many of these new oxygenated fatty acids possess physiological activities and can be used as biomedicals, in addition to their known applications such as specialty chemicals. The position of hydroxyl groups on the fatty acyl carbon chain plays an important role in the activity against certain specific plant pathogenic fungi. Bacillus megaterium ALA2 converted polyunsaturated fatty acids (PUFA) in different ways. It converted omega-6 PUFAs to a mixture of diepoxy bicyclic, tetrahydrofuranyl rings, and/or trihydroxy groups in their molecules while the products from omega-3 PUFAs produced only hydroxyl tetrahydrofuranyl ring products. The monooxygenase gene of strain ALA2 was sequenced and is a soluble, self-sufficient P450(BM-3) subclass that was highly homologous with the wild-type protein. This new enzyme also possessed a significant high homology in all of the expected reductase regions as well. Fat and oil represent an area with tremendous opportunity for new biotechnology to explore.

New bioactive fatty acids.

Many oxygenated fatty acids are bioactive compounds. Nocardia cholesterolicum and Flavobacterium DS5 convert oleic acid to 10 hydroxy stearic acid and linoleic acid to 10-hydroxy-12(Z)-octadecanoic acid. Pseudomonas aeruginosa PR3 converts oleic acid to the new compounds, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) through 10-hydroxy-8-octadecenoic acid, and racinoleic acid to 7,10,12-trihydroxy-8-octadecenoic acid. DOD showed antibacterial activity including against food-borne pathogens. Bacillus megaterium ALA2 converted n-6 and n-3 PUFAs to many new oxygenated fatty acids. For example: linoleic acid was converted to12,13-epoxy-9-octadecenoic acid and then to 12,13-dihydroxy-9-octadecenoic acid (12,13-DHOA). From here, there are two bioconversion pathways. The major pathway is: 12,13-DHOA --> 12,13,17-trihydroxy-9(S)-octadecenoic acid (THOA) --> 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid (DEOA) --> 7-hydroxy-DEOA. The minor pathway is: 12,13-DHOA --> 12,13,16-THOA --> 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid. 12,13,17-THOA has anti-plant pathogenic fungal activity. The tetrahydrofuranyl moiety is known in anti cancer drugs. Strain ALA2 also converts other n-3 and n-6 PUFAs such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) to many new oxygenated unsaturated fatty acid products. All of these new products have high potential for antimicrobial agents or biomedical applications. We also screened 12 Mortierella fungal strains from the ARS Culture Collection for the production of bioactive fatty acids such as dihomo-gama-linolenic acid (DGLA) and arachidonic acid. All of the strains tested produced AA and DGLA from glucose or glycerol. The top five AA producers (mg AA/g CDW) were in the following order: M. alpina > M. zychae > M. hygrophila > M. minutissima > M. parvispora. Both AA and DGLA are important natural precursors of a large family of prostaglandin and thromboxane groups.

Production of arachidonic acid and dihomo-gamma-linolenic acid from glycerol by oil-producing filamentous fungi, Mortierella in the ARS culture collection.

The filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose, and the species alpina is currently used in industrial production of arachidonic acid in Japan. In anticipation of a large excess of the co-product glycerol from the national biodiesel program, we are trying to find new uses for bioglycerin. We screened 12 Mortierella species: M. alpina NRRL 6302, M. claussenii NRRL 2760, M. elongata NRRL 5246, M. epigama NRRL 5512, M. humilis NRRL 6369, M. hygrophila NRRL 2591, M. minutissima NRRL 6462, M. multidivaricata NRRL 6456, M. nantahalensis NRRL 5216, M. parvispora NRRL 2941, M. sepedonioides NRRL 6425, and M. zychae NRRL 2592 for their production of arachidonic acid (AA) and dihomo-gamma-linolenic acid (DGLA) from glycerol. With glucose as substrate all of the strains tested produced AA and DGLA. The total fatty acid content of 125 mg/g cell dry weight (CDW) and fatty acid composition for AA (19.63%) and DGLA (5.95%) in the mycelia of M. alpina grown on glucose were comparable with those reported by Takeno et al. (Appl Environ Microbiol 71:5124-5128, 2005). With glycerol as substrate all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced were comparable with those obtained with glucose-grown mycelia. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora>M. claussenii>M. alpina>M. zychae>M. minutissima. The top five dry mycelia weights were: M. zychae>M. epigama>M. hygrophila>M. humilis>M. minutissima. The top five species for total fatty acids production (mg/g CDW) were: M. claussenii>M. parvispora>M. minutissima>M. hygrophila>M. maltidivaricata. We selected two species, M. alpina and M. zychae for further studies with glycerol substrate. Their optimum production conditions were determined. Time course studies showed that the maximum cell growth and AA production for both species were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(E)-octadecenoic acid by Pseudomonas aeruginosa PR3.

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301-306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25 degrees C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.

New finding and optimal production of a novel extracellular alkaline lipase from Yarrowia lipolytica NRRL Y-2178.

Lipases are industrially useful versatile enzymes that catalyze numerous different reactions including hydrolysis of triglycerides, transesterification, and chiral synthesis of esters under natural conditions. Although lipases from various sources have been widely used in industrial applications, such as in food, chemical, pharmaceutical, and detergent industries, there are still substantial current interests in developing new microbial lipases, specifically those functioning in abnormal conditions. We screened 17 lipase-producing yeast strains, which were prescreened for substrate specificity of lipase from more than 500 yeast strains from the Agricultural Research Service Culture Collection (Peoria, IL, U.S.A.), and selected Yarrowia lipolytica NRRL Y-2178 as a best lipase producer. This report presents new finding and optimal production of a novel extracellular alkaline lipase from Y. lipolytica NRRL Y-2178. Optimal c ulture conditions f orlipase production by Y. lipolytica NRRL Y-2178 were 72 h incubation time, 27.5 degrees C, pH 9.0. Glycerol and glucose were efficiently used as the most efficient carbon sources, and a combination of yeast extract and peptone was a good nitrogen source for lipase production by Y. lipolytica NRRL Y-2178. These results suggested that Y. lipolytica NRRL Y-2178 showsgood industrial potential as a new alkaline lipase producer.

Production and identification of a novel compound, 7,10-dihydroxy-8(E)-hexadecenoic acid from palmitoleic acid by Pseudomonas aeruginosa PR3.

Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.

Production of 7, 10-dihydroxy-8(E)-octadecenoic acid from triolein via lipase induction by Pseudomonas aeruginosa PR3.

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.