- Email: [email protected]
Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms
Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
51
Research News
Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms Johnathan A. Napier Polyunsaturated fatty acids (PUFAs) are involved in determining the biophysical properties of membranes as well as being precursors for signalling molecules. C20++ PUFA biosynthesis is catalysed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of polyunsaturated fatty acids is now known to exist that is analogous to polyketide synthases (PKS). These novel PKS genes could be used to direct the synthesis of PUFAs in heterologous hosts, as well as exploiting the combinatorial chemistry of PKSs to make unusual fatty acids.
The biosynthetic pathway of polyunsaturated fatty acids (PUFAs; usually defined as fatty acids of 18 carbons or more that contain two or more double bonds) has been the subject of much research activity recently because there is good clinical evidence that PUFAs play a vital role in multiple aspects of human health, development and nutrition [1]. Metabolism of 20 carbon (C20) PUFAs by oxygenases yields a range of important molecules (generically known as the eicosanoids), such as prostaglandins, leukotrienes and thromboxanes [2], that modulate many biological processes including vasodilation, platelet aggregation and blood pressure [2]. Because PUFAs play a central role in regulating cellular and organismal homeostasis, these compounds are potential pharmaceutical (or nutraceutical) targets for manipulation [1,3]. There are several additional considerations regarding PUFAs that need to be highlighted. First, mammals need the essential fatty acids linoleic acid and α-linolenic acid in their diet to serve as precursors for subsequent downstream PUFA synthesis (Fig. 1). If appropriate amounts of these fatty acids are ingested, they are then used as substrates for further desaturation and elongation as part of normal metabolism, producing C20 (and C22) http://plants.trends.com
PUFAs through the action of multiple enzymes [3]. Alternatively, in the case of compromised or degraded metabolism, production of C20+ PUFAs might be reduced, resulting in an alteration in eicosanoid levels. Second, C20+ PUFAs are also synthesized by a variety of microorganisms that are cultured as sources of PUFAs and, more recently, have served as sources of genes encoding this metabolic pathway [4]. Over the past few years, sequences encoding virtually all the enzyme activities involved in microsomal PUFA biosynthesis have been cloned [3]. These include the PUFA-specific ‘front-end’ desaturases, where the enzyme desaturates between pre-existing double bonds and the ‘front’ (∆) carboxyl group of the fatty acid. Examples of this class of enzyme are the ∆5- and ∆6-desaturases (where the superscript number indicates the desaturated carbon relative to the carboxyl end) (Fig. 1), with homologues being functionally identified from many species [5]. More recently, transcripts encoding the C22 ∆4-desaturase [6] and the C18 and C20 elongases [7,8] have been cloned and functionally characterized. Thus, although all the components of this pathway have been identified, the longterm goal of reconstituting PUFA biosynthesis in a suitable heterologous host (such as a transgenic oilseed) is still a daunting task, requiring the transfer and regulation of multiple genes. Initial attempts to express PUFA biosynthetic enzymes in heterologous transgenic systems have met with some success, notably the accumulation of γ-linolenic acid (GLA) in canola (Brassica napa) to levels in excess of 40% of total fatty acid [9], although these are (literally) only the first steps towards efficient pathway reconstitution. Novel PUFA biosynthetic pathway
Recently, Jim Metz and colleagues [10] have described a novel alternative pathway for the biosynthesis of C20+ PUFAs. This system does not require the multiple desaturase and elongase enzymes outlined above, but instead uses a polyketide synthase-like gene cluster to synthesize
PUFAs. Certain species of marine bacteria such as Shewanella sp. synthesize C20+ PUFAs and, as such, probably represent the primary input of these fatty acids into the marine food web (which culminates in the accumulation of these PUFAs in fish oils). Previously, a 38 kb genomic fragment from Shewanella, identified by Haruko Takeyama and colleagues, resulted in the production of C20 PUFAs when expressed in E. coli or Synechococcus [11]. Sequencing of this Shewanella fragment predicted the presence of eight openreading-frames (ORFs), three of which were similar to fatty acid synthases (FAS). The remaining five were suggested to be the effectors of C20 PUFA biosynthesis [11]. Subsequent biochemical studies of this pathway in Shewanella led to the hypothesis that synthesis of C20 PUFAs probably requires aerobic desaturases and elongases, presumably encoded for by these five genes present within the 38 kb genomic fragment [12]. However, Metz and colleagues observed that several of the PUFAsynthesizing Shewanella ORFs were more closely related to polyketide synthases (PKS) than to FAS, aerobic desaturases or elongases. In silico analysis of four of these Shewanella ORFs revealed several regions with similarity to product synthesis domains of polyketide synthase (such as 3-ketoacyl synthases, 3-ketoacyl reductase, acyl carrier protein, chain length factor and acyl transferases). This also indicated putative accessory enzyme activity in the form of phosphopantetheine transferase (required for activation of the acyl carrier protein domain), and the presence of FAS-related sequences (in the form of enoyl reductase and FabA-like dehydratases) as previously reported [11,13]. Definitive demonstration of the PKS-like nature of these ORFs was obtained by heterologous expression. When E. coli cells expressing the Shewanella ORFs were cultured under aerobic or anaerobic conditions, the synthesis of C20 PUFAs was apparent under both conditions, ruling out a role for
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Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
Animals
Plants
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∆4-Desaturase 22:6 Docosahexaenoic acid CO2H Fig. 1. Generalized pathway for the aerobic synthesis of polyunsaturated fatty acids (PUFAs) in plants (shown in green) and in animals (shown in blue). The different enzymatic reactions required for aerobic PUFA biosynthesis are shown. Because of a genetically defined lack of an oleate ∆12- desaturase, animals have a dietary requirement for linoleic acid and α-linolenic acid. These two essential fatty acids serve as substrates for the n-6 and n-3 pathways, respectively, leading to the formation of polyunsaturated very long chain (C20) fatty acids. The biosynthesis of C22 PUFAs is considered to proceed in a similar manner (i.e. by elongation and desaturation). In higher plants, fatty acid desaturation usually results in the formation of linoleic acid and αlinolenic acid, but no subsequent synthesis of C20+ PUFAs is observed. CO2H is the ‘front’ carboxyl group of the fatty acid; superscript numbers indicate the desaturated carbon relative to the ‘front’ carboxyl end.
(aerobic) desaturases, indicating instead a PKS-like system [10]. Moreover, expression of these Shewanella PKS-like ORFs in a fabB− (β-ketoacyl-acyl carrier protein synthase I) mutant of E. coli rescued the cells from needing exogenously supplied unsaturated fatty acids. Similar PKS-like gene clusters were also observed http://plants.trends.com
in other C20+ PUFA-accumulating marine bacteria (such as Vibrio marinus), indicating that this biosynthetic pathway is probably conserved among these prokaryotic organisms. Polyketide synthase PUFA biosynthesis in eukaryotes
The discovery of analogous PUFA PKS-like ORFs in eukaryotes was perhaps more surprising. Although PUFA-accumulating lower eukaryotes have been used as sources from which to isolate genes encoding enzymes of this biosynthetic pathway, these organisms were assumed to use the aerobic desaturase–elongase system. Isolation of the C22 PUFA front-end ∆4-desaturase from the marine microheterotroph Thraustochytrium sp., a member of the Thraustochytriidae, was thought to corroborate this [8]. However, when Metz et al. characterized the marine protist Schizochytrium via random cDNA
TRENDS in Plant Science
sequencing, the resulting 8500 ESTs failed to include the expected number of desaturases, whereas sequences related to the Shewanella PKS-like ORFs were well represented. Subsequent cloning of these Schizochytrium PKS-like genes revealed three ORFs compared with the five characterized for Shewanella, although the common presence of conserved PKS domains indicated a clear evolutionary relationship between the two species. Therefore, it is likely that a PKS-like system for the biosynthesis of PUFAs exists in some lower eukaryotes as well as in bacteria. Although the exact mechanism by which very long chain unsaturated fatty acids are assembled by the reiterative reaction of a PKS-like system needs to be unravelled, it probably involves precise double-bond isomerization. It is likely that such an isomerase activity is contained within the two conserved domains of the PUFA PKS-like ORFs that show homology
Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
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Processive polyunsaturated fatty acids Fig. 2. Generalized scheme for the processive synthesis of polyunsaturated fatty acids (PUFAs) by a polyketide synthase (PKS) system. In the case of PUFAs, it is envisaged that a primer molecule (in the form of acetylCoA) undergoes several rounds of sequential reactions (keto-synthase, keto-reductase, dehydratase and enoyl reductase), resulting in repeated synthesis and fatty acyl chain (esterified to the acyl carrier protein) elongation by two carbons per cycle. Because PUFAs contain methylene-interrupted double bonds (i.e. at the third carbon), it is likely that a dehydratase (FabA-like) module in the PKS also simultaneously carries out a trans–cis isomeration to generate this configuration.
to the E. coli FabA enzyme, a component of FAS, which is known to have both dehydratase and isomerase activity [13]. Importantly, the trans–cis double-bond isomerase activity represents new possibilities in the manipulation and exploitation of the PKS biosynthetic pathway, not least of all in the production of novel antibiotics (Fig. 2).
TRENDS in Plant Science
pathway [14]. It will be interesting to determine just how efficiently a heterologous PUFA–PKS can compete for endogenous substrates, and also if this results in any pleiotropic effects on the transgenic host. Several ‘front-end’ PUFA aerobic desaturases from Thraustochytrium have been identified recently [8], but the prevalence of this newly discovered PKS-like biosynthetic pathway is not known. Because Schizochytrium is also a member of the Thraustochytriidae, it is perhaps surprising to find these two distinct biosynthetic pathways represented in the same family, but molecular characterization of PUFA biosynthesis in the Thraustochytriidae might provide insights into the evolution of this important pathway. Conclusions
Engineering PUFAs in transgenic plants
In terms of producing very long chain PUFAs in transgenic oilseeds, these novel PKS-like ORFs potentially represent an attractive alternative route. For example, introducing and regulating the three Schizochytrium PKS-like ORFs in a transgenic plant is relatively simple compared with the more than five desaturase and elongase genes required for the previously characterized aerobic http://plants.trends.com
A novel polyketide synthase-like system has been identified as the PUFA biosynthetic pathway for both prokaryotic and eukaryotic marine organisms. The relative simplicity of this PKS-like system makes it attractive in terms of transgenic production of PUFAs. In addition, the identification of new (PUFA-specific) PKS activities such as double-bond isomerization might help in the bioengineering of new types of antibiotics.
Acknowledgements
IACR-Long Ashton Research Station receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. I would like to thank John Crosby and Tom Simpson (University of Bristol) for their assistance in the production of the graphics, and for helpful discussions. References 1 Broun, P. et al. (1999) Genetic engineering of plant lipids. Annu. Rev. Nutr. 19, 197–216 2 Gill, I. and Valivety, R. (1997) Polyunsaturated fatty acids: occurrence, biological activities and applications. Trends Biotechnol. 15, 401–409 3 Napier, J.A. et al. (1999) Plant desaturases: harvesting the fat of the land. Curr. Opin. Plant Biol. 2, 123–127 4 Michaelson, L.V. et al. (1998) Isolation of a ∆5-fatty acid desaturase gene from Mortierella alpina. J. Biol. Chem. 273, 19055–19059 5 Napier, J.A. et al. (1999) A growing family of cytochrome b5-domain fusion proteins. Trends Plant Sci. 4, 2–4 6 Qiu, X. et al. (2001) Identification of a ∆4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. J. Biol. Chem. 276, 31561–31566 7 Beaudoin, F. et al. (2000) Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proc. Natl. Acad. Sci. U. S. A. 97, 6421–6426 8 Leonard, A.E. et al. (2000) Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids. Biochem. J. 350, 765–770
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9 Liu, J-W. et al. (2001) Evaluation of the seeds oils from a canola plant genetically transformed to produce high levels of γ-linolenic acid. In γ-Linolenic Acid: Recent Advance in Biotechnology and Clinical Applications (Huang, Y-S. and Ziboh, V.A., eds), pp. 61–71, American Oil Chemists’ Society Press, Champaign, IL, USA 10 Metz, J.G. et al. (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290–293
11 Takeyama, H. et al. (1997) Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 143, 2725–2731 12 Watanabe, K. et al. (1997) Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation systems. J. Biochem. 122, 467–473 13 Bentley, R. and Bennett, J.W. (1999) Constructing polyketides: from Collie to combinatorial biosynthesis. Annu. Rev. Microbiol. 53, 411–446
14 Abbadi, A. et al. (2001) Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids? Eur. J. Lipid Sci. Technol. 103, 106–113
Johnathan A. Napier IACR-Long Ashton Research Station, Long Ashton, Bristol, UK BS41 9AF. e-mail: [email protected]
Meeting Report
Protein phosphorylation in and around signal transduction John Mundy and Kay Schneitz Plant Protein Phosphorylation, 12–15 September 2001, Vienna, Austria.
Metabolism
The recent Plant Protein Phosphorylation meeting was exciting for two reasons. First, it was focused so that people could share results and techniques. Second, it had a broad scope because phosphorylation is involved in regulating just about everything. Cell cycle
Dénes Dudits (Hungarian Academy of Sciences, Szeged, Hungary) has identified an alfalfa cyclin-dependent kinase inhibitor (CKI) that itself becomes phosphorylated by a calmodulin-like domain kinase (CDPK), suggesting a link between calcium-based signaling and cell-cycle complexes in vivo. What is the role of individual mitogen-activated protein kinases (MAPKs), and some of their regulators, such as MAPK kinase kinase (MAPKKK), in plant development? Patrick Krysan (University of Wisconsin, USA) addressed this issue with a reversegenetics approach. He has analyzed mutants carrying T-DNA insertions in any of three Arabidopsis MAPKKK genes, ANP-1, ANP-2 and ANP-3. There appears to be some redundancy because single mutants showed no apparent phenotype. Genome-wide expression analysis of anp2 anp3 double mutants suggests a role for these genes in repressing stress responses. Cathal Wilson (University of Vienna, Austria) provided evidence that the MAP kinase p43Ntf6, the MAPK kinase NtMEK1, and NPK1, a previously known MAPKKK, constitute a MAP kinase module that functions in cytokinesis. http://plants.trends.com
Tatjana Kleinow (Max-Planck-Institut für Züchtungsforschung, Cologne, Germany) has identified Arabidopsis orthologs of the β- and γ-activator-subunit of the SNF1related kinase complex. Surprisingly plants seem to have two different kinase complex forms: one complex with an α, β and γ subunit and one with just α and the new combined βγ subunit. Several new regulatory components of the SNF1related kinase signaling cascade have been identified using the two-hybrid system and in vitro binding assays. Stress responses
Kazuo Shinozaki (RIKEN, Tsukuba, Japan) discussed the signal crosstalk and integration that probably occurs between MAPKs given their large numbers (~24 MPKs, 18 MPKKs and 30 MPKKKs in Arabidopsis) and potential redundancy and partner promiscuity. Claudia Jonak and Irute Meskiene (University of Vienna, Austria) presented work on alfalfa WIG (wound-induced glycogen synthase kinase 3 or GSK3) and SIMK (woundand salt-inducible MAPK). Further characterization of WIG is warranted because GSK3s modulate the downstream specificity of other kinase signaling pathways. Biochemical analyses indicate that activity of the SIMK MPK is attenuated by the PP2C-type phosphatase MP2C, and that MP2C interaction with SIMK is mediated by a conserved MAPK docking motif in the MP2C N-terminus. Systems to assign functions to calcium (Ca2+-) dependent protein kinases (CPDKs, including one that functions upstream of a pathway required for Avr9-induced reactive
oxygen species (ROS), were described by Tina Romeis (John Innes Centre, Norwich, UK). Epitope tagging has shown that tobacco NtCDPK2 is shared between pathways triggered by biotic and abiotic stresses. Jörg Kudla (University of Ulm, Germany) showed that Ca2+ regulates interactions between calcineurin B-like proteins (CBL) and NAF kinase domains. Molecular and reverse genetic approaches indicate that differential CBL–calcineurin B-like interacting protein kinase (CIPK) affinities impose specificity upon Ca2+ signals. Two research groups have linked MAP kinases to genotoxic stress responses. Johannes Stratmann (University of South Carolina, Columbia, SC, USA) showed that the systemin receptor is involved in ultraviolet B (UV-B) signaling via activation of a tomato MAP kinase. It is also possible that UV-B responses are mediated non-specifically via stress pathways, including the systemin receptor. A genetic screen described by Roman Ulm (Friedrich Miescher Institute, Basel, Germany) revealed that loss of function of an Arabidopsis MAP kinase phosphatase (AtMKP1) results in ultraviolet C (UV-C) and methyl methanesulfonate (MMS)hypersensitivity. AtMKP1 interacts with several MPKs, therefore AtMPK1 must be an important regulator of MAP kinase activities in vivo. Pathogen responses
Three research groups linked pathogen responses to MAP kinase signaling. Dierk Scheel (Institute of Plant Biochemistry, Halle, Germany) described responses to the peptide elicitor PEP-13 in parsley (Petroselinum crispum). PEP-13
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02192-6
TRENDS in Plant Science Vol.7 No.2 February 2002
51
Research News
Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms Johnathan A. Napier Polyunsaturated fatty acids (PUFAs) are involved in determining the biophysical properties of membranes as well as being precursors for signalling molecules. C20++ PUFA biosynthesis is catalysed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of polyunsaturated fatty acids is now known to exist that is analogous to polyketide synthases (PKS). These novel PKS genes could be used to direct the synthesis of PUFAs in heterologous hosts, as well as exploiting the combinatorial chemistry of PKSs to make unusual fatty acids.
The biosynthetic pathway of polyunsaturated fatty acids (PUFAs; usually defined as fatty acids of 18 carbons or more that contain two or more double bonds) has been the subject of much research activity recently because there is good clinical evidence that PUFAs play a vital role in multiple aspects of human health, development and nutrition [1]. Metabolism of 20 carbon (C20) PUFAs by oxygenases yields a range of important molecules (generically known as the eicosanoids), such as prostaglandins, leukotrienes and thromboxanes [2], that modulate many biological processes including vasodilation, platelet aggregation and blood pressure [2]. Because PUFAs play a central role in regulating cellular and organismal homeostasis, these compounds are potential pharmaceutical (or nutraceutical) targets for manipulation [1,3]. There are several additional considerations regarding PUFAs that need to be highlighted. First, mammals need the essential fatty acids linoleic acid and α-linolenic acid in their diet to serve as precursors for subsequent downstream PUFA synthesis (Fig. 1). If appropriate amounts of these fatty acids are ingested, they are then used as substrates for further desaturation and elongation as part of normal metabolism, producing C20 (and C22) http://plants.trends.com
PUFAs through the action of multiple enzymes [3]. Alternatively, in the case of compromised or degraded metabolism, production of C20+ PUFAs might be reduced, resulting in an alteration in eicosanoid levels. Second, C20+ PUFAs are also synthesized by a variety of microorganisms that are cultured as sources of PUFAs and, more recently, have served as sources of genes encoding this metabolic pathway [4]. Over the past few years, sequences encoding virtually all the enzyme activities involved in microsomal PUFA biosynthesis have been cloned [3]. These include the PUFA-specific ‘front-end’ desaturases, where the enzyme desaturates between pre-existing double bonds and the ‘front’ (∆) carboxyl group of the fatty acid. Examples of this class of enzyme are the ∆5- and ∆6-desaturases (where the superscript number indicates the desaturated carbon relative to the carboxyl end) (Fig. 1), with homologues being functionally identified from many species [5]. More recently, transcripts encoding the C22 ∆4-desaturase [6] and the C18 and C20 elongases [7,8] have been cloned and functionally characterized. Thus, although all the components of this pathway have been identified, the longterm goal of reconstituting PUFA biosynthesis in a suitable heterologous host (such as a transgenic oilseed) is still a daunting task, requiring the transfer and regulation of multiple genes. Initial attempts to express PUFA biosynthetic enzymes in heterologous transgenic systems have met with some success, notably the accumulation of γ-linolenic acid (GLA) in canola (Brassica napa) to levels in excess of 40% of total fatty acid [9], although these are (literally) only the first steps towards efficient pathway reconstitution. Novel PUFA biosynthetic pathway
Recently, Jim Metz and colleagues [10] have described a novel alternative pathway for the biosynthesis of C20+ PUFAs. This system does not require the multiple desaturase and elongase enzymes outlined above, but instead uses a polyketide synthase-like gene cluster to synthesize
PUFAs. Certain species of marine bacteria such as Shewanella sp. synthesize C20+ PUFAs and, as such, probably represent the primary input of these fatty acids into the marine food web (which culminates in the accumulation of these PUFAs in fish oils). Previously, a 38 kb genomic fragment from Shewanella, identified by Haruko Takeyama and colleagues, resulted in the production of C20 PUFAs when expressed in E. coli or Synechococcus [11]. Sequencing of this Shewanella fragment predicted the presence of eight openreading-frames (ORFs), three of which were similar to fatty acid synthases (FAS). The remaining five were suggested to be the effectors of C20 PUFA biosynthesis [11]. Subsequent biochemical studies of this pathway in Shewanella led to the hypothesis that synthesis of C20 PUFAs probably requires aerobic desaturases and elongases, presumably encoded for by these five genes present within the 38 kb genomic fragment [12]. However, Metz and colleagues observed that several of the PUFAsynthesizing Shewanella ORFs were more closely related to polyketide synthases (PKS) than to FAS, aerobic desaturases or elongases. In silico analysis of four of these Shewanella ORFs revealed several regions with similarity to product synthesis domains of polyketide synthase (such as 3-ketoacyl synthases, 3-ketoacyl reductase, acyl carrier protein, chain length factor and acyl transferases). This also indicated putative accessory enzyme activity in the form of phosphopantetheine transferase (required for activation of the acyl carrier protein domain), and the presence of FAS-related sequences (in the form of enoyl reductase and FabA-like dehydratases) as previously reported [11,13]. Definitive demonstration of the PKS-like nature of these ORFs was obtained by heterologous expression. When E. coli cells expressing the Shewanella ORFs were cultured under aerobic or anaerobic conditions, the synthesis of C20 PUFAs was apparent under both conditions, ruling out a role for
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02191-4
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Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
Animals
Plants
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∆4-Desaturase 22:6 Docosahexaenoic acid CO2H Fig. 1. Generalized pathway for the aerobic synthesis of polyunsaturated fatty acids (PUFAs) in plants (shown in green) and in animals (shown in blue). The different enzymatic reactions required for aerobic PUFA biosynthesis are shown. Because of a genetically defined lack of an oleate ∆12- desaturase, animals have a dietary requirement for linoleic acid and α-linolenic acid. These two essential fatty acids serve as substrates for the n-6 and n-3 pathways, respectively, leading to the formation of polyunsaturated very long chain (C20) fatty acids. The biosynthesis of C22 PUFAs is considered to proceed in a similar manner (i.e. by elongation and desaturation). In higher plants, fatty acid desaturation usually results in the formation of linoleic acid and αlinolenic acid, but no subsequent synthesis of C20+ PUFAs is observed. CO2H is the ‘front’ carboxyl group of the fatty acid; superscript numbers indicate the desaturated carbon relative to the ‘front’ carboxyl end.
(aerobic) desaturases, indicating instead a PKS-like system [10]. Moreover, expression of these Shewanella PKS-like ORFs in a fabB− (β-ketoacyl-acyl carrier protein synthase I) mutant of E. coli rescued the cells from needing exogenously supplied unsaturated fatty acids. Similar PKS-like gene clusters were also observed http://plants.trends.com
in other C20+ PUFA-accumulating marine bacteria (such as Vibrio marinus), indicating that this biosynthetic pathway is probably conserved among these prokaryotic organisms. Polyketide synthase PUFA biosynthesis in eukaryotes
The discovery of analogous PUFA PKS-like ORFs in eukaryotes was perhaps more surprising. Although PUFA-accumulating lower eukaryotes have been used as sources from which to isolate genes encoding enzymes of this biosynthetic pathway, these organisms were assumed to use the aerobic desaturase–elongase system. Isolation of the C22 PUFA front-end ∆4-desaturase from the marine microheterotroph Thraustochytrium sp., a member of the Thraustochytriidae, was thought to corroborate this [8]. However, when Metz et al. characterized the marine protist Schizochytrium via random cDNA
TRENDS in Plant Science
sequencing, the resulting 8500 ESTs failed to include the expected number of desaturases, whereas sequences related to the Shewanella PKS-like ORFs were well represented. Subsequent cloning of these Schizochytrium PKS-like genes revealed three ORFs compared with the five characterized for Shewanella, although the common presence of conserved PKS domains indicated a clear evolutionary relationship between the two species. Therefore, it is likely that a PKS-like system for the biosynthesis of PUFAs exists in some lower eukaryotes as well as in bacteria. Although the exact mechanism by which very long chain unsaturated fatty acids are assembled by the reiterative reaction of a PKS-like system needs to be unravelled, it probably involves precise double-bond isomerization. It is likely that such an isomerase activity is contained within the two conserved domains of the PUFA PKS-like ORFs that show homology
Research Update
TRENDS in Plant Science Vol.7 No.2 February 2002
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OH CH2R
DH
Processive polyunsaturated fatty acids Fig. 2. Generalized scheme for the processive synthesis of polyunsaturated fatty acids (PUFAs) by a polyketide synthase (PKS) system. In the case of PUFAs, it is envisaged that a primer molecule (in the form of acetylCoA) undergoes several rounds of sequential reactions (keto-synthase, keto-reductase, dehydratase and enoyl reductase), resulting in repeated synthesis and fatty acyl chain (esterified to the acyl carrier protein) elongation by two carbons per cycle. Because PUFAs contain methylene-interrupted double bonds (i.e. at the third carbon), it is likely that a dehydratase (FabA-like) module in the PKS also simultaneously carries out a trans–cis isomeration to generate this configuration.
to the E. coli FabA enzyme, a component of FAS, which is known to have both dehydratase and isomerase activity [13]. Importantly, the trans–cis double-bond isomerase activity represents new possibilities in the manipulation and exploitation of the PKS biosynthetic pathway, not least of all in the production of novel antibiotics (Fig. 2).
TRENDS in Plant Science
pathway [14]. It will be interesting to determine just how efficiently a heterologous PUFA–PKS can compete for endogenous substrates, and also if this results in any pleiotropic effects on the transgenic host. Several ‘front-end’ PUFA aerobic desaturases from Thraustochytrium have been identified recently [8], but the prevalence of this newly discovered PKS-like biosynthetic pathway is not known. Because Schizochytrium is also a member of the Thraustochytriidae, it is perhaps surprising to find these two distinct biosynthetic pathways represented in the same family, but molecular characterization of PUFA biosynthesis in the Thraustochytriidae might provide insights into the evolution of this important pathway. Conclusions
Engineering PUFAs in transgenic plants
In terms of producing very long chain PUFAs in transgenic oilseeds, these novel PKS-like ORFs potentially represent an attractive alternative route. For example, introducing and regulating the three Schizochytrium PKS-like ORFs in a transgenic plant is relatively simple compared with the more than five desaturase and elongase genes required for the previously characterized aerobic http://plants.trends.com
A novel polyketide synthase-like system has been identified as the PUFA biosynthetic pathway for both prokaryotic and eukaryotic marine organisms. The relative simplicity of this PKS-like system makes it attractive in terms of transgenic production of PUFAs. In addition, the identification of new (PUFA-specific) PKS activities such as double-bond isomerization might help in the bioengineering of new types of antibiotics.
Acknowledgements
IACR-Long Ashton Research Station receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. I would like to thank John Crosby and Tom Simpson (University of Bristol) for their assistance in the production of the graphics, and for helpful discussions. References 1 Broun, P. et al. (1999) Genetic engineering of plant lipids. Annu. Rev. Nutr. 19, 197–216 2 Gill, I. and Valivety, R. (1997) Polyunsaturated fatty acids: occurrence, biological activities and applications. Trends Biotechnol. 15, 401–409 3 Napier, J.A. et al. (1999) Plant desaturases: harvesting the fat of the land. Curr. Opin. Plant Biol. 2, 123–127 4 Michaelson, L.V. et al. (1998) Isolation of a ∆5-fatty acid desaturase gene from Mortierella alpina. J. Biol. Chem. 273, 19055–19059 5 Napier, J.A. et al. (1999) A growing family of cytochrome b5-domain fusion proteins. Trends Plant Sci. 4, 2–4 6 Qiu, X. et al. (2001) Identification of a ∆4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. J. Biol. Chem. 276, 31561–31566 7 Beaudoin, F. et al. (2000) Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proc. Natl. Acad. Sci. U. S. A. 97, 6421–6426 8 Leonard, A.E. et al. (2000) Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids. Biochem. J. 350, 765–770
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9 Liu, J-W. et al. (2001) Evaluation of the seeds oils from a canola plant genetically transformed to produce high levels of γ-linolenic acid. In γ-Linolenic Acid: Recent Advance in Biotechnology and Clinical Applications (Huang, Y-S. and Ziboh, V.A., eds), pp. 61–71, American Oil Chemists’ Society Press, Champaign, IL, USA 10 Metz, J.G. et al. (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290–293
11 Takeyama, H. et al. (1997) Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 143, 2725–2731 12 Watanabe, K. et al. (1997) Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation systems. J. Biochem. 122, 467–473 13 Bentley, R. and Bennett, J.W. (1999) Constructing polyketides: from Collie to combinatorial biosynthesis. Annu. Rev. Microbiol. 53, 411–446
14 Abbadi, A. et al. (2001) Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids? Eur. J. Lipid Sci. Technol. 103, 106–113
Johnathan A. Napier IACR-Long Ashton Research Station, Long Ashton, Bristol, UK BS41 9AF. e-mail: [email protected]
Meeting Report
Protein phosphorylation in and around signal transduction John Mundy and Kay Schneitz Plant Protein Phosphorylation, 12–15 September 2001, Vienna, Austria.
Metabolism
The recent Plant Protein Phosphorylation meeting was exciting for two reasons. First, it was focused so that people could share results and techniques. Second, it had a broad scope because phosphorylation is involved in regulating just about everything. Cell cycle
Dénes Dudits (Hungarian Academy of Sciences, Szeged, Hungary) has identified an alfalfa cyclin-dependent kinase inhibitor (CKI) that itself becomes phosphorylated by a calmodulin-like domain kinase (CDPK), suggesting a link between calcium-based signaling and cell-cycle complexes in vivo. What is the role of individual mitogen-activated protein kinases (MAPKs), and some of their regulators, such as MAPK kinase kinase (MAPKKK), in plant development? Patrick Krysan (University of Wisconsin, USA) addressed this issue with a reversegenetics approach. He has analyzed mutants carrying T-DNA insertions in any of three Arabidopsis MAPKKK genes, ANP-1, ANP-2 and ANP-3. There appears to be some redundancy because single mutants showed no apparent phenotype. Genome-wide expression analysis of anp2 anp3 double mutants suggests a role for these genes in repressing stress responses. Cathal Wilson (University of Vienna, Austria) provided evidence that the MAP kinase p43Ntf6, the MAPK kinase NtMEK1, and NPK1, a previously known MAPKKK, constitute a MAP kinase module that functions in cytokinesis. http://plants.trends.com
Tatjana Kleinow (Max-Planck-Institut für Züchtungsforschung, Cologne, Germany) has identified Arabidopsis orthologs of the β- and γ-activator-subunit of the SNF1related kinase complex. Surprisingly plants seem to have two different kinase complex forms: one complex with an α, β and γ subunit and one with just α and the new combined βγ subunit. Several new regulatory components of the SNF1related kinase signaling cascade have been identified using the two-hybrid system and in vitro binding assays. Stress responses
Kazuo Shinozaki (RIKEN, Tsukuba, Japan) discussed the signal crosstalk and integration that probably occurs between MAPKs given their large numbers (~24 MPKs, 18 MPKKs and 30 MPKKKs in Arabidopsis) and potential redundancy and partner promiscuity. Claudia Jonak and Irute Meskiene (University of Vienna, Austria) presented work on alfalfa WIG (wound-induced glycogen synthase kinase 3 or GSK3) and SIMK (woundand salt-inducible MAPK). Further characterization of WIG is warranted because GSK3s modulate the downstream specificity of other kinase signaling pathways. Biochemical analyses indicate that activity of the SIMK MPK is attenuated by the PP2C-type phosphatase MP2C, and that MP2C interaction with SIMK is mediated by a conserved MAPK docking motif in the MP2C N-terminus. Systems to assign functions to calcium (Ca2+-) dependent protein kinases (CPDKs, including one that functions upstream of a pathway required for Avr9-induced reactive
oxygen species (ROS), were described by Tina Romeis (John Innes Centre, Norwich, UK). Epitope tagging has shown that tobacco NtCDPK2 is shared between pathways triggered by biotic and abiotic stresses. Jörg Kudla (University of Ulm, Germany) showed that Ca2+ regulates interactions between calcineurin B-like proteins (CBL) and NAF kinase domains. Molecular and reverse genetic approaches indicate that differential CBL–calcineurin B-like interacting protein kinase (CIPK) affinities impose specificity upon Ca2+ signals. Two research groups have linked MAP kinases to genotoxic stress responses. Johannes Stratmann (University of South Carolina, Columbia, SC, USA) showed that the systemin receptor is involved in ultraviolet B (UV-B) signaling via activation of a tomato MAP kinase. It is also possible that UV-B responses are mediated non-specifically via stress pathways, including the systemin receptor. A genetic screen described by Roman Ulm (Friedrich Miescher Institute, Basel, Germany) revealed that loss of function of an Arabidopsis MAP kinase phosphatase (AtMKP1) results in ultraviolet C (UV-C) and methyl methanesulfonate (MMS)hypersensitivity. AtMKP1 interacts with several MPKs, therefore AtMPK1 must be an important regulator of MAP kinase activities in vivo. Pathogen responses
Three research groups linked pathogen responses to MAP kinase signaling. Dierk Scheel (Institute of Plant Biochemistry, Halle, Germany) described responses to the peptide elicitor PEP-13 in parsley (Petroselinum crispum). PEP-13
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