Characterization and Application of Marine Microbial Omega-3 Polyunsaturated Fatty Acid Synthesis

Allemann, Marco N.; Allen, Eric E.

doi:10.1016/bs.mie.2018.02.018

Cited by 20 publications

(20 citation statements)

References 97 publications

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“…VLCPUFAs occur widely in microorganisms including bacteria, microalgae, fungi and protists (Hauvermale et al ., 2006; Nichols et al ., 2014; Yoshida et al ., 2016; Allemann and Allen, 2018). Biosynthesis of these fatty acids goes through either an aerobic pathway or anaerobic pathway (Qiu, 2003).…”

Section: Introductionmentioning

confidence: 99%

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

Xie

Sun

Meesapyodsuk

et al. 2020

Environmental Microbiology

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Thraustochytrium is a unicellular marine protist for the commercial production of very long-chain polyunsaturated fatty acids (VLCPUFAs). Biosynthesis of these VLCPUFAs in the protist is catalysed by a PUFA synthase comprising three subunits, each with multiple catalytic domains. Among these domains, two tandem FabA-like dehydratase domains (DH1 and DH2) in subunit-C together are responsible for introducing double bonds in VLCPUFAs. Domain swapping analysis in yeast showed that the defective phenotype of a Scfas1 mutant could be complemented by expressing an engineered ScFAS1 gene in which the DH domain was replaced by a single DH1 or mutated DH2 of the two. Heterologous expression of the PUFA synthase in E. coli showed that the mutation of DH1 of the two or deletion of DH1 or substitution of DH1 with DH2 resulted in the complete loss of activity in the biosynthesis of VLCPUFAs. Mutation of DH2 of the two or deletion of the DH2 domain produced a small amount of DPA, but not docosahexaenoic acid (DHA). These results indicate that each of the two FabA-like domains of the PUFA synthase possesses distinct function. DH1 domain is essential for the biosynthesis of VLCPUFAs, but DH2 domain is required for the biosynthesis of DHA.

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Section: Introductionmentioning

confidence: 99%

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

Xie

Sun

Meesapyodsuk

et al. 2020

Environmental Microbiology

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“…A subset of marine Gammaproteobacteria, particularly strains isolated from cold and/or high-pressure environments, produce omega-3 polyunsaturated fatty acids (PUFA) such as EPA (20:5n-3) and DHA (22:6n-3), that are incorporated into phospholipid membranes (13,14). The biosynthesis of these unique fatty acids is linked to the pfaABCDE operon, which encodes for a type I FAS/polyketide synthase (15).…”

Section: Introductionmentioning

confidence: 99%

“…The biosynthesis of these unique fatty acids is linked to the pfaABCDE operon, which encodes for a type I FAS/polyketide synthase (15). In these bacteria, the Pfa synthase pathway co-exists with the Type II FAS, which produces saturated (SFA) and monounsaturated fatty acids (14,16). Given that both pathways utilize the same precursor substrates (15,17,18) and their respective end products are destined for phospholipids (13,14,(19)(20)(21)(22), an interesting question arises as to how these pathways are physiologically coordinated in the cell.…”

Section: Introductionmentioning

confidence: 99%

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

Allemann

Allen

2020

J Bacteriol

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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.

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“…For conjugation into P. profundum , biparental matings were performed using the E. coli donor strain S17-1λ pir containing the desired plasmid to be mobilized into SS9R. Selection for exconjugants was performed on 2216 agar containing rifampicin and kanamycin as described previously (Lauro et al, 2005; Allemann and Allen, 2018). After colony purification of exconjugants, colonies were grown without selection and dilutions plated onto 2216 agar supplemented with 5% sucrose to select for a second recombination event.…”

Section: Methodsmentioning

confidence: 99%

“…The pathway for biosynthesis of omega-3 polyunsaturated fatty acids (PUFAs) in certain marine bacteria occurs via a polyketide synthase type mechanism encoded by five genes pfaABCDE (Metz et al, 2001; Allen and Bartlett, 2002; Shulse and Allen, 2011; Yoshida et al, 2016; Allemann and Allen, 2018). The “Pfa synthase” has been identified in several bacterial lineages and shown to synthesize a variety of PUFA products, most notably the long-chain omega-3 PUFAs, such as eicosapentaenoic acid (20:5 n -3, EPA) and docosahexaenoic (22:6 n -3) acids (Shulse and Allen, 2011; Yoshida et al, 2016; Allemann and Allen, 2018). The Pfa synthase multienzyme complex contains all of the required enzymatic domains for lipid biosynthesis and these activities reside on either multi-domain or stand-alone proteins (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Linkage of Marine Bacterial Polyunsaturated Fatty Acid and Long-Chain Hydrocarbon Biosynthesis

2019

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Various marine gamma-proteobacteria produce omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (20:5, EPA) and docosahexaenoic acid (22:6, DHA), which are incorporated into membrane phospholipids. Five genes, designated pfaABCDE , encode the polyketide/fatty acid synthase necessary for production of these long-chain fatty acids. In addition to de novo biosynthesis of EPA and DHA, the “Pfa synthase” is also involved with production of a long-chain polyunsaturated hydrocarbon product (31:9, PUHC) in conjunction with the oleABCD hydrocarbon biosynthesis pathway. In this work, we demonstrate that OleA mediates the linkage between these two pathways in vivo . Co-expression of pfaA-E along with oleA from Shewanella pealeana in Escherichia coli yielded the expected product, a 31:8 ketone along with a dramatic ∼10-fold reduction in EPA content. The decrease in EPA content was independent of 31:8 ketone production as co-expression of an OleA active site mutant also led to identical decreases in EPA content. We also demonstrate that a gene linked with either pfa and/or ole operons in diverse bacterial lineages, herein designated pfaT , plays a role in maintaining optimal production of Pfa synthase derived products in Photobacterium and Shewanella species.

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Characterization and Application of Marine Microbial Omega-3 Polyunsaturated Fatty Acid Synthesis

Cited by 20 publications

References 97 publications

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

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

Linkage of Marine Bacterial Polyunsaturated Fatty Acid and Long-Chain Hydrocarbon Biosynthesis

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