UC San Diego

Bacterial fatty acid biosynthesis can occur utilizing one of three "types" of biosynthesis systems. This thesis focuses on the genetic characterization of the pfa gene cluster (pfaA-E) involved in the synthesis of the omega-3 polyunsaturated fatty acid (PUFA) docohexaenoic acid (DHA, C22:6n-3) in the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H. The research presented is an attempt to interrogate bacterial DHA biosynthesis via molecular genetic methods so as to advance our understanding of the physiological significance and biotechnological potential of bacterial omega-3 PUFA biosynthesis. Through the combined use of Gas Chromatography-Mass Spectrometer (GC-MS) analysis, total fatty acid production was compared in C. psychrerythraea cultivated at: 4°C, 8°C, and 16°C. When compared to fatty acid profiles of the mesophile Escherichia coli, the profiles of C.psychrerythraea do not show evidence of thermal compensation. This was apparent by the minimal production of cis-vaccenic acid (C18:1n-7), a result of FabF activity, at all temperatures examined. To examine the functional role of the Pfa Synthase system, a mutant defective in DHA synthesis is currently in the process of being engineered. Specifically, a 516 bp internal fragment of the Ketoacyl Reductase domain of pfaA is being used to disrupt the pfa operon in C.psychrerythraea. Conjugal transfer of the suicide vector pEE3 into wild-type C.psychrerythraea and selection for kanamycin resistant exconjugants will allow, for the first time, an assessment of the functional significance of DHA biosynthesis in the growth and fitness of an obligate psychrophilic bacterium