Omega-3 fatty acid production in deep-sea bacteria
Introduction
Omega-3 fatty acid type and structure
Omega-3 fatty acids are a group of fats called alpha-linoleic acid (ALA ; 18 carbons and 3 double bonds), eicosapentaenoic acid (EPA; 20 carbons and 5 double bonds), and docosahexaenoic acid (DHA; 22 carbons and 6 double bonds).
These fatty acids are termed “essential” fatty acids because the human body cannot efficiently synthesize omega-3 fatty acids from precursor molecules, and therefore, sufficient amounts must be consumed through fish products [1].
Documented functions and health benefits
Omega-3 polyunsaturated fatty acids (PUFAs) are an important component in the cell membranes of animals [1]. In addition, pharmacological benefits of two omega-3 PUFAs, EPA and DHA, are well characterized [2]. Pregnancy supplemented with EPA and DHA has been implicated in numerous benefits for the infant [3]. Also, these fatty acids have been demonstrated to deliver significant health benefits in reduction of coronary vascular diseases, such as stroke, high blood pressure, and arrhythmia [4], as well as in treating depression, arthritis and asthma [5][6].
Bacterial Production of EPA and DHA
The origin of EPA in marine food chain has been viewed as microalgae, which is the single primary food source in marine and freshwater food chains [7]. It was also once thought that PUFAs were absent in bacterial membranes because mesophilic species such as E. coli, whose biochemistry and molecular biology had been well-studied, was found to have no PUFAs [8]. However, in addition to this food chain, search for PUFA-producing bacteria was initiated, and some species of Shewanella that are pressure- and cold-adapted, were discovered to produce large amounts of EPA [7]. This was a unique discovery as most bacteria produce C16 and C18 saturated and monounsaturated fatty acids, rather than PUFAs [8]. Out of 50 000 bacterial strains isolated from the intestines of marine animals, one strain, Shewanella sp. SCRC-2738, was discovered to have the highest EPA productivity [7].
Polyketide synthase (PKS)-like pathway and evolutionary perspective
Omega-3 fatty acids in PUFA-producing eukaryotes are synthesized via iterative chain of elongation and desaturation, as shown in Figure 3 for omega-6 and omega-3 pathways [7]. However, surprisingly, the deduced proteins encoded by EPA biosynthesis genes in Shewanella sp., as well as in other marine bacteria that were shown to synthesize PUFAs, did not match with any conserved sequences of fatty acid desaturase genes [7][9]. This suggests that these bacteria possess a novel fatty acid synthesis system, employing polyketide synthase (PKS)-like module, as depicted in Figure 3 [7][9].
In Shewanella EPA gene cluster, putative enzyme domains in ORFs corresponded to well-conserved domains in polyketide synthase (PKS) proteins, indicating that the gene cluster was a PKS-like module for EPA production [7][9]. In other PUFA-producing marine bacteria, such as Photobacteria profundum, genes homologous to PKS-like gene cluster of Shewanella were discovered [9]. Recent works with genome sequencing has shown abundant and wide distribution of deduced proteins in PKS-like gene cluster among various marine bacteria, including Colwellia and Moritella, a DHA-producing bacteria [8]. These findings indicated that this PKS-like pathway is likely common to and responsible for all bacterial EPA and DHA synthesis in marine bacteria that are high pressure- and cold-adapted [8][10].
EPA biosynthesis gene cluster from Shewanella SCRC-2738 was isolated for analysis, and a homology search of amino acid sequences from PKS gene cluster showed similarity with several enzymes involved in fatty acid synthesis of Escherichia coli, Saccharopolyspora erythraea, and plant chloroplast [7]. By analyzing EPA productivity of deletion mutants of genomic fragment from Shewanella cloned into plasmid and expressed in E. coli, five ORFs in gene cluster were identified to be necessary and sufficient for EPA biosynthesis in E. coli [9].
Genetic analysis has also suggested an origin of these PKS gene clusters in Shewanella and other marine bacteria [9]. Schizochytrium is a marine protist that is a recognized producer docosapentaenoic acid (DPA) and DHA [9]. PKS enzyme domains in Schizochytrium and Shewanella showed a high degree of sequence similarity; this suggests that a lateral gene transfer may have been involved in acquiring PKS gene clusters in the marine environment [9][11].
Role of omega-3 synthesis in bacterium
Production of omega-3 PUFAs in deep-sea bacteria is likely to play a role in regulating membrane fluidity [12]. In Shewanella strain SCRC-2378, growth temperature has been shown to inversely affect cellular EPA contents [7]. For many marine bacteria, there is a need to tolerate hydrostatic pressure and reduced temperature, the factors which change the fluid membrane to a non-fluid state [13].
Deep-sea bacteria incorporate greater amounts of PUFAs, such as EPA, into membrane phospholipids to counter such problematic changes in the membrane [13]. Shewanella species that are adapted to high pressure and low temperature produce much greater amounts of EPA, whereas mesophilic and pressure-sensitive members of the group only produce scant amounts [14].
Significance of marine omega-3 production
Issues with current source of EPA and DHA
Larger organisms, including humans, cannot synthesize omega-3 fatty acids [1]. Currently, the principal nutritional source of omega-3 fatty acids for humans is seafood [1]. However, decline in global fish stocks, overfishing and chemical contamination of fish stock (for example, by mercury) call for an alternative source of these essential fatty acids [15]. Also, in response to global warming, microalgae may reduce the production of omega-3 fatty acids, suggesting an impending decline in overall omega-3 fatty acids [16]. Because of these current challenges and concerns, omega-3 fatty acid supply for human consumption has emerged as a relevant issue.
Genetic engineering prospects of PKS-module of marine bacterias
Microalgae are known producers of omega-3 PUFAs [15]. However, direct harvest of microalgae for omega-3 fatty acids may be costly and ineffective [17]. This makes genetic engineering of bacterial genes and industrial bacterial production of omega-3 PUFAs an attractive alternative. EPA and DHA biosynthesis gene clusters from deep-sea bacteria have been cloned into E. coli, which may help with the goal of expression in heterologous host organisms, such as plants, yeast and cyanobacteria, to produce industrially significant products [8]. DNA fragment has been cloned from Shewanella sp. and expressed in E. coli, with successful production of EPA [7]. Further genomic analysis has resolved ORFs in Shewanella that are necessary and sufficient for EPA production in E. coli [9]. Successful recombinant production of DHA has been also recently demonstrated in E. coli [8].
Heterologous expression of PKS-like gene cluster of marine bacteria to produce valuable omega-3 fatty acids has many benefits, such as the reduced need for reducing equivalents like NADPH [18][19]. Also, preparation of pure DHA or EPA is difficult with fish oils, but with PKS-like modules of marine bacteria, just one of DHA and EPA can be produced, which presents high pharmaceutical and commercial value [18][19].
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