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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2013)
To interrogate the possible interactions between the polyunsaturated fatty acid (PUFA) and hydrocarbon yielding pathways in Shewenalla pealeana, the pfaA-E genes along with oleB, an annotated thioesterase enzyme, were heterologously expressed in a BL21 DE3 fadD::kan strain of Escherichia coli. The gene knockout in E. coli was performed using lambda red recombination in the TOP 10 strain and P1 phage transduction was used to move the mutation into BL21 DE3. Strains containing various combinations of pfaA-E and oleB expression constructs were cultured at 16°C and analyzed for fatty acid content using gas chromatography-mass spectrometry (GC/MS). Expression of oleB correlated with increases in 18:1 and 16:1 content along with decreases in 16:0 content, which overall contributed to higher unsaturated to saturated fatty acid ratios (UFA/SFA). Eicosapentaenoic acid (EPA) content varied from 5% to 13% in strains carrying the pfaA -E genes and variations in EPA content were not correlated with oleB expression. The proposed 16:4 fatty acid precursor of 31:9 polyunsaturated hydrocarbons found in species of Shewanella was not detected in any of the strains analyzed in this study
    Cover page: Heterologous expression of an uncharacterized thioesterase in E. coli and its relationship to polyunsaturated fatty acid synthesis /
    • Thesis
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2019)

    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.

      Cover page: Genetic investigation of marine microbial polyunsaturated fatty acid biosynthesis
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2020)
      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.
        Cover page: Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2021)
        The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
          Cover page: Genetic Suppression of Lethal Mutations in Fatty Acid Biosynthesis Mediated by a Secondary Lipid Synthase
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2017)
          Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2024)
            The accumulation of microplastics in various ecosystems has now been well documented and recent evidence suggests detrimental effects on various biological processes due to this pollution. Accumulation of microplastics in the natural environment is ultimately due to the chemical nature of widely used petroleum-based plastic polymers, which typically are inaccessible to biological processing. One way to mitigate this crisis is adoption of plastics that biodegrade if released into natural environments. In this work, we generated microplastic particles from a bio-based, biodegradable thermoplastic polyurethane (TPU-FC1) and demonstrated their rapid biodegradation via direct visualization and respirometry. Furthermore, we isolated multiple bacterial strains capable of using TPU-FC1 as a sole carbon source and characterized their depolymerization products. To visualize biodegradation of TPU materials as real-world products, we generated TPU-coated cotton fabric and an injection molded phone case and documented biodegradation by direct visualization and scanning electron microscopy (SEM), both of which indicated clear structural degradation of these materials and significant biofilm formation.
              Cover page: Rapid biodegradation of microplastics generated from bio-based thermoplastic polyurethane.
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