Abstract:
The present invention relates to the use of a novel enzyme and its encoding gene for transformation. More specifically, the invention relates to the use of a gene encoding an enzyme with acyl-CoA:diacylglycerol acyltransferase activity. This gene expressed alone in transgenic organisms will increase the total amount of oil (i.e. triacylglycerols) that is produced.

Description:
This application claims the benefit of U.S. Provisional Application No. 60/164,859 filed Nov. 12, 1999. should appear as the first sentence of the description. 
     FIELD OF THE INVENTION 
     The present invention relates to the use of a novel enzyme and its encoding gene for transformation. More specifically, the invention relates to the use of a gene encoding an enzyme with acyl-CoA : diacylglycerol acyltransferase activity. This gene expressed alone in transgenic organisms will increase the total amount of oil (i.e. triacylglycerols) that is produced. 
     BACKGROUND OF THE INVENTION 
     In oil crops like rape, sunflower, oilpalm etc., the oil (i.e. triacylglycerols) is the most valuable product of the seeds or fruits and other compounds such as starch, protein and fiber is regarded as by-products with less value. Enhancing the quantity of oil per weight basis at the expense of other compounds in oil crops would therefore increase the value of the crop. If enzymes regulating the allocation of reduced carbon into the production of oil can be upregulated by overexpression, the cells will accumulate more oil at the expense of other products. This approach could not only be used to increase the oil content in already high oil producing organisms such as oil crops, they could also lead to significant oil production in moderate or low oil containing crops such as soy, oat, maize, potato, sugar beats, and turnips as well as in microorganisms. 
     Development in genetic engineering technologies combined with greater understanding of the biosynthesis of triacylglycerols now makes it possible to transfer genes coding for key enzymes involved in the synthesis of triacylglycerols from a wild plant species or organisms of other kingdoms into domesticated oilseed crops. In this way, triacylglycerols can be produced in high purity and quantities at moderate costs. 
     It is known that the biosynthesis of triacylglycerols (TAG) in fat-accumulating tissues in animals (Bell &amp; Coleman, 1983) and plants (Cao &amp; Huang, 1986, Martin &amp; Wilson 1983) as well as the accumulation of oil in microbial organisms such as bacteria (Ekundayo &amp; Packler, 1994), yeast and other fungi (Ratledge 1989) can be catalyzed by acyl-CoA : diacylglycerol acyltransferases (DAGATs), enzymes that transfer an acyl-group from acyl-CoA to diacylglycerol, thus forming TAG. 
     During the past few years genes coding for DAGATs, have been identified in animals (Cases et al., 1998), plants (Hobbs et al., 1999; Lardizabal et al., 2000) and in microbes (Lardizabal et al., 1999). These DAGATs belong to two unrelated protein families. 
     The first type of DAGAT that was characterized, DAGAT A, has so far been found only in animals (Cases et al., 1998) and plants (Hobbs et al. 1999). These genes show sequence similarities to genes encoding acyl-CoA : cholesterol acyltransferase (ACAT). The mouse DAGAT A has 20% amino acid sequence identity to the mouse ACAT (Cases et al., 1998). However, DAGATs A from plants and animals are more similar to each other than to ACAT. Thus, the mouse DAGAT A has 38% amino acid sequence identity to the  Arabidopsis thaliana  DAGAT A (Hobbs et al., 1999). It is also approximately 80% identical to the human ACAT like protein ARGP1, which was suggested to be involved in TAG synthesis (Oelkers et al., 1998), indicating that ARGP1 is a DAGAT A. 
     The yeast  S. cerevisiae  contain 2 genes with sequence similarity to ACAT, ARE1 and ARE2 (Yang et al., 1996). The encoded proteins have approximately 24% overall amino acid sequence identity to the mouse ACAT and 15% identity to the DAGAT A from mouse. It should be noted that they are both more similar to each other (45% amino acid sequence identity) than to either ACATs or DAGATs from higher eukaryotes. It is not possible to classify them as putative ACATs or DAGATs based on sequence similarities alone, since their evolutionary distances from both groups of higher eukaryotic enzymes are similar. However, experiments have shown that both Are1 and Are2 are ACATs, which together are responsible for all of the sterol ester synthesis that occurs in yeast (Yang et al., 1996; Yu et al., 1996). The possible involvement of Are1 and Are2 in the synthesis of TAG has also been studied (Yang et al., 1996; Yu et al., 1996). From these studies, it was concluded that Are1 and Are2 are not involved in TAG synthesis. Thus, these is no prior art to show that Are1 is a TAG synthesizing enzyme, nor can it be concluded, on the basis of homologies to ACAT like sequences already published, that Are1 is a DAGAT (Lassner and Ruezinsky, 1999). 
     The second family of DAGAT enzymes, the DAGAT B family, is unrelated to any other known proteins. These enzymes show no sequence homology to the mouse and plant ACAT like DAGAT A proteins (Lardizabal et al., 2000) or to any other known proteins. 
     DAGAT A and B are not the only enzymes that contribute to TAG biosynthesis. TAG can also be synthesized by an acyl-CoA independent reaction. Thus, the newly discovered enzyme phospholipid : diacylglycerol acyltransferase (PDAT) catalyses the formation of TAG by transferring an acryl group from the sn-2 position of a phospholipid to DAG (Dahlqvist et al., 1999; St{dot over (a)}hl, 1999). 
     SUMMARY OF INVENTION 
     This invention describes the identification of a gene encoding an enzyme that is partly responsible for TAG accumulation in yeast. 
     In a first embodiment, this invention is directed to the TAG synthesising enzyme comprising an amino acid sequence as set forth in SEQ ID NO 2 or a functional fragment, derivative, variant, ortologue or isoenzyme thereof. 
     The present invention further includes the nucleotide sequence as set forth in SEQ ID NO 1, as well as portions of the genomic sequence, the cDNA sequence, allelic variants, synthetic variants and mutants thereof. This includes sequences that codes for variants of the polypeptide set forth in the sequence listing including biologically active triacylglycerol synthesising enzymes as well as sequences that are to be used as probes, vectors for transformation or cloning intermediates. 
     Another aspect of the present invention relates to hose polypeptides, which have at least 60% identity to SEQ ID NO 2. Preferred embodiments are polynucleotides that encode polypeptides with diacrylglycerol acyltransferase activity. 
     In a different aspect, this invention relates to the use of these nucleotide sequences in recombinant DNA constructs to direct the transcription and translation of the diacylglycerol acyltransferase sequence of the present invention in a host organisms or progeny thereof, including oil seeds, yeast and other fungi, as well as other oil accumulating organisms. Cells and organisms containing the diacylglycerol acyltransferase as a result of the production of the acyltransferase encoding sequence are also included within the scope of the invention. 
     Of particular interest is the expression of the nucleotide sequences of the present invention from transcription initiation regions that are preferentially expressed in plant seed tissues. It is contemplated that the gene sequence may be synthesized, especially when there is interest to provide plant-preferred codons. 
     In a different embodiment, this invention also relates to methods of using a DNA sequence encoding a said protein of the present invention for increasing the oil-content within the cells of different organisms. 
     Further, the invention makes possible a process for the production of triacylglycerol, which comprises growing transgenic cells or organisms under conditions whereby any of the nucleotide sequences discussed above are expressed in order to produce an enzyme in these cells with the ability to transfer a fatty acid from acyl-CoA to diacylglycerol, thus forming triacylglycerol. 
     Moreover, triacylglycerols produced by the aforementioned process are included in the scope of the present invention. 
     The present invention can be essentially characterized by the following aspects: 
     1. Use of a nucleic acid sequence encoding an enzyme catalysing the transfer of a fatty acid from acyl-CoA to diacylglycerol for the production of triacylglycerol (TAG) by genetic transformation of an oil-producing organism with said sequence in order to be expressed in this organism and result in an active enzyme in order to increase the oil content of the organism. 
     The nucleic acid sequence is derived from the sequence shown in SEQ ID NO. 1, from the  Saccharomyces cerevisiae  ARE1 gene (genomic clone or cDNA), or from a nucleic acid sequence or cDNA that contain nucleotide sequences coding for a protein with an amino acid sequence that is 60% or more identical to the amino acid sequence as presented in SEQ. ID. NO. 2. 
     2. Transgenic organisms comprising, in their genome or on a plasmid, a nucleic acid sequence according to the above, transferred by recombinant DNA technology. The transgenic organisms are selected from the group consisting of fungi, plants and animals. Preferably the transgenic organisms agricultural plants and preferably said nucleotide sequence is expressed under the control of a storage organ specific promoter. Alternatively, the nucleotide, the nucleotide sequence is expressed under the control of a seed-specific promoter. 
     3. Oils from organism according to aspect 2. 
     4. A protein encoded by a DNA molecule according to SEQ ID NO. 1 or a functional (enzymatically active) fragment thereof. Alternatively, the protein produced in an organism as specified in aspect 2, which has the amino acid sequence set forth in 
     SEQ ID NO. 2 or an amino acid sequence with at least 60% homology to said amino acid sequence. Preferably the protein is isolated from  Saccharomyces cerevisiae.    
     5. Use of a protein as specified in aspect 4 in the production of triacylglycerols. 
     6. Triacylglycerols according to aspect 5. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention now having generally described will be more readily understood by reference to the following drawings and examples, which are included for the purpose of illustration only, and are not intended to limit scope of the present invention. 
     Description of the figures: 
     FIG.  1 . In vitro DAGAT activity in a yeast strain (SCY62) that overexpresses the ARE1 gene. Aliquots of microsomal membranes prepared from the control strain (lane A) or the ARE1 overexpressing strain (lane B) were assayed for DAGAT activity according to Method A described in Material and Methods. The radioactive triacylglycerol synthesised was visualised and quantified as cpm (figures in brackets) on the TLC plate by electronic autoradiography (Instant Imager, Packard, US). Abbreviations used in the figure: triacylglycerol (TAG) and unesterified fatty acids (FA). 
     FIG.  2 . In vitro DAGAT activity in a PDAT DAGAT B double mutant, a PDAT DAGAT B ARE1 triple mutant, and in the same triple mutant containing a plasmid that overexpresses the ARE1 gene. 
    
    
     The radioactive triacylglycerols (TAG) synthesized in microsomes from the double mutant, H1226 (lane A), the triple mutant, H1236 (lane B) and the same triple mutant containing a plasmid that overexpresses the ARE1 gene (lane C) were visualised in a TLC plate by electronic autoradiography (Instant Imager, Packard, US). 
     BRIEF DESCRIPTION OF THE SEQ ID: 
     SEQ ID NO. 1: Genomic DNA sequence of the  Saccharomyces cerevisiae  ARE1 gene, ORF YCR048W. 
     SEQ ID NO. 2: The amino acid sequence of the open reading frame YCR048W from  Saccharomyces cerevisiae.    
     EXAMPLES 
     Example 1 
     Triacylglycerol accumulation is reduced in yeast cells that lack the ARE1 gene. 
     Materials and Methods 
     Yeast strains. Yeast strains used in this study were congenic to the W303-1A (Thomas &amp; Rothstein, 1989) background. An are1 mutant strain, H1111, with the genotype MATα are1-Δ::IIIS3 ADE2 can 1-100 leu2-3, 112 trip1-1 ura3-1, was generated by crossing the two strains SCY60 (MATα are1-Δ::IIIS3 ade2-1 can 1-100 leu2-3, 112 trp1-1 ura3-1) and SCY61 (MATα are2-Δ::LEU2 ADE2 can 1-100 his3-11,15 trp1-1 ura3-1) (Yang et al., 1996) and dissecting tetrads. As a wild type control, we used SCY62 (MATα ADE2 can 1-100 his3-11,15 leu2-3 trp1-1 ura3-1) (Yang et al., 1996). Yeast mutant strains disrupted in YNR008w and YOR245c encoding yeast DAGAT B and PDAT, respectively, and the ARE1 gene were constructed through a series of yeast transformations using the lithium acetate method. Linear DNA fragments used for the disruption of the YOR245c and YNR008w genes were created as follows. Printers specific for YOR245c (300 bases upstream, CAGCATTGACGTAATGGGAA, and downstream, AAAGCCAAAAAGAGAAGGACA, of the gene) were constructed and the gene was synthesised using PCR from SCY62 genomic DNA. The PCR-fragment was blunt-ended and ligated into pUC119 previously cleaved with the restriction enzyme SmaI. The resulting plasmid, YOR245c-pUC119, was then digested with ClaI/StuI and dephosphorylated to prevent religation. The marker KanMX4 was obtained by digestion of the plasmid pFA6a by SmaI/SacI. The blunted KanMX4 fragment was then ligated into the YOR245c-pUC119 vector between the ClaI and SluI sites within the YOR245c open reading frame. A linear fragment containing the resulting YOR245c::KanMX4 disruption cassette was finally obtained through cleavage by BamIII/NdeI. The linear fragment used to disrupt the YNR008w gene was constructed in a similar manner as the YOR245c::KanMX4 fragment. The YNR008w gene was constructed in a similar manner as the YOR245c::KanMX4 fragment. The YNR008w gene was amplified from SCY62 genomic DNA, cloned into the pBluescript vector (Dahlqvist et al., 2000) and digested with restriction enzyme BbsI/MunI. The TRP1 marker was then ligated between the BbsI and MunI sites in the YNR008w-pBluescript plasmid, and a linear fragment containing the disruption cassette was obtained by BamHI/PstI digestion. The single PDAT mutant, H1079, with the genotype MATa pdat-Δ::TRP1 ADE2 leu2-3, 112 ura3-1 his-3-11,15 trip1-1, was generated by transforming the wild type strain SCY62 with the linear YNR008w::TRP1 fragment. The PDAT DAGAT B double mutant, H1226, with the genotype MATa pdat-Δ::TRP1 dagat B-Δ::KanMX4 ADE2 leu2-3,112 ura3-1 his3-11,15 trp1-1, was constructed in an identical manner by transforming H1079 with the linear YOR245c::KanMX4 fragment. An ARE1 PDAT double mutant, H1224, with the genotype MATα are1-Δ::TRP1 ADE2 can 1-100 leu2-3,112 ura3-1 trp1-1, was generated by transforming II1111 with the linear YNR008w::TRP1 fragment. The triple mutant strain, H1236, with the genotype MATα are1-Δ::HIS3 pdat-Δ::TRP1 dagat B-Δ::KanMX4 ADE2 leu2-3, 112 ura3-1 trp-1-1, was constructed by transforming II1224 with the linear YOR245c::KanMX4 fragment. Yeast Cultivations. Yeast cells were cultivated at 28 or 30° C. on a rotary shaker in liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose). Transformed cells were grown in synthetic medium (Sherman et al., 1986) lacking uracil and supplemented with 2% (vol/vol) glycerol and 2% (vol/vol) ethanol. 
     Lipid Analysis. The lipid content of the yeast cells was determined as described by Dahlqvist et al. (2000) and is presented as nmol of fatty acid (FA) per mg dry weight yeast. 
     Results 
     The lipid content of a mutant yeast strain (SCY60), in which the ARE1 gene was disrupted, was analyzed and compared to wild type yeast cells (SCY62) at different stages of growth. In are1 mutant cells, harvested in exponential phase after 10 hours of cultivation, the total amount of lipid, measured as nmol FA per dry weight yeast, was not significantly different from the wild type yeast (table 1), nor did the amount of fatty acids accumulated into TAG differ strongly between the wild-type and the are1 mutant. The effect of the are1 disruption on oil accumulation in stationary phase cells was analysed in an experiment were the yeast cells were pre-cultivated for 24 h in liquid YPD medium. The cells were then harvested and re-suspended in minimal medium (Meesters et al., 1996), supplemented with 16 g/l glycerol, to the original volume of the growth culture. In this glycerol supplemented minimal medium the yeast cells will enter stationary phase under conditions suitable for TAG accumulation. After further cultivation for 24 h, the cells were harvested and their lipid composition was determined. The total lipid content in the are1 mutant was 15% less than in the wild type. The TAG amount in the are1 mutant was almost 40% lower than in the wild type, whereas the polar lipid content did not differ significantly between the are1 mutant and the wild type yeast (table 1). 
     Two other genes, YNR008w and YOR254c (St{dot over (a)}hl, 1999; Dahlqvist, et al., 2000; Lardizabal et al., 2000) have recently been shown to be involved in TAG synthesis in yeast. These genes encode a PDAT and a DAGAT B protein, respectively. A yeast strain disrupted in all three genes (ARE1, YNR008w and YOR254c) and a yeast strain with disruptions in only the PDAT and DAGAT B genes were made and they are here named the tripe and double mutant, respectively. The TAG content of the double mutant was 48% of the wild type (table 2), whereas the amount of TAG accumulated in the triple mutant was only 4% of the level in the wild type yeast. By comparing the amounts of TAG accumulated in the double triple mutants it is clear that Are1 protein contributes to TAG synthesis in yeast. 
     In summary, these experiment clearly show that the product of the ARE1 gene contributes to TAG accumulation in yeast. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Lipid content in ARE1 mutant (SCY60) 
               
               
                 and wild type (SCY62) yeast cells. The 
               
               
                 lipid accumulation in yeast disrupted in 
               
               
                 the ARE1 gene (are1 mutant) was analysed 
               
               
                 at different stages of growth and compared 
               
               
                 to the control wild type yeast. The lipid 
               
               
                 composition of cells in exponential growth 
               
               
                 was analysed after 10 hours of cultivation 
               
               
                 in YPD medium at 28° C. Yeast cells in 
               
               
                 stationary phase was prepared by pre-cultivating 
               
               
                 the cells on liquid YPD medium for 24 hours at 
               
               
                 28° C., after which the cells were harvested, 
               
               
                 re-suspended in minimal medium (Meesters et al, 
               
               
                 1996) supplemented with 16 g/l glycerol, and 
               
               
                 cultivated for an additional 24 hours at 28° C. 
               
               
                 The content of sterol esters, TAG, other 
               
               
                 neutral lipids, and polar lipids was determined 
               
               
                 as nmol fatty acids (FA) per mg of dry yeast 
               
               
                 weight. 
               
             
          
           
               
                   
                 SCY62 
                   
                 SCY60 
                   
               
               
                   
                 (nmol FA/mg) 
                   
                 (nmol FA/mg) 
               
             
          
           
               
                   
                 10 h 
                 48 h 
                 10 h 
                 48 h 
               
               
                   
                   
               
             
          
           
               
                   
                 Sterol esters 
                 15 
                 24 
                 12 
                 19 
               
               
                   
                 Triacylglycerol 
                 6 
                 44 
                 8 
                 28 
               
               
                   
                 Other neutral lipids 
                 4 
                 6 
                 4 
                 5 
               
               
                   
                 Polar lipids 
                 65 
                 74 
                 63 
                 74 
               
               
                   
                 Total lipids 
                 90 
                 148 
                 87 
                 126 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Lipid content in the PDAT DAGATB double 
               
               
                 mutant strain (H1226), if the PDAT DAGATB 
               
               
                 ARE1 triple mutant strain (II1236) and in 
               
               
                 wild type yeast cells (SCY62). The different 
               
               
                 yeast stains, all of which contained the 
               
               
                 empty expression plasmid pJN92 (Ronne et al., 
               
               
                 1991), were cultivated in YNB medium to which 
               
               
                 2% (v/v) of galactose was added at an A 600   
               
               
                 of 4. The cells were harvested after an 
               
               
                 additional 22 hours growth and the content of 
               
               
                 sterol esters, TAG, other neutral lipids, and 
               
               
                 polar lipids was determined as nmol fatty 
               
               
                 acids (FA) per mg of dry yeast weight. 
               
             
          
           
               
                   
                 SCY62 
                 H1226 
                 II1236 
               
               
                   
                 (nmol FA/mg) 
                 (nmol FA/mg) 
                 (nmol FA/mg) 
               
               
                   
                   
               
             
          
           
               
                 Sterol esters 
                 13 
                 10 
                 1 
               
               
                 Triacylglycerol 
                 163 
                 78 
                 7 
               
               
                 Other neutral lipids 
                 17 
                 16 
                 41 
               
               
                 Polar lipids 
                 58 
                 66 
                 44 
               
               
                 Total lipids 
                 251 
                 170 
                 87 
               
               
                   
               
             
          
         
       
     
     Example 2 
     Triacylglycerol accumulation is increased in yeast cells that overexpress the ARE1 gene. 
     Material and Methods 
     For induced overexpression of the ARE1 gene, a 2001 bp EheI/Ecl136II fragment from the plasmid YEP 3-16 (Yang et al., 1996) was cloned into the BamHI site of the GAL1 expression vector pHN92 (Ronne et al., 1991), thus generating pUS5. The wild type yeast strain SCY62 (MATα ADE2 can 1-100 his3-11,15 leu2-3 trp1-1 ura3-1) (Yang et al., 1996), was transformed with the pUS5 and cultivated at 28° C. on a rotary shaker in synthetic medium (Sherman et al., 1986) lacking uracil and supplemented with 2% (vol/vol) glycerol and 2% (vol/vol) ethanol. The GAL1 promoter was induced after 43 h of growth by the addition of 2% (wt/vol) final concentration of galactose. Cells were harvested after an additional 24 hours of growth. Wild type (SCY62) cells transformed with the empty vector, pJN92, and cultivated under identical conditions were used as a control. The lipid content of the yeast cells was determined as described by Dahlqvist et al. (2000) and is presented as nmol of fatty acid (FA) per mg dry weight yeast. 
     Results 
     The effect of overexpression of the ARE1 gene on lipid accumulation was studied by transforming the wild-type yeast (strain SCY62) with a plasmid containing the ARE1 gene under control of the galactose-induced GAL1 promoter (Table 3). Overexpression of the ARE1 gene from this promoter had no strong effect on the growth rate as determined by optical density measurements. However, the total lipid content in yeast cells that overexpressed ARE1 was 1.4 fold higher than in the control yeast transformed with an empty expression vector (Table 3). The elevated lipid content in yeast cells overexpressing ARE1 is mostly due to a 50% increase in the TAG content, but the amount of sterol esters also increased significantly in these cells, as compared to the control. These results clearly demonstrate that the gene product of ARE1, in addition to its earlier reported involvement in the synthesis of sterol esters (Yang et al., 1996), also is involved in TAG synthesis. The elevated levels of TAG achieved in the ARE1 overexpressing cells also clearly demonstrate the potential use of the ARE1 gene in increasing the oil content in transgenic organisms. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Lipid content in yeast cells that overexpress 
               
               
                 the ARE1 gene. Yeast cells (SCY62) transformed 
               
               
                 with the ARE1 gene under the control of the GAL1 
               
               
                 promotor in the pJN92 vector were cultivated as 
               
               
                 described in the Material and Method section. 
               
               
                 Yeast cells (SCY62), transformed with an empty 
               
               
                 vector, cultivated under identical conditions 
               
               
                 were used as control. The cells were harvested 
               
               
                 and the content of sterol esters, triacylglycerols, 
               
               
                 other neutral lipids and polar lipids was 
               
               
                 determined as nmol fatty acids (FA) per mg dry yeast 
               
               
                 weight. 
               
             
          
           
               
                   
                   
                 SCY62 overexpressing 
               
               
                   
                 SCY62 
                 ARE1 
               
               
                   
                 (nmol FA/mg) 
                 (nmol FA/mg) 
               
               
                   
                   
               
             
          
           
               
                   
                 Sterol esters 
                 19 
                 27 
               
               
                   
                 Triacylglycerol 
                 160 
                 239 
               
               
                   
                 Other neutral lipids 
                 30 
                 32 
               
               
                   
                 Polar lipids 
                 48 
                 56 
               
               
                   
                 Total lipids 
                 257 
                 354 
               
               
                   
                   
               
             
          
         
       
     
     Example 3 
     The ARE1 gene product has diacylglycerol acyltransferase activity. 
     Materials and Methods 
     In vitro diacylglycerol acyltransferase (DAGAT) activity was analyzed, in microsomal fractions prepared from yeast cells, by using one of the following methods. 
     Method A: A wild type yeast (strain SCY62) was transformed with a plasmid (pUS5) containing the ARE1 gene under the control of a GAL1 promoter (described in Material and Methods in Example 2). The transformed yeast was cultivated at 28° C. in defined YNB medium lacking uracil. The expression of the ARE1 gene was inducted by the addition of 2% (v/v) galactose after 8 hours growth and the cells were harvested after an additional 17 hours. Microsomal membranes were prepared from the transformed yeast by resuspending 1 g of yeast (fresh weight) in 8 ml of ice-cold buffer (20 mM Tri-Cl, pH 7.9, 10 mM MgCl 2 , 1 mM EDTA, 5% (v/v) glycerol, 1 mM DTT, 0.3 M ammonium sulphate) in a 12 ml glass tube to which 4 ml of glass bends (diameter 0.45-0.5 mm) were added. The glass tube was heavily shaken (3×60 s) with a MSK cell homogenizer (B. Braun Melsungen AG, Germany). The suspension was centrifuged at 20 000 g for 15 min at 6° C. and the resulting supernatant was centrifuged at 100 000 g for 2 h at 6° C. The resulting pellet, containing microsomal membranes, was resuspended in 0.1 M K-phosphate (pH 7.2) buffer and stored at −80° C. DAGAT activity was analyzed in aliquots of microsomal membranes (50 μl), corresponding to 10 nmol phosphatidylcholine, to which 1 μmol of dioleoyl-PG and 0.25 μmol of dioleoyl-DAG emulsified in 50 μl of buffer containing 190 mM HEPES-NaOH, pH 7.5, 125 mM MgCl 2 , 30 mM CHAPS, 2.5 mg/ml BSA and 2 nmol [ 14 C]-palmitoyl-CoA (2775 dpm/nmol), were added. The reaction mixture was incubated at 30° C. for 30 min. The lipids were then extracted in chloroform and separated using thin layer chromatography on silica gel 60 plates in hexane/diethyl ether/acetic acid (80:20:1). The radioactive lipids were visualized and quantified on the plates by electronic autoradiography (Instant Imager, Packard, US). 
     Method B: The PDAT DAGAT B double mutant (H1226) and the PDAT DAGAT B ARE1 triple mutant (II1236), described in Material and Methods in Example 1, were transformed with the empty expression plasmid (pJN92). A transformant expressing the ARE1 gene under the control of the GAL1 promoter was generated by transforming the triple mutant H1236 with the plasmid pUS5 (described in Material and Methods in Example 2). All yeast transformants were cultivated in YNB medium to which 2% (v/v) of galactose was added at an A 600  of 4. The cells were harvested after an additional 6 hours growth and microsomes were prepared using a modification of the procedure of Dahlqvist et al. (2000). Yeast cells (0.2 g) were resuspended in 1.5 ml of ice-cold buffer (20 mM Tris-Cl pH 7.9, 10 mM MgCl 2 , 1 mM EDTA, 5% (vol/vol) glycerol, 1 mM DTT, 0.3 M ammonium sulfate) in a 2 ml Eppendorf tube containing 0.2 ml glass beads (0.45-0.5 mm in diameter). The tube was heavily shaken (3×60 s) in a cell homogenizer (Mini Bead Beater). The homogenized yeast was centrifuged at 1350×g for 20 min at 4° C., and the resulting supernatant was subsequently centrifuged at 150 000×g for 1 h at 4° C. The pellet was re-suspended in 0.1 M potassium phosphate (pH 7.2), and stored at −80° C. Dihexanoyl-DAG (5 nmol) dissolved in chloroform was added to micro tubes and the chloroform was evaporated under a stream of N 2 . Aliquots (90 μl) of microsomal fractions corresponding to 150 μg protein, in a buffer consisting of 50 mM HEPES (pH 7.2), 5 mM MgCl 2 , and 1 mg/ml BSA were added to the tubes and the suspension was thoroughly mixed. Finally, 10 μl of [ 14 C]-palmitoyl-CoA (20 nmol, 5000 dpm/nmol) was added, and the mixtures were incubated at 30° C. for 15 min. Lipids were extracted from the reaction mixture into chloroform (Bligh &amp; Dyer, 1959) and separated by TLC on silica gel 60 plates (Merck). The TLC plate was first developed in chloroform/methanol/acetic acid/water (85:15:10:3.5) for 80 mm. The dried plate was then developed in hexane/diethyl ether/acetic acid (80:20:1.5) for 180 mm. The radioactive lipids were visualized and quantified on the plates by electronic autoradiography (Instant Imager, Packard). 
     Results 
     Microsomal membranes prepared from the transformed yeast overexpressing the ARE1 gene and from control yeast transformed with an empty plasmid (pJN92) were assayed for DAGAT activity according to Method A in Materials and Methods. The amount of radiolabelled TAG synthesized from [ 14 C]palmitoyl-CoA in microsomal membranes prepared from the ARE1 overexpressor was increased with 66% as compared to the control yeast (FIG.  1 ). DAGAT activity was also assayed in microsomal membranes prepared from the PDAT DAGAT B double mutant strain (H1226) and the PDAT DAGAT B ARE1 triple mutant strain (H1236) cells (method B). In the double mutant, with a functional ARE1 gene, TAG with two hexanoyl and one [ 14 C]palmitoyl chain, was synthesized from added dihexanoyl-DAG and [ 14 C]palmitoyl-CoA. This synthesis was barely detectable in the triple mutant (FIG. 2) where the ARE1 gene was disrupted. However, the in vitro synthesis of TAG was restored in triple mutant cells transformed with a plasmid expressing the ARE1 gene. This clearly shows that the in vitro synthesis of TAG in these yeast mutants correlates with the presence of a functional ARE1 gene and that the protein encoded by the ARE1 gene possesses DAGAT activity. 
     Example 4 
     Triacylglycerol accumulation is increased in the seeds of  Arabidopsis thaliana  that express the ARE1 gene. 
     Material and Methods 
     The ARE1 gene was amplified from the yeast genome using the proof reading enzyme polymerase pfu (Promega). An EcoR1 and Xba1 restriction enzyme site was introduced respectively into the 5′ and 3′ ends of this fragment to allow directional cloning of the fragment. The PCR fragment was cloned into the vector pBluescript (Stratagene). The insert derived from this plasmid was then cloned downstream of a napin promoter fragment (St{dot over (a)}lberg et al., 1993) in the vector pPGTV-KAN (Becker et al., 1993). This plasmid was transformed into Agrobacterium strain GV3301. Transformed Agrobacterium cells were then used to transform root explants from  Arabidopsis thaliana  (Valvekens et al., 1992). The lipid content in Arabidopsis seeds was determined by methylation of fatty acids. Fatty acids in the oil of proximately 2-3 mg of seeds were methylated in 2 ml 2% (vol/vol) H 2 SO 4  in dry methanol for 90 min at 90° C. The fatty acid method esters were extracted with hexane and analyzed by GLC through a 50 m×0.32 mm CP-Wax58-CB fused-silica column (chrompack), methylheptadecanoic acid was used as internal standard. 
     Results 
       A. thaliana  was transformed with the ARE1 gene under the control of a napin promoter, which is seed specific and active during the major phase of oil accumulation. The oil content was analyzed in seeds from single T2 plants derived from four independent transformation events (Table 4). The results showed that in three lines between 50% and 100% of the T2 plants generated seeds with statistically significant elevated oil content as compared to the oil content in the seeds from the control plants. The oil content was elevated with up to 18% in the seeds expressing ARE1. One line (28-1) had the same oil content as the seeds from the control plants. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Accumulation of oil in seeds from  Arabidopsis thaliana   
               
               
                 transformed with the ARE1 gene. 
               
               
                 T2 plants transformed with the ARE1 gene under 
               
               
                 the control of the napin promotor and control 
               
               
                 plants transformed with an empty vector were 
               
               
                 cultivated in a growth chamber under controlled 
               
               
                 conditions. The oil content in mature seeds of 
               
               
                 these plants was determined by GLC analyses 
               
               
                 and is presented as nmol fatty acids (FA) 
               
               
                 per mg seed. 
               
             
          
           
               
                   
                 Transformants 
               
             
          
           
               
                   
                 control 
                 28-1 
                 28-2 
                 28-3 
                 28-4 
               
               
                   
                   
               
             
          
           
               
                 Number of T2 
                 4 
                 6 
                 2 
                 6 
                 11 
               
               
                 plants analyzed 
               
               
                 Number of T2 
                 — 
                 0 
                 2 
                 3 
                  9 
               
               
                 plants with 
               
               
                 significant 
               
               
                 increased seed 
               
               
                 oil content* 
               
               
                 nmol FA per mg 
                 1535 ± 
                 1562 ± 
                 1753 ± 
                 1641 ± 
                 1818 ± 
               
               
                 seed in T2 plant 
                 114 
                 28 
                 53 
                 82 
                 18 
               
               
                 with highest oil 
               
               
                 content 
               
               
                   
               
               
                 *Calculated with the mean difference two-sided test at α = 5 and based on the average oil content of 4 control plants.  
               
             
          
         
       
     
     REFERENCES 
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     Bell, R. M., and Coleman, R. A. (1983) in  The Enzymes  (Boyer, P. D., ed.) Vol. 16, pp. 87-111, Academic Press, Orlando 
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     Dahlqvist, A., St{dot over (a)}hl, U., Lenman, M., Banas, A., Ronne, H., and Stymne, S. (1999)  Enzymes catalysing a transacylation reaction involved in triacylglycerol synthesis.  Presented at Biochem, Mol. Biol. Plant Fatty Acids Glycerolipids Symp., South Lake Tahoe, Calif., USA 
     Dahlqvist, A., St{dot over (a)}hl, U., Lenman, M., Banas, A., Lee, M., Sandager, L., Ronne, H., and Stymne, S. (2000)  Proc. Natl. Acad. Sci. USA.  97, 6487-6492 
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     Yu, C., Kennedy, N. J., Chang, C. Y. G., and Rothblatt, J. A. (1996)  J. Biol. Chem.  271, 24157-24163 
     
       
         
           
             2 
           
           
             1 
             1833 
             DNA 
             Saccharomyces cerevisiae 
           
            1
atgacggaga ctaaggattt gttgcaagac gaagagtttc ttaagatccg cagactcaat     60
tccgcagaag ccaacaaacg gcattcggtc acgtacgata acgtgatcct gccacaggag    120
tccatggagg tttcgccacg gtcgtctacc acgtcgctgg tggagccagt ggagtcgact    180
gaaggagtgg agtcgactga ggcggaacgt gtggcaggga agcaggagca ggaggaggag    240
taccctgtgg acgcccacat gcaaaagtac ctttcacacc tgaagagcaa gtctcggtcg    300
aggttccacc gaaaggatgc tagcaagtat gtgtcgtttt ttggggacgt gagttttgat    360
cctcgcccca cgctcctgga cagcgccatc aacgtgccct tccagacgac tttcaaaggt    420
ccggtgctgg agaaacagct caaaaattta cagttgacaa agaccaagac caaggccacg    480
gtgaagacta cggtgaagac tacggagaaa acggacaagg cagatgcccc cccaggagaa    540
aaactggagt cgaacttttc agggatctac gtgttcgcat ggatgttctt gggctggata    600
gccatcaggt gctgcacaga ttactatgcg tcgtacggca gtgcatggaa taagctggaa    660
atcgtgcagt acatgacaac ggacttgttc acgatcgcaa tgttggactt ggcaatgttc    720
ctgtgcactt tcttcgtggt tttcgtgcac tggctggtga aaaagcggat catcaactgg    780
aagtggactg ggttcgttgc agtgagcatc ttcgagttgg ctttcatccc cgtgacgttc    840
cccatttacg tctactactt tgatttcaac tgggtcacga gaatcttcct gttcctgcac    900
tccgtggtgt ttgttatgaa gagccactcg tttgcctttt acaacgggta tctttgggac    960
ataaagcagg aactcgagta ctcttccaaa cagttgcaaa aatacaagga atctttgtcc   1020
ccagagaccc gcgagattct gcaaaaaagt tgcgactttt gccttttcga attgaactac   1080
cagaccaagg ataacgactt ccccaacaac atcagttgca gcaatttctt catgttctgt   1140
ttgttccccg tcctcgtgta ccagatcaac tacccaagaa cgtcgcgcat cagatggagg   1200
tatgtgttgg agaaggtgtg cgccatcatt ggcaccatct tcctcatgat ggtcacggca   1260
cagttcttca tgcacccggt ggccatgcgc tgtatccagt tccacaacac gcccaccttc   1320
ggcggctgga tccccgccac gcaagagtgg ttccacctgc tcttcgacat gattccgggc   1380
ttcactgttc tgtacatgct cacgttttac atgatatggg acgctttatt gaattgcgtg   1440
gcggagttga ccaggtttgc ggacagatat ttctacggcg actggtggaa ttgcgtttcg   1500
tttgaagagt ttagcagaat ctggaacgtc cccgttcaca aatttttact aagacacgtg   1560
taccacagct ccatgggcgc attgcatttg agcaagagcc aagctacatt atttactttt   1620
ttcttgagtg ccgtgttcca cgaaatggcc atgttcgcca ttttcagaag ggttagagga   1680
tatctgttca tgttccaact gtcgcagttt gtgtggactg ctttgagcaa caccaagttt   1740
ctacgggcaa gaccgcagtt gtccaacgtt gtcttttcgt ttggtgtctg ttcagggccc   1800
agtatcatta tgacgttgta cctgacctta tga                                1833
 
           
             2 
             610 
             PRT 
             Saccharomyces cerevisiae 
           
            2
Met Thr Glu Thr Lys Asp Leu Leu Gln Asp Glu Glu Phe Leu Lys Ile
  1               5                  10                  15
Arg Arg Leu Asn Ser Ala Glu Ala Asn Lys Arg His Ser Val Thr Tyr
             20                  25                  30
Asp Asn Val Ile Leu Pro Gln Glu Ser Met Glu Val Ser Pro Arg Ser
         35                  40                  45
Ser Thr Thr Ser Leu Val Glu Pro Val Glu Ser Thr Glu Gly Val Glu
     50                  55                  60
Ser Thr Glu Ala Glu Arg Val Ala Gly Lys Gln Glu Gln Glu Glu Glu
 65                  70                  75                  80
Tyr Pro Val Asp Ala His Met Gln Lys Tyr Leu Ser His Leu Lys Ser
                 85                  90                  95
Lys Ser Arg Ser Arg Phe His Arg Lys Asp Ala Ser Lys Tyr Val Ser
            100                 105                 110
Phe Phe Gly Asp Val Ser Phe Asp Pro Arg Pro Thr Leu Leu Asp Ser
        115                 120                 125
Ala Ile Asn Val Pro Phe Gln Thr Thr Phe Lys Gly Pro Val Leu Glu
    130                 135                 140
Lys Gln Leu Lys Asn Leu Gln Leu Thr Lys Thr Lys Thr Lys Ala Thr
145                 150                 155                 160
Val Lys Thr Thr Val Lys Thr Thr Glu Lys Thr Asp Lys Ala Asp Ala
                165                 170                 175
Pro Pro Gly Glu Lys Leu Glu Ser Asn Phe Ser Gly Ile Tyr Val Phe
            180                 185                 190
Ala Trp Met Phe Leu Gly Trp Ile Ala Ile Arg Cys Cys Thr Asp Tyr
        195                 200                 205
Tyr Ala Ser Tyr Gly Ser Ala Trp Asn Lys Leu Glu Ile Val Gln Tyr
    210                 215                 220
Met Thr Thr Asp Leu Phe Thr Ile Ala Met Leu Asp Leu Ala Met Phe
225                 230                 235                 240
Leu Cys Thr Phe Phe Val Val Phe Val His Trp Leu Val Lys Lys Arg
                245                 250                 255
Ile Ile Asn Trp Lys Trp Thr Gly Phe Val Ala Val Ser Ile Phe Glu
            260                 265                 270
Leu Ala Phe Ile Pro Val Thr Phe Pro Ile Tyr Val Tyr Tyr Phe Asp
        275                 280                 285
Phe Asn Trp Val Thr Arg Ile Phe Leu Phe Leu His Ser Val Val Phe
    290                 295                 300
Val Met Lys Ser His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Asp
305                 310                 315                 320
Ile Lys Gln Glu Leu Glu Tyr Ser Ser Lys Gln Leu Gln Lys Tyr Lys
                325                 330                 335
Glu Ser Leu Ser Pro Glu Thr Arg Glu Ile Leu Gln Lys Ser Cys Asp
            340                 345                 350
Phe Cys Leu Phe Glu Leu Asn Tyr Gln Thr Lys Asp Asn Asp Phe Pro
        355                 360                 365
Asn Asn Ile Ser Cys Ser Asn Phe Phe Met Phe Cys Leu Phe Pro Val
    370                 375                 380
Leu Val Tyr Gln Ile Asn Tyr Pro Arg Thr Ser Arg Ile Arg Trp Arg
385                 390                 395                 400
Tyr Val Leu Glu Lys Val Cys Ala Ile Ile Gly Thr Ile Phe Leu Met
                405                 410                 415
Met Val Thr Ala Gln Phe Phe Met His Pro Val Ala Met Arg Cys Ile
            420                 425                 430
Gln Phe His Asn Thr Pro Thr Phe Gly Gly Trp Ile Pro Ala Thr Gln
        435                 440                 445
Glu Trp Phe His Leu Leu Phe Asp Met Ile Pro Gly Phe Thr Val Leu
    450                 455                 460
Tyr Met Leu Thr Phe Tyr Met Ile Trp Asp Ala Leu Leu Asn Cys Val
465                 470                 475                 480
Ala Glu Leu Thr Arg Phe Ala Asp Arg Tyr Phe Tyr Gly Asp Trp Trp
                485                 490                 495
Asn Cys Val Ser Phe Glu Glu Phe Ser Arg Ile Trp Asn Val Pro Val
            500                 505                 510
His Lys Phe Leu Leu Arg His Val Tyr His Ser Ser Met Gly Ala Leu
        515                 520                 525
His Leu Ser Lys Ser Gln Ala Thr Leu Phe Thr Phe Phe Leu Ser Ala
    530                 535                 540
Val Phe His Glu Met Ala Met Phe Ala Ile Phe Arg Arg Val Arg Gly
545                 550                 555                 560
Tyr Leu Phe Met Phe Gln Leu Ser Gln Phe Val Trp Thr Ala Leu Ser
                565                 570                 575
Asn Thr Lys Phe Leu Arg Ala Arg Pro Gln Leu Ser Asn Val Val Phe
            580                 585                 590
Ser Phe Gly Val Cys Ser Gly Pro Ser Ile Ile Met Thr Leu Tyr Leu
        595                 600                 605
Thr Leu
    610