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Production of Polyunsaturated Fatty Acids by Polyketide Synthases in Both Prokaryotes and Eukaryotes

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Science  13 Jul 2001:
Vol. 293, Issue 5528, pp. 290-293
DOI: 10.1126/science.1059593

Abstract

Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules. Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids. These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism. Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics. It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes.

PUFAs are critical components of membrane lipids in most eukaryotes (1,2) and are the precursors of certain hormones and signaling molecules (3, 4). Known pathways of PUFA synthesis involve the processing of the saturated 16:0 (5) or 18:0 products of fatty acid synthase (FAS) by elongation and aerobic desaturation reactions (6–8). The synthesis of docosahexeanoic acid (DHA, 22:6ω3) from acetyl–coenzyme A (acetyl-CoA) requires approximately 30 distinct enzyme activities and nearly 70 reactions, including the four repetitive steps of the fatty acid synthesis cycle. PKSs carry out some of the same reactions as FAS (9, 10) and use the same small protein (or domain), acyl carrier protein (ACP), as a covalent attachment site for the growing carbon chain. However, in these enzyme systems, the complete cycle of reduction, dehydration, and reduction seen in FAS is often abbreviated, so that a highly derivatized carbon chain is produced, typically containing many keto and hydroxy groups as well as carbon-carbon double bonds in the trans configuration. The linear products of PKSs are often cyclized to form complex biochemicals that include antibiotics, aflatoxins, and many other secondary products (9–11).

Very-long-chain PUFAs such as DHA and eicosapentaenoic acid (EPA, 20:5ω3) have been reported from several species of marine bacteria, including Shewanella sp. (12–14). Analysis of a genomic fragment (cloned as plasmid pEPA) from Shewanella sp. strain SCRC2738 identified five open reading frames (ORFs) that are necessary and sufficient for EPA production in Escherichia coli(13). Several of the predicted protein domains are homologs of FAS enzymes, and it was suggested that PUFA synthesis inShewanella involved the elongation of 16- or 18-carbon fatty acids produced by FAS and the insertion of double bonds by undefined aerobic desaturases (15). We identified at least 11 regions within the five ORFs as putative enzyme domains (Fig. 1A). When compared with sequences in the nonredundant database (16), eight of these were more strongly related to PKS proteins than to FAS proteins (Fig. 1B). However, three regions were homologs of bacterial FAS proteins. One of these was similar to triclosan-resistant enoyl reductase (ER) fromStreptococcus pneumoniae (17). Two regions were homologs of the E. coli FAS protein encoded byfabA, which catalyzes the synthesis oftrans-2-decenoyl-ACP and the reversible isomerization of this product to cis-3-decenoyl-ACP (18). Thus, at least some of the double bonds in EPA from Shewanella are probably introduced by a dehydrase-isomerase mechanism catalyzed by the FabA-like domains in ORF7.

Figure 1

Genomics analysis of Shewanellagenes encoding enzymes of EPA synthesis. (A) Dark gray areas indicate proposed enzymatic domains. Hatched areas designate regions whose amino acid sequences are highly conserved amongst the EPA- and DHA-synthesizing proteins of Shewanella, Moritella marina (GenBank accession number AB025342.1), andSchizochytrium but for which potential enzymatic functions are not evident. Light gray indicates regions whose amino acid sequences are not conserved among these organisms. (B) Summary of sequence analysis data for the 11 domains designated a to k in (A) (30, 31). a.a., amino acid.

To exclude the involvement of an oxygen-dependent desaturase in EPA synthesis, we cultured E. coli harboring the pEPA plasmid anaerobically. Anaerobically grown cells accumulated EPA to the same level as aerobic cultures (Fig. 2A). When pEPA was introduced into afabB mutant of E. coli, which is unable to synthesize unsaturated fatty acids that are required for growth, the resulting cells lost their fatty acid auxotrophy. They also accumulated higher concentrations of EPA than other pEPA-containing strains (Fig. 2A), suggesting that EPA competes with endogenously produced monounsaturated fatty acids for transfer to glycerolipids. Carbon–13 nuclear magnetic resonance (13C-NMR) analysis of purified EPA from the cells grown in [13C]acetate confirmed its structure and indicated that this fatty acid was synthesized from molecules derived from acetate (presumably acetyl-CoA and malonyl-CoA) (19). A cell-free homogenate from pEPA-containingfabB cells synthesized both EPA and saturated fatty acids from [1-14C]malonyl-CoA (Fig. 2B). High-speed centrifugation of the homogenate indicated that saturated fatty acid synthesis was confined to the supernatant, which is consistent with the soluble nature of the type II FAS enzymes (20). Synthesis of EPA was found only in the 200,000g pellet fraction, indicating that EPA synthesis does not rely on FAS or soluble intermediates (such as 16:0ACP) from the cytoplasmic fraction. Because the proteins encoded by the Shewanella genes are not particularly hydrophobic, restriction of EPA synthesis to this fraction may reflect a requirement for a membrane-associated acceptor molecule. In contrast to the E. coli FAS, EPA synthesis is specifically dependent on the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and does not require NADH (Fig. 2C). These results are consistent with the pEPA genes encoding a multifunctional PKS that acts independently of FAS, elongase, and desaturase activities to synthesize EPA directly. Genes with homology to the Shewanella EPA gene cluster have been found in other PUFA-containing marine bacteria (21, 22), indicating that the PKS pathway may be common in these organisms.

Figure 2

Biochemical analysis of EPA synthesis in E. coli. (A) EPA accumulation in whole cells (quantified by gas chromatography analysis of methyl-ester derivatives) of E. coli containing a plasmid vector alone (control) or vector plus the Shewanella EPA genes [nucleotides 7708 to 35559 of GenBank accession number U73935.1 in the pNEB vector (New England BioLabs, Beverly, MA)]. Results were qualitatively similar with a variety of E. coli host strains; control cells never produced EPA, whereas the expression of the five EPA genes resulted in EPA accumulation. Cells were grown at 22°C (13) on media supplemented with glucose. EPA production in afadE mutant (deficient in fatty acid degradation) was compared when cells were grown under aerobic (pEPA-aerobic) and anaerobic (pEPA-anaerobic) growth conditions. Anaerobic conditions were achieved in Gas Pak Anaerobic Jars (Becton Dickinson Diagnostic Systems, Sparks, MD). The last column (pEPA-fabB ) shows a level obtained when pEPA was expressed in thefabB mutant. (B) Synthesis of EPA and saturated fatty acids from [1-14C]malonyl-CoA in subcellular fractions fromfabB cells expressing the EPA genes. Control cells contained a vector alone and were maintained by the supplementation of growth medium with oleic acid. Cells were disrupted by passage through a French pressure cell and centrifuged (at 8000g for 10 min) to yield a cell-free homogenate (CFH). High-speed centrifugation (at 200,000g for 1 hour) was used to generate supernatant (H-S super) and membrane (H-S pellet) fractions. The H-S pellet was resuspended in buffer equivalent to the CFH volume. Aliquots were incubated in 50 mM phosphate buffer (pH 7.2), containing 20 μM acetyl-CoA, 100 μM [1-14C]malonyl-CoA (0.9 GBq/mol), 2 mM dithiothreitol (DTT), 2 mM NADH, and 2 mM NADPH. Lipids were extracted and fatty acids were converted to methyl esters before separation by TLC (19). The figure shows radioactivity detected (with a radioanalytic scanner) on the TLC plate in the region in which saturated (16:0) and EPA methyl esters migrate. (C) Reductant requirement for in vitro EPA synthesis was tested with the H-S pellet fraction described in (B). Assays were carried out in reaction buffer containing 2 mM NADH and/or NADPH, or neither, as indicated. Incorporation of radioactivity into EPA was measured as described in (B).

Schizochytrium is a thraustochytrid marine protist that accumulates large quantities of triacylglycerols rich in DHA and docosapentaenoic acid (DPA, 22:5ω6) (23). In eukaryotes that synthesize 20- and 22-carbon PUFAs by an elongation-desaturation pathway, the pools of 18-, 20-, and 22-carbon intermediates are relatively large, so that in vivo labeling experiments with [14C]acetate reveal clear precursor-product kinetics for the intermediates (24). In addition, radiolabeled intermediates provided exogenously to such organisms are converted to the final PUFA products (25). [1-14C]acetate was rapidly taken up by Schizochytrium cells and incorporated into fatty acids (Fig. 3A). At 1 min, DHA contained 31% of the label recovered in fatty acids, and this percentage remained constant during the 10 to 15 min of [1-14C]acetate incorporation and the subsequent 24 hours of culture growth. Similarly, DPA represented 10% of the label throughout the experiment. There is no evidence for a precursor-product relation between 16- or 18-carbon fatty acids and the 22-carbon polyunsaturated fatty acids (25). These results are consistent with rapid synthesis of DHA from [1-14C]acetate involving very small (possibly enzyme-bound) pools of intermediates. A cell-free homogenate derived from Schizochytrium cultures incorporated [1-14C]malonyl-CoA into DHA, DPA, and saturated fatty acids (Fig. 3C). The same biosynthetic activities were retained in a 100,000g supernatant fraction but were not present in the membrane pellet (Fig. 3C). Thus, DHA and DPA synthesis inSchizochytrium does not involve membrane-bound desaturases or fatty acid elongation enzymes such as those described for other eukaryotes (7, 8). These fractionation data contrast with the data obtained from the Shewanella enzyme (Fig. 2B) and may indicate the use of a soluble acceptor molecule, such as CoA, by the Schizochytrium enzyme.

Figure 3

Biochemical analysis of DHA synthesis inSchizochytrium. (A) and (B) In vivo labeling ofSchizochytrium cells. [1-14C]acetate was supplied to a 2-day-old culture as a single pulse at zero time. Samples of cells were then harvested by centrifugation and the lipids were extracted. (A) [1-14C]acetate uptake by the cells was estimated by measuring the radioactivity of the sample before and after centrifugation. (B) Fatty acid methyl esters derived from the total cell lipids were separated by AgNO3-TLC (solvent, hexane:diethyl ether:acetic acid, 70:30:2 by volume) (32). The identity of the fatty acid bands was verified by gas chromatography, and the radioactivity in them was measured by scintillation counting. (C) Synthesis of fatty acids from [1-14C]malonyl-CoA in subcellular fractions fromSchizochytrium cells. Cells were disrupted in 100 mM phosphate buffer (pH 7.2), containing 2 mM DTT, 2 mM EDTA, and 10% glycerol, by vortexing with glass beads. The cell-free homogenate was centrifuged at 100,000g for 1 hour. Equivalent aliquots of total homogenate, pellet (H-S pellet), and supernatant (H-S super) fractions were incubated in homogenization buffer supplemented with 20 μM acetyl-CoA, 100 μM [1-14C]malonyl-CoA (0.9 GBq/mol), 2 mM NADH, and 2 mM NADPH for 60 min at 25°C. Assays were extracted and fatty acid methyl esters were prepared and separated as described above before detection of radioactivity with an Instantimager (Packard Instruments, Meriden, CT). The migration of fatty acid standards is indicated.

Additional support for a PKS-based pathway was provided from the sequencing of 8500 randomly selected clones from aSchizochytrium cDNA library (26). Within this data set, only one moderately expressed gene (0.3% of all sequences) was identified as a fatty acid desaturase, although a second putative desaturase was represented by a single clone (0.01%). In contrast, sequences that exhibited homology to 8 of the 11 domains of the Shewanella PKS genes (Fig. 1) were all identified at frequencies of 0.2 to 0.5%. Further sequencing of cDNA and genomic clones allowed the identification of three ORFs containing domains with homology to those in Shewanella (Fig. 4). These proteins may constitute a PKS that catalyzes DHA and DPA synthesis. The homology between the prokaryotic Shewanellaand eukaryotic Schizochytrium genes suggests that the PUFA PKS has undergone lateral gene transfer.

Figure 4

Comparison of putative PKS enzyme domains inShewanella and Schizochytrium ORFs (GenBank accession numbers: AF378327, AF378328, and AF378329).Shewanella domains were compared with the predictedSchizochytrium gene products by means of the LALIGN program (29) with matrix file BLOSUM50 and gap penalties of14/4 (opening/elongation). Boundaries and levels of protein sequence identity for regions identified by LALIGN are indicated. KS, 3-ketoacyl synthase; MAT, malonyl-CoA; ACP, acyl carrier protein; KR, 3-ketoacyl-ACP reductase; AT, acyl transferase; CLF, chainlength factor; ER, enoyl reductase; DH, dehydrase.

The primary structures of the Shewanella andSchizochytrium PKSs do not conform to any known class of PKS proteins (9, 10). Instead, the data suggest that the PKSs synthesize PUFAs from malonyl-CoA (perhaps using acetyl-CoA as a primer) with an enzyme complex that carries out iterative processing of the fatty acyl chain but also performs trans-cis isomerization and enoyl reduction reactions in selected cycles. Although the exact sequence of reactions involved in PUFA synthesis remains to be determined, schemes can be envisioned that accommodate many aspects of the data (27).

The identification of the PUFA PKSs and of putative dehydrase-isomerases may provide new tools to engineer the production of additional polyketide antibiotics. In addition, characterization of PUFA synthesis in Shewanella, Schizochytrium, and their relatives has implications for understanding food web dynamics in aquatic ecosystems (28). Because these organisms are important primary producers of 20- and 22-carbon PUFAs in cold-water oceans (12), the PKS pathway may be a significant source of PUFAs for fish and mammals.

  • * To whom correspondence should be addressed. E-mail: jmetz{at}omegadha.com (J.G.M.); jab{at}wsu.edu (J. B.)

  • For the present addresses of these authors, contact J.G.M.

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