Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway

Abstract

AbstractA characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. Biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is accomplished using a polyketide/fatty acid synthase mechanism encoded by a set of five genespfaABCDE.This unique fatty acid synthesis (FAS) pathway co-exists with the canonical Type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of thepfagenes in the model marine bacteriumPhotobacterium profundumSS9. Using a reporter gene fusion, we showed that expression of thepfaoperon is down regulated in response to exogenous fatty acids, particularly long chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present inP. profundumSS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designatedpfaF, to be responsible for the observed regulation of thepfaoperon inP. profundumSS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds thepfaApromoter and identified the PfaF binding site.ImportanceThe production of polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid responsive transcriptional regulator specific to the PUFA biosynthesis pathway.