UC San Diego

To interrogate the possible interactions between the polyunsaturated fatty acid (PUFA) and hydrocarbon yielding pathways in Shewenalla pealeana, the pfaA-E genes along with oleB, an annotated thioesterase enzyme, were heterologously expressed in a BL21 DE3 fadD::kan strain of Escherichia coli. The gene knockout in E. coli was performed using lambda red recombination in the TOP 10 strain and P1 phage transduction was used to move the mutation into BL21 DE3. Strains containing various combinations of pfaA-E and oleB expression constructs were cultured at 16°C and analyzed for fatty acid content using gas chromatography-mass spectrometry (GC/MS). Expression of oleB correlated with increases in 18:1 and 16:1 content along with decreases in 16:0 content, which overall contributed to higher unsaturated to saturated fatty acid ratios (UFA/SFA). Eicosapentaenoic acid (EPA) content varied from 5% to 13% in strains carrying the pfaA -E genes and variations in EPA content were not correlated with oleB expression. The proposed 16:4 fatty acid precursor of 31:9 polyunsaturated hydrocarbons found in species of Shewanella was not detected in any of the strains analyzed in this study