Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/8/2005
Publication Date: 6/15/2006
Citation: Hou, C.T. 2005. Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2. Applied Microbiology and Biotechnology. 69:463-468. Interpretive Summary: Previously, we discovered a new microbial strain ALA2 that produced seven new polyhydroxy unsaturated fatty acids from linoleic acids. Hydroxy fatty acids are useful as starting materials for the synthesis of specialty chemicals, special military nylon, plastisizers, and coating materials. Polyhydroxy unsaturated fatty acids are also valuable precursors for prostaglandins and the synthesis of other hormones, can act as fungicides in agricultural applications, are precursors of flavor ingredients, and are chiral synthons of possible drug discovery interest. To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products was studied. Optimum amount of carbon source, nitrogen source and metal ions as well as pH, and temperature were determined. These data provide the basis for engineering scale-up production of these new products.
Technical Abstract: We identified (Hou 2003) many novel oxygenated fatty acids produced from linoleic acid by Clavibacter sp. ALA2: 12,13-dihydroxy- 9(Z)-octadecenoic acid (12, 13-DHOA); 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid (12-hydroxy-THFA); 7,12-dihydroxy-13,16-epoxy-9(Z)-octadecenoic acid (7,12-dihydroxy-THFA); 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid (DEOA); and 7-hydroxy-12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid (7-hydroxy-DEOA), with 12,13,17-THOA as the main product. 12,13,17-THOA has anti plant pathogenic fungal activity. Strain ALA2 was recently reclassified as Bacillus megaterium ALA2 (Hou et al 2004). To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products were studied. Dextrose at 5 g/l was the optimum amount for the carbon source. Yeast extract was found better than tryptone as a nitrogen source, and showed no difference between the concentrations from 10 - 25 g/l. A combination of 15 g yeast extract and 10 g tryptone also showed good results. Among the metal ions tested, magnesium 2.0 mM, iron 0.5 mM, zinc 0.1 mM, nickel 0.05 mM, and cobalt 0.05 mM were the optimum concentration for the reaction. Copper ions did not affect the production of oxygenated products, however, manganese ions inhibited the reaction. Addition of these metal ions did not alter the distribution of products. The optimum temperature and pH for the production of THOAs were 30 deg C and pH 6.5. The optimum conditions for conversion of 12,13,17-THOA to DEOA were temperature 35 deg C and pH 6.5. And the optimum conditions for the conversion of DEOA to 7-hydroxy-DEOA were temperature 20 deg C and pH 6.5. Time course studies showed forty-eight hours is the optimum for the production of both 12,13,16-THOA and 12,13,17-THOA. The rest of the products: 12-hydroxy-THFA; 7,12-dihydroxy-THFA; DEOA; and 7-hydroxy-DEOA were relatively in small amounts at between 1 - 5 mg per 50 ml reaction mixture. These data provide the basis for engineering scale-up production of these new products.