Genetic Regulation of the Bacterial Omega-3 Polyunsaturated Fatty Acid Biosynthesis Pathway

Abstract

A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is mediated by a polyketide/fatty acid synthase mechanism encoded by a set of five genes, pfaABCDE. This unique fatty acid synthesis pathway co-exists with the principal type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of core 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 the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon 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 in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. Importance The production of long-chain omega-3 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 PUFA biosynthesis.

Genetic Suppression of Lethal Mutations in Fatty Acid Biosynthesis Mediated by a Secondary Lipid Synthase

“…While genetic disruption of either set of genes proved to be possible, the resulting strains displayed various degrees of auxotrophy for unsaturated fatty acids. As expected from previous work ( 7 , 27 ), providing exogenous oleic acid in the form of Tween 80 (18:1) reversed this growth impairment.…”

“…Similar to previous work in Pseudomonas aeruginosa ( 22 ), deletion of both fabA and desA led to an obligate requirement for exogenous MUFA supplementation. Supplementing the growth medium with 18:1 fatty acid in the form of Tween 80, which has been shown to be an effective means of delivering fatty acids to SS9 ( 7 , 27 ), fully or partially complemented the growth phenotypes of both mutants as expected ( Fig. 2B, D , and F .)…”

A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571

“…The reading region of rihR was cloned into pET28a (Novagen, Madison, WI, USA) to express His-tagged RihR in E. coli BL21 (DE3) [ 46 ] following standard procedures [ 42 , 56 , 57 ]. E. coli BL21 carrying pET28a- rihR was grown in 500 mL LB medium with the appropriate antibiotic and then induced with the addition of 0.5 mM β- d -1-thiogalacto-pyranoside (IPTG) at OD 600 ≈ 0.6.…”

Genome‐enabled exploration of microbial ecology and evolution in the sea: a rising tide lifts all boats

“…That bacteria could synthesize PUFAs at all was somewhat controversial, since de novo PUFA biosynthesis was thought to be restricted to select eukaryote groups, in particular, microalgae at the time. It is now broadly recognized, however, that deep-sea and other psychrophilic bacteria commonly synthesize and incorporate PUFAs in their membrane lipids, using a unique polyketide biosynthetic pathway (Metz et al, 2001;Allen and Bartlett, 2002;Allemann and Allen, 2020).…”