Objective
All living organisms contain lipids, more generally known as fats. These consist of long chain fatty acids esterified to glycerol and in some cases to sugars, amino acids, phosphate, sulphate and other similar molecules. However, wide differences are seen between the fatty acids found in various organisms. They may differ in terms of their chain length (number of carbon atoms), degree of saturation (number and position of double bonds) and inclusion of unusual stereochemical configurations or chemical groups, such as epoxides. This project aims to build a better understanding of lipid metabolism in order to develop methods of enhancing the production of desirable compounds.
In eukaryotic micro-organisms and plants, acetyl-CoA is a precursor for the synthesis of many compounds including fatty acids and polyketides, compounds that are finding increasing application in the food and chemical industries. Some polyketides are food-associated toxins, whereas others are widely used as antibiotics and pharmaceuticals. The fatty acid synthase (FAS) and polyketide synthase (PKS) enzymes are remarkably similar in their function with differences apparent primarily in the detailed programming. These two enzymes constitute a metabolic branch point between primary and secondary metabolism. Fatty acids enter a second branch point between membrane lipid biosynthesis (functional fats) and fat accumulation (storage fats) and this switch is controlled by the acyl-CoA; diacylglycerol transferase (DAGAT) enzyme.
Activities
These are based on a series of tasks as follows:
Task 1 Metabolic switching between polyketide synthase and fatty acid synthase in Aspergillus nidulans.
Fatty acid synthase (FAS) genes encoding the alpha and beta subunits of the enzymes have been cloned from Aspergillus nidulans in the US. They have identified constitutive and inducible FAS genes. This project has independently isolated the constitutive FAS2 gene from the gene libraries previously constructed. In addition, FAS 1 and FAS2 genes were isolated from the libraries which were distinct in homologies from the inducible and constitutive FAS genes. These genes are being sequenced. Specific DNA probes for hybridisation studies with each of the known A.nidulans FAS genes are being constructed so that their differential regulation of transcription can be investigated. The new FAS genes are being incorporated into gene replacement vectors so that the phenotypes of gene-deleted transformants can be examined.
The gene (ACC1) encoding acetyl-CoA carboxylase has been cloned from A.nidulans. It is present in the genome as a single copy and transcript analysis showed a 7kb mRNA. The gene has been partially sequenced and construction of a gene replacement vector for its disruption is underway. Analysis of the ACC promoter has been initiated. The ACC gene is potentially a key factor in regulating the supply of malonyl-CoA for the synthesis of fatty acids and polyketides. Cloning of the gene, enables the synthesis of fatty acids and polyketides to be manipulated.
Task 2: Metabolic flux to cytosolic acetyl-CoA in moulds.
Substantial progress has been made in the aims of cloning genes from A.nidulans that encode ATP:citrate lyase (ACL) and carnitine acetyl transferase (CAT). Following on from the partial purification of the ACL protein, it has now been purified by 43-fold yielding a product that appears to be pure on polyacrylamide gels. N-terminal and internal protein sequences have been obtained from this and used in attempts to clone the encoding gene by reverse genetics techniques. Putative gene fragments, obtained by Polymerase Chain Reaction, have been cloned and sequencing is underway. An alternative method for cloning the ACL gene has also been adopted. Antibodies have been raised against the purified ACL protein and will be used to screen expression libraries. An A.nidulans mutant deficient in CAT activity (and consequently unable to grow on acetate as a carbon source) was transformed to acetate-utilisation with a cosmid library from A.nidulans. This will be rescued, the region of the cosmid that is responsible for transformation to acetate utilisation will be determined and sequenced. This should encode the CAT gene. Having either the ACL or CAT genes, or preferably both, will enable manipulation of the supply of cytosolic acetyl-CoA and, thereby, provide the means to regulate the synthesis of fatty acids.
Task 3: Fatty acid desaturation and chain length modification in Saccharomyces cerevisiae.
Here the objective is to manipulate Saccharomyces cerevisiae for modification of its fatty acid composition. Two thiolase genes have been cloned and deleted from S cerevisiae: FOX3 (encodes 3-oxoacyl-CoA thiolase) and ERG10 (encodes acetoacetyl-CoA thiolase). Analysis of the fatty acids in the strain lacking FOX3 suggests that the proportion of short chain fatty acids is enhanced. In an attempt to identify genes the products of which are involved in the elongation of C13:0 fatty acids, seven temperature sensitive putative fatty acid elongase mutants unable to grow in the presence of C13:0 at the restrictive temperature were selected. One of these mutant strains is now being used to clone the genes involved in elongation of C13:0. S. cerevisiae strains overexpressing the yeast FAS 1, FAS2 and FAS l/FAS2 genes were grown to identical stages of growth and analysed for their fatty acid compositions. The lipid and fatty acid contents of Saccharomyces cerevisiae overexpressing FAS 1, FAS2 and FAS 1/FAS2 genes were rneasured. Cells were harvested at the same stage of growth for the analytical comparisons. No differences in the total lipid contents were found in the various transformed strains in relation to wild-type yeast. However, transformants over-expressing FAS2 appeared to have significantly higher levels of C18 fatty acids at the expense of C16 fatty acids. This did not occur in the FAS 1/FAS2 over-expressing transformants.
Task 4: Fatty acid desaturation and chain length modification in Apiotrichum curvatum.
The overall aim of this part of the project is to genetically manipulate genetically fatty acid biosynthesis in Apiotrichum curvatum (syn. Cryptococcus curvatus). This continues on from the cloning and sequencing of the delta 9 stearoyl-CoA desaturase gene, resulting in a transformation system for A. curvatum based on a vector constructed using the homologous glyceraldehyde 3' phosphate dehydrogenase promoter to drive expression of the ble gene from Streptoalloteichus hindustanus which confers resistance to phleomycin. Whole cell electroporation gave 50-100 transformants per micro g DNA. The transforming DNA is stably integrated into the host genome. Use of this vector in co-transformation studies has shown that other genes can be introduced into A.curvatum.
Task 5: The regulatory role of acyl-CoA:diacylglycerol acyl transferase (DAGAT) in yeasts and moulds.
This Task has continued to make progress in the purification of diacylglycerol acyltransferase (DAGAT). This enzyme is necessary for the synthesis of triacylglycerol and is the only unique step because the immediate precursor (diacylglycerol) is also used in the synthesis of phospholipids. DAGAT is an integral membrane protein which presents obstacles to its purification. So far, a 20-fold enrichment of DAGAT activity has been achieved through a two-step chromatography purification of detergent-solubilised protein. Although such conditions caused a loss of activity, activity was restored by addition of anionic lipids. Methods for further purifying DAGAT are being pursued.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences biochemistry biomolecules lipids
- medical and health sciences basic medicine pharmacology and pharmacy pharmaceutical drugs antibiotics
- natural sciences chemical sciences organic chemistry amines
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
NR4 7UA Norwich
United Kingdom
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