Genetic and functional diversity of microbial secondary lipid biosynthetic pathways

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine Gammaproteobacteria. The genes responsible for de novo PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain enzyme complexes akin to type I iterative fatty acid and polyketide synthases. Likewise, the heterocyst glycolipids produced by nitrogen-fixing cyanobacteria and the phenolic lipids of Azotobacter vinelandii are produced by similar complexes. The prevalence of these "secondary lipid synthases," so named because their products have thus far not been shown to be essential to cell growth and survival under laboratory conditions, in both cultured and uncultured bacteria is completely unknown. Bioinformatic methods were used to identify homologous type I FAS/PKS gene clusters in diverse microbial lineages representing 10 phyla. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Next, PCR primers targeting the keto-acyl synthase (KS) domain of the pfaA gene involved in PUFA biosynthesis were used to construct environmental clone libraries to investigate the potential for microbial secondary lipid synthesis in disparate marine habitats. Of the 446 sequences recovered, 27.6% clustered with KS sequences involved in the synthesis of EPA, DHA and arachadonic acid (AA, 20:4n-6). The remaining 72.4% of clones formed environmental-only clades or clustered with KS domains of pfaA homologs from organisms producing unidentified products. Lastly, the production of two distinct secondary lipid products at various temperatures was analyzed in six strains of Shewanella in order to provide insight into the factors governing secondary lipid synthesis. The current dissertation significantly expands the known genetic and ecological prevalence of microbial processes involved in secondary metabolism and delivers new opportunities to explore the physiological basis and biotechnological value of novel lipid molecules