Genetic investigation of marine microbial polyunsaturated fatty acid biosynthesis
- Author(s): Allemann, Marco Nicholas
- Advisor(s): Allen, Eric E
- et al.
Biosynthesis of fatty acids and incorporation into phospholipid membranes is an essential process that underpins all life. The biophysical properties of the phospholipid membrane can be modulated in response to various parameters such as temperature and hydrostatic pressure by the incorporation of unsaturated fatty acids, which act to maintain the fluidity of the membrane by dispersing acyl chain packing. A subset of marine Gamma-proteobacteria utilize a hybrid/fatty acid synthase polyketide pathway to produce polyunsaturated fatty acids (PUFA); eicosapentaenoic (EPA, 20:5n-3) or docosahexaenoic (DHA, 22:6n-3) and incorporate them into their phospholipid membranes. This dissertation provides new insights into a variety of aspects of PUFA biosynthesis with particular emphasis on various interactions with other metabolic pathways in the native producing strains.
Chapter 2 examines the relationship between production of EPA and PUHC biosynthetic pathways and describes how these pathways are linked by OleA, a non-decarboxylative ketosynthase, responsible for initiation of the PUHC pathway. The linkage of these pathways is
further emphasized by the phenotypic characterization of pfaT, a thioesterase, which is required for optimal production of both pathway end products.
Chapter 3 presents new insights into how the pfa operon of Photobacterium profundum SS9 is controlled at the genetic level. Of the culture conditions tested, addition of exogenous unsaturated fatty acids was shown to dramatically down-regulate the operon. A subsequent transposon screen identified a transcriptional regulator of the pfa operon, designated pfaF, and subsequent genetic and biochemical experiments validated that PfaF is a fatty acid responsive transcriptional regulator of the pfa operon.
Chapter 4 examines the relationship between polyunsaturated and monounsaturated fatty acid biosynthetic pathways and examines how these pathways are functionally related to one another. Derivatives of P. profundum SS9, with varying severities of monounsaturated fatty acid synthesis impairments were subjected to high-pressure growth conditions to enrich for suppressor mutants. Strains capable of growth at high pressure and in the absence of exogenous fatty acid supplementation were isolated and sequenced to identify candidate suppressor mutations. Transpositions of insertion sequences into the fabD that abolished function or impaired transcription were uncovered. Genetic experiments confirmed that fabD is not an essential gene in P. profundum SS9 and that fabD and pfaA constitute a synthetically lethal pair. Heterologous expression of Pfa synthases in Escherichia coli complemented a temperature sensitive fabD mutant, suggesting that the malonyl-CoA transacylase (MAT) domain found in PfaA can compensate for loss of FabD activity in vivo.