Scanning electron micrograph of oil droplets purified from the oleaginous yeast Apiotrichum curvatum

SUMMARY

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.

ACTIVITIES

Task 1 is an investigation of metabolic switching between polyketide and fatty acid synthase in Aspergillus nidulans. In the first year, two genomic libraries of the sterigmatocystin producing strain of A.nidulans FGSC26 were made in order to initiate the isolation of genes encoding fatty acid synthase (FAS) and polyketide synthase (PKS) through direct gene cloning using heterologous gene probing and polymerase chain reaction (PCR). Isolation of the inducible FAS and PKS genes was attempted by heterologous probing using various cosmids. This approach yielded the targeted genes. Although FAS genes have been cloned by other workers, this group independently isolated the constitutive FAS2 gene from the gene libraries. In addition, they isolated FAS1 and FAS2 genes which are distinct in homologies to the inducible and constitutive FAS genes. Sequencing of these genes is not yet complete. Specific DNA probes for hybridisation studies with each of the known A. nidulans FAS genes are being developed so that the 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 (ACCl) encoding acetyl CoA carboxylase has been cloned from A. nidulans. The gene has been partially sequenced and analysis of the ACC promoter has been initiated. The ACC gene is a key factor in regulating the supply of malonyl CoA for the synthesis of fatty acids and polyketides. Having cloned the gene, the group is in a strong position to be able to manipulate the synthesis of these compounds.

Task 2 looks at the metabolic flux to cytosolic acetyl CoA. The aim was to clone the gene encoding ATP: citrate lyase and thus to manipulate the levels of cytosolic acetyl CoA in A. nidulans. Attempts to isolate an ACL negative mutant and heterologous probing proved unsuccessful. A third method, purification of the ACL was more promising. Initial problems were overcome and a 40-fold purification achieved. N terminal and internal protein sequences have been obtained from the purified ACL and used in attempts to clone the encoding gene. Putative gene fragments, obtained by PCR, 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. It has not yet proved possible to rescue the cosmid from the transformed strain but this work is continuing. Having either the ACL or CAT genes, or preferably both, will enable manipulation of the supply of cytosolic acetyl CoA and thereby enable regulation of the synthesis of fatty acids.

Task 3 involves fatty acid desaturation and chain length modification in the yeast Saccharomyces cerevisiae. The complete rat FAS cDNA was assembled from constituent overlapping cDNA clones and incorporated into the yeast expression vector pVT100 U behind the yeast alcohol dehydrogenase (ADHI) promoter. To ensure efficient transcription/translation of the rat FAS gene in yeast, some manipulation of the 5' untranslated region of the cDNA clone was then necessary. The functionality of the constructed vectors was assessed by transforming the plasmids produced into two yeast strains which had been previously disrupted for FAS1 and FAS2 genes. It was confirmed that the introduced rat FAS gene was transcribed in yeast. 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, although further studies are underway. In an attempt to identify genes whose products 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. FAS1, FAS2 and FASl/FAS2 genes were grown to identical stages of growth and analysed for their fatty acid compositions.

Task 4 The broad aim of this Task is to manipulate genetically fatty acid biosynthesis in Apiotrichum curvatum (syn. Cryptococcus curvatus). In the first year, the stearoyl CoA desaturase gene was cloned and sequenced. A major advance in the second year has been the development of a transformation system for A. curvatum. A transforming vector was constructed that used the homologous glyceraldehyde 3' phosphate dehydrogenase promoter to drive expression of the ble gene from Streptoalloteichus hindustanus which confers resistance to phleomycin. 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 concerns the regulatory role of acyl CoA: diacylglycerol acyl transferase (DAGAT) in yeasts and moulds. 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. Methods for further purifying DAGAT are being pursued.

The lipid and fatty acid contents of Saccharomyces cerevisiae overexpressing FAS1, FAS2 and FAS1/FAS2 genes were measured. 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 FASl/FAS2 over expressing transformants.

Introduction

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.

Objective

The objective is to investigate metabolic switching and pathway manipulation in yeasts, filamentous fungi and plants with a focus on the production of fatty acids and polyketides. Research effort is concentrated on FAS, PKS, DAGAT, supply of acetyl-CoA and the ability to produce fatty acids of predetermined chain length and degree of desaturation. Target species have been chosen because of their detailed genetic history (Saccharomyces cerevisiae, Aspergillus nidulans and Brassica napus), their ability to accumulate fats/oils (Apiotrichum curvatum and Brassica napus) and moulds which are important in food biotechnology (Aspergillus niger).

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.

Introduction
Fatty acids and polyketides are important metabolites with practical applications. Yeasts and filamentous fungi provide a system for their production that is amenable for study and manipulation. Some oleaginous yeasts and filamentous fungi are suited to commercial use for production of these compounds. Several crops are already widely used for production of oils but there is a need for continued research to fully exploit their potential in yield improvement and providing novel oils. Oil crops remain the cheapest EU source of many oils and derived fatty acids. This position is unlikely to be challenged in the foreseeable future except for the supply of polyunsaturated fatty acids. The work in this programme is therefore significant in its expected impact in scientific terms and in application. The activity is divided into six main tasks covering the following aspects of lipid metabolism:

• Metabolic switching between polyketide synthase and fatty acid synthase in Aspergillus nidulans
• Metabolic flux to cytosolic acetyl-CoA in moulds.
• Fatty acid desaturation and chain length modification in Saccharomyces cerevisiae
• Fatty acid desaturation and chain length modification in Apiotrichum curvatum.
• The regulatory role of acyl-CoA:diacylglycerol acyl transferase (DAGAT) in yeasts and moulds.
• Metabolic engineering of lipid metabolism in plants.

The overall pattern of organisation of this programme is, in the first phase, to isolate important genes involved in the biosynthesis of fatty acids, storage lipids and polyketides and then to use these genes to manipulate the biosynthesis of lipids, fatty acids and polyketides in fungi and plants. The FAS and polyketide synthase (PKS) genes from A. nidulans were original targets of the project, but were obtained and described from outside sources. Genes encoding fatty acid elongases in S. cerevisiae, carnitine acetyl transferase (CAT) in A. nidulans, ATP citrate lyase (ACL) in A. nidulans and a plant diacylglycerol acyltransferase (DAGAT) remain targets. It is unlikely that any of these genes will be available for expression studies in plants within the time frame of the project. However, a cDNA version of the ACC gene will be constructed for expression in plants.

Achievements

Task 1 In last year's report a clone of the gene, described as single copy, that encodes acetyl-CoA carboxylase (ACC) in Aspergillus nidulans had been obtained. During this year, we have shown that A. nidulans contains two ACC-encoding genes, accA and accB. The accA gene has been fully sequenced and accB partially sequenced. Measurement of ACC activity has been made under various conditions and reporter constructs based on the accA and accB promoters are being made. ACC is a key enzyme in regulating the supply of malonyl-CoA for synthesis of fatty acids and, possibly, of polyketides and we are well placed to study the regulation of ACC at transcriptional and translational levels.

Task 2 This task is aimed at the purification of the two candidate enzymes for supply of cytosolic acetyl-CoA: carnitine acetyl transferase (CAT) and ATP: citrate lyase (ACL). The purified enzymes will then be used to clone the respective encoding genes. Both CAT and ACL proteins have been highly purified but this has not yet facilitated successful gene cloning. Attempts to clone both genes will continue, with emphasis on the ACL-encoding gene because recent evidence favours CAT being more important in transferring cytosolic acetyl-CoA into mitochondria.

Task 3 The yeast Saccharomyces cerevisiae is a suitable model lower eukaryote for gene manipulation studies because the genomic sequence facilitates gene cloning and most gene manipulations are straightforward. The FOX3 (encoding 3-oxoacyl-CoA thiolase) and ERG10 (encoding acetoacetyl-CoA thiolase) genes had previously been disrupted. Double disrupted strains have now been produced, including double disruptants in a background lacking FAS2 (encoding a subunit of the fatty acid synthase). Fatty acid analysis is underway. Fatty acid elongation is a particular focus of this Task. A gene (ELO1) that putatively encodes an elongase has been reported and we have cloned a gene (GNS1) with substantial homology to ELO1. The function of GNS1 is now being assessed. Mutant strains deficient in fatty acid elongation have been obtained and potential complementing genes are being characterised.

Task 4 Work on the oleaginous yeast Cryptococcus curvatus focused on manipulation of the fatty acid content by regulation of fatty acid desaturase activity and introduction of a heterologous thioesterase-encoding gene. The delta-9-desaturase gene was cloned earlier in the project and anti-sense transformants have been produced to study down-regulation. Other transformants containing the rat thioesterase II gene (under control of C. curvatus promoters) have also been produced. The impact of these constructs on fatty acid accumulation by C. curvatus is being assessed.

Task 5 Diacylglycerol (DAG) is a common precursor for the synthesis of both phospholipids and triacylglycerol (TAG). Hence, the acylation of DAG yielding TAG is the only unique step in the TAG assembly. The regulation of this enzymatic step, catalysed by diacylglycerol acyltransferase (DAGAT), is therefore of particular interest. The objective of this project is to purify the DAGAT in order to isolate the gene and thereby study the regulatory role of this enzyme in oil accumulation. In this period a partial purification of DAGAT was achieved. A further purification of DAGAT with the use of chromatography has not yet been successful due to severe loss in yield. Data was also obtained on the localisation of in vitro DAGAT activity in developing seeds from oat and sunflower seeds, Avocado mesocarp and the oleaginous yeast Cryptococcus curvatus. These data show that at least 80% of the activity resides in the microsomal fraction irrespective of organism and that the highest specific activity was measured in microsomes from sunflower seeds. Initial attempts to solubilise the DAGAT activity from C. curvatus failed.

Task 6 This Task became active for the first time in year 3. Its aim is to examine the impact of expressing genes, cloned in other Tasks, in plants on the yield and type of fatty acids produced. Tobacco plants were transformed with the rat FAS cDNA gene in expression constructs with and without plastid tarP getting sequences. The transformed tobacco plants are being analysed. Tobacco was chosen as a model plant in initial work rather than oilseed rape.