Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway

ω3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Δ6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds. ω3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Δ6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds. Human development and health depend in many respects on the availability of long chain multiply unsaturated fatty acids of 20 or 22 carbons in length that contain up to 6 methylene-flanked cis-double bonds. These fatty acids are classified under the designation very long chain polyunsaturated fatty acids (VLCPUFAs). 2The abbreviations used are:VLCPUFAvery long chain polyunsaturated fatty acid(s)ARAarachidonic acidCLdiphosphatidylglycerolEPAeicosapentaenoic acidFAMEfatty acid methyl esterLPCATacyl-CoA:lysophosphatidylcholine acyltransferasePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineTAGtriacylglycerolRACErapid amplification of cDNA endsMES2-(N-morpholino)ethanesulfonic acidocsoctopine synthase. Nutritionally important VLCPUFAs include arachidonic acid (ARA, 20:4Δ5,8,11,14), an ω6-fatty acid, and the ω3-fatty acids eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19). ω3-VLCPUFAs are of particular interest from a nutritional standpoint since the uptake of these fatty acids is considered to be low in Western diets (1Simopoulos A.P. Am. J. Clin. Nutr. 1991; 54: 438-463Crossref PubMed Scopus (1869) Google Scholar). ω3-VLCPUFAs have long been investigated for their importance during human fetal development and the formation and function of the central nervous system, brain, and retina. In addition to their structural functions in membranes, several medical studies indicate that ω3-fatty acids have anti-inflammatory properties and, therefore, might be useful in the management of inflammatory and autoimmune diseases such as cardiovascular disease, major depression, arthritis, inflammatory bowel disease, asthma, and psoriasis (2Simopoulos A.P. J. Am. Coll. Nutr. 2002; 21: 495-505Crossref PubMed Scopus (1533) Google Scholar). very long chain polyunsaturated fatty acid(s) arachidonic acid diphosphatidylglycerol eicosapentaenoic acid fatty acid methyl ester acyl-CoA:lysophosphatidylcholine acyltransferase phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine triacylglycerol rapid amplification of cDNA ends 2-(N-morpholino)ethanesulfonic acid octopine synthase. Whereas mammals, including humans, can convert the essential precursors ω6–18:2Δ9,12 or ω3–18:3Δ9,12,15 (α-linolenic acid) to VLCPUFAs, a considerable proportion of VLCPUFA has to be taken up directly as components of the diet (3Goyens P.L. Spilker M.E. Zock P.L. Katan M.B. Mensink R.P. Am. J. Clin. Nutr. 2006; 84: 44-53Crossref PubMed Scopus (291) Google Scholar). Naturally occurring producers and sources of ω3-VLCPUFAs are microorganisms, including marine bacteria and microalgae. These organisms represent the starting point of the aquatic food chain by which VLCPUFAs ultimately accumulate in fish oils. At the moment the main source of ω3-VLCPUFAs for human consumption are fatty ocean fish, such as salmon, mackerel, tuna, or herring. Unfortunately, the increased demand for fish and fish oils has led to a depletion of fish stocks in vast ocean areas worldwide. Thus, as an exclusive source of VLCPUFAs, fish cannot cover the needs of a growing world population (4Hites R.A. Foran J.A. Schwager S.J. Knuth B.A. Hamilton M.C. Carpenter D.O. Environ. Sci. Technol. 2004; 38: 4945-4949Crossref PubMed Scopus (255) Google Scholar). Altogether, the increasing interest to find alternative sources of ω3-VLCPUFAs has led to various attempts to produce ω3-VLCPUFAs via the biotechnological introduction of new biosynthetic pathways in plants, ultimately aiming for VLCPUFA-production in annual oilseeds, e.g. in rapeseed or linseed. VLCPUFA synthesis in transgenic plants established so far starts from the plant endogenous fatty acids ω6–18:2Δ9,12 or ω3–18:3Δ9,12,15 and requires two distinct catalytic activities, desaturases and elongases. Both activities work together in an alternating manner to establish the final product. Genes for the biosynthesis of VLCPUFAs have been isolated from organisms of different kingdoms (algae, amoeba, fungi, moss, and flowering plants) and have been characterized by heterologous expression in various model systems regarding the properties of the encoded enzymes. In recent years a range of tests was done to establish the biosynthesis of VLCPUFAs in plants by introducing a variety of different front-end desaturases and elongases (5Graham I.A. Larson T. Napier J.A. Curr. Opin. Biotechnol. 2007; 18: 142-147Crossref PubMed Scopus (82) Google Scholar, 6Napier J.A. Annu. Rev. Plant Biol. 2007; 58: 295-319Crossref PubMed Scopus (217) Google Scholar). Two previous approaches are of particular relevance for the study presented here. First, the so called alternative pathway for the biosynthesis of VLCPUFAs was successfully tested in Arabidopsis leaves (7Qi B. Fraser T. Mugford S. Dobson G. Sayanova O. Butler J. Napier J.A. Stobart A.K. Lazarus C.M. Nat. Biotechnol. 2004; 22: 739-745Crossref PubMed Scopus (378) Google Scholar). This pathway is based on the sequential action of a Δ9 elongase (from B. Fraser T. Stobart A.K. Napier J.A. Lazarus C.M. 2002; PubMed Scopus Google a (from J. PubMed Scopus Google and a (from the Lazarus C.M. G. Napier J.A. Stobart A.K. J. Biol. PubMed Scopus Google Scholar). The Δ9 elongase which be to and via and desaturation, In a rate-limiting was and of up to of the of Arabidopsis was be that is and that the fatty acids are directly lipids as oils as is the in seeds and as be for VLCPUFA a was The and to very in the acyl-CoA pool of the transgenic Arabidopsis plants O. B. Lazarus C.M. Napier J.A. 2006; PubMed Scopus Google an of these fatty acids of the acyl-CoA pool. In the the of and desaturases of the with a elongase from the in showed that the and desaturation on in the plant with a for the of phosphatidylcholine the elongase as J. Napier J.A. Plant 2004; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). acyl-CoA and of the transgenic plants that production of VLCPUFAs in plants requires the of desaturases and elongases the of acyl from the pool the pool and This is very by the enzymatic of an acyl-CoA:lysophosphatidylcholine acyltransferase The endogenous to the Δ6-desaturated fatty and thus, the Δ6-desaturated fatty acids to In the of fatty acids between the and by the action of a rate-limiting for VLCPUFA production by transgenic in J. Napier J.A. Plant 2004; PubMed Scopus Google Scholar). In the research as as in several e.g. G. J. T. Nat. Biotechnol. PubMed Scopus Google and and Scholar, desaturases from various organisms for their feasibility to produce The that the front-end desaturases characterized from fungi, and are desaturases and substrates. In to these a of front-end desaturases acyl-CoA as PubMed Scopus Google Scholar, T. T. PubMed Scopus Google Scholar, PubMed Google Scholar). acyl-CoA-dependent from the microalga was J. PubMed Scopus Google Scholar). fatty acid on O. be that desaturases with a for acyl-CoA can be isolated from acyl-CoA-dependent with the the rate-limiting and exchange of of VLCPUFA synthesis between desaturation in the pool and elongation steps in the acyl-CoA pool. plants and in their oils produce or VLCPUFAs with chain or triacylglycerol produce the ω3-fatty acid, in is to elongation and two desaturation the of of desaturation, the be for substrates. known to is the rate-limiting of fatty acids between and by J. Napier J.A. Plant 2004; PubMed Scopus Google Scholar). to a for these we report on the and of acyl-CoA-dependent desaturases from the Mantoniella and O. the new enzymes, an acyl-CoA-dependent biosynthetic pathway of and was successfully established in and in seeds of plants. This pathway a during VLCPUFA biosynthesis and avoids the bottleneck desaturation by J. Napier J.A. 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Plant 2004; PubMed Scopus Google a of the acyl-CoA-dependent the bottleneck and the the in yeast was is that yeast be different from in the used and that the of the thus, be in the heterologous The of from the elongation of the by as This to in the expression with the biosynthesis In the fatty acid was in expression which is in with the The establishment of an acyl-CoA-dependent pathway for production in yeast of data for the feasibility of the with the yeast model cannot be to the in plants, the of experiments at acyl-CoA-dependent production in Arabidopsis seeds. of in acyl-CoA-dependent biosynthesis be established in plants, and with in plants, subsequently to as plants. comparison of the of production by different enzymes, a and with was which be to as provide a the of and with in plants, which the previously (2Simopoulos A.P. J. Am. Coll. Nutr. 2002; 21: 495-505Crossref PubMed Scopus (1533) Google Scholar, J. J. G. J. J. of the in S. and which be to as plants. The plant encoded under the of the 1991; PubMed Scopus Google Scholar). The fatty acid of seeds of and plants showed several new fatty acids that as and and for and as and that in plants the pathway and in and plants the and the pathways been successfully The fatty acid of the endogenous fatty acids as for the VLCPUFA pathway, and are or in the transgenic Arabidopsis seeds and the new fatty acids in the and the is In the fatty acids and at the first desaturation of the The of Δ6-desaturated fatty acids in the plants is with with in rapeseed, or (2Simopoulos A.P. J. Am. Coll. Nutr. 2002; 21: 495-505Crossref PubMed Scopus (1533) Google Scholar, J. J. G. J. J. of the in S. and the of the bottleneck in VLCPUFA biosynthesis. In in the and or of the desaturation was and was to the with very by the elongase in of the plants. 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The low with acyl-CoA-dependent is a of of the bottleneck with and studies be the of acyl-CoA-dependent which have been the in the fatty acids of seeds of plants that the of desaturation has been successfully the from important for VLCPUFA production is the the fatty acids in or in phospholipids, the of the various fatty acid of the established biosynthetic pathways was and the of with different lipids of the of different transgenic seeds that transgenic of The comparison of plants the and a of fatty acids fatty acids in The of and in different of Arabidopsis seeds showed that the VLCPUFA by acyl-CoA-dependent pathways to a in by the lipid-linked pathway These on the of biosynthesis in Arabidopsis seeds. on the in to VLCPUFAs, VLCPUFAs phospholipids, we that is by an acyltransferase by a acyltransferase in Arabidopsis seeds. is to that of was that of in for the of be a of the acyltransferase acyltransferase for the of of from biosynthesis of Arabidopsis of of of in a new plant studies is known that the of in the plant an important for biotechnological In with between different The low of in the transgenic Arabidopsis plants thus, in be a of and for low are or by the for that are in the plants, as was for In VLCPUFA can be in plants an exclusively acyl-CoA-dependent pathway that avoids rate-limiting of fatty acid between lipids and such as low catalytic activities of the desaturases or low expression of in plants VLCPUFA in the experiments the data that in is for VLCPUFA by an acyl-CoA-dependent are in from the fatty acid that acyl-CoA-dependent VLCPUFA biosynthesis approaches to the of plant oils. and for and for of the with