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AIR2-CT94-0967
Manipulation of Lipid Metabolism aimed at Production of Fatty Acids and Polyketides - Final Report |
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Contract No | AIR2-CT94-0967 |
| Total Cost | 3 004 540 ECU | |
| EC Contribution | 1 769 770 ECU | |
| Start Date | 01/09/1994 | |
| Duration | 50 months |
FINAL PROGRESS REPORT
Commercial quantities of oils are obtained from animals, plants, algae, yeasts, filamentous fungi and bacteria although plants are the major quantitative sources. Oil-bearing crops are widely grown in Europe and are by far the most common sources of oils. Improvements to the specific yield and also the types of fatty acids esterified to glycerol in the triacylglycerols would be beneficial and explains the considerable research effort to understand and then manipulate lipid biosynthesis in oil crops. Non-plant sources of oils are used commercially primarily because oils with different properties, determined by the fatty acids, are available. For example, long-chain polyunsaturated fatty acids (PUFAs) are found in the oils of some fungi and algae but not all of these fatty acids can yet be produced from plants. Oils are also accumulated by some yeasts and filamentous fungi; these are the oleaginous species. The fungus Mortierella alpina, for example, can accumulate oil to more than half it weight, as can the yeast C. curvatus. A. nidulans is also sometimes considered to be oleaginous as it accumulates up to a quarter of its weight as lipid. M. alpina is used to produce oil containing arachidonic acid (C20:4, w-6). C. curvatus accumulates oil during growth on cheap carbon sources such as whey permeate, grows rapidly and is a potentially useful commercial producer of oils. In this project, we have focused on two model fungi: the filamentous fungus A. nidulans and the yeast S. cerevisiae. In addition, C. curvatus and two plants (tobacco and rape) have been part of the project. A. nidulans and S. cerevisiae were chosen because they represent the best developed systems for gene cloning and manipulation among the filamentous fungi and yeasts. The genome sequence is available for S. cerevisiae and this proved to be useful in the project. Although the A. nidulans genome is almost now completely sequenced, the sequence is not yet accessible and use could not therefore be made of it in the project.
The overall objectives of this project were (1) to study aspects of lipid synthesis in yeast, filamentous fungi and plants, (2) to apply the knowledge gained in the manipulation of lipid synthesis in these organisms and, (3) to clone genes important in lipid synthesis and to use these genes to manipulate lipid synthesising species, especially plants and the oleaginous yeast C. curvatus. The use of genes derived from non-plant sources may prove advantageous in the resulting transgenic plants because the regulation of plant, yeast and filamentous fungal genes can vary and in a heterologous host, may give a new and possibly unexpected phenotype. The differences between plant and fungal lipid synthesis are exemplified by primary and cellular sites of oleic acid (C18:1) synthesis; plant plastids and fungal cytoplasm. Thus, the environment for function of these pathways will be different and subject to differences in regulation.
Our understanding of the carbon flux to triacylglycerols, in terms of the metabolic pathways, their interconnections and regulation, is incomplete. Part of the project was aimed at gaining more knowledge in these areas with a focus on the supply of cytosolic acetyl-CoA, synthesis of malonyl-CoA, elongation and desaturation of fatty acids and synthesis of triacylglycerols. Acetyl-CoA is synthesised in the mitochondria of fungi and must be transported to the cytoplasm for use in fatty acid synthesis. Transport of acetyl-CoA to the cytoplasm is thought to be mediated by ATP: citrate lyase (ACL) and/or carnitine acetyl transferase (CAT). Assessment of which of these two transport routes was more important in A. nidulans was a Task of the project to be followed up by cloning the encoding gene. The approaches used to achieve this Task relied upon the use of mutant strains of A. nidulans and purification of the enzymes followed by gene cloning by reverse genetics. During the course of this project, it became apparent that malic enzyme was a major source of NADPH for the synthesis of storage lipid by A. nidulans. For this reason, further investigation of the role of malic enzyme in storage lipid synthesis by A. nidulans was made. Both ACL and CAT enzymes were purified to apparent homogenicity and cloning of the ACL-encoding gene is underway.
Cytosolic acetyl-CoA is metabolised to malonyl-CoA by acetyl-CoA carboxylase (ACC). ACC is the first committed step in the synthesis of fatty acids providing two of its three carbons to the fatty acid synthase (FAS). Malonyl-CoA is also necessary for polyketide synthesis although it has not formally been shown in A. nidulans that ACC is the source of malonyl-CoA for synthesis of the polyketide sterigmatocystin. A. nidulans is known to contain genes encoding the two subunits of the constitutive FAS used in synthesis of C16 and C18 fatty acids and also genes encoding an inducible FAS dedicated to the supply of C6 fatty acid for ST synthesis. We cloned the accA gene from A. nidulans and studied its regulation. Because ACC is such a key enzyme in fatty acid synthesis, the encoding gene was a target for introduction into plants, particularly as it has already been shown that over-expression of a cytosolic ACC from Arabidopsis thaliana targeted to the plastids of Brassica napus increased the oil content of seeds. A cDNA version of accA was therefore constructed and introduced into rape. The transformants are being studied.
S. cerevisiae is the model lower eukaryote for gene manipulation studies. The accessible genome sequence presents an opportunity for gene cloning and functional analysis of cloned genes is relatively straightforward compared to other yeasts and filamentous fungi. S. cerevisiae was used to examine fatty acid elongation, with three distinct metabolic pathways being the focus of the work: FAS, fatty acid elongation (non-FAS) and b-oxidation component enzymes operating in reverse. A limited number of fatty acid elongation associated genes in S. cerevisiae are known and we aimed to extend understanding in this area. S. cerevisiae was investigated as a possible host for expression of the rat FAS cDNA and thioesterase genes to test if medium chain (e.g. C12) fatty acids could be produced. S. cerevisiae was also used as a host for the heterologous expression of plant genes encoding a fatty acid acetylase and hydroxylase to produce novel fatty acids. We showed that introduction of the A12 acetylenase gene from Crepis alpina into S. cerevisiae enabled the yeast to synthesise the novel fatty acid crepenynic acid.
The focus of our studies with C. curvatus was fatty acid desaturation and elongation. In contrast to S. cerevisiae, at the inception of this project it was not possible to transform C. curvatus and no genes were cloned. The attractions of C. curvatus as an oleaginous yeast with a capacity for growth on cheap carbon sources such as whey permeate led us to develop a transformation system and to assess the suitability of C. curvatus as a host for expressing heterologous genes involved in lipid biosynthesis. Many species of yeasts and filamentous fungi can be transformed although many species require homologous promoters to drive the expression of selectable resistance genes. This proved to be so with C. curvatus and therefore we cloned promoters which we used in transforming vectors. We also cloned a gene encoding a fatty acid desaturase. The genes and transformation system will enable us to manipulate the fatty acid profile of C. curvatus
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