Patent Application: US-53771000-A

Abstract:
the present invention relates to the production of triacylglycerol in a transformed organism or host cell , by means of a nucleotide sequence from s . cerevisiae , whereby said enzyme catalyzes the transfer of fatty acids from phospholipids to diacylglycerol in any acyl - coa - independent reaction . the transformation of the organism or host cell results in increased oil content .

Description:
yeast strains and plasmids . the wild type yeast strains used were either fy1679 ( matα his3 - δ200 leu2 - δ1 trp1 - δ6 ura3 - 52 ) or w303 - 1a ( mata ade2 - 1 can1 - 100 his3 - 11 , 15 leu2 - 3 , 112 trp1 - 1 ura3 - 1 ) ( 7 ). the ynr008w :: kanmx2 disruption strain fvkt004 - 04c ( al ), which is congenic to fy1679 , was obtained from the euroscarf collection ( 8 ). a 2751 bp fragment containing the ynr008w gene with 583 bp of 5 ′ and 183 bp of 3 ′ flanking dna was amplified from w303 - 1a genomic dna using taq polymerase with 5 ′- tctccatcttctgcaaaacct - 3 ′ and 5 ′- cctgtcaaaaaccttctcctc - 3 ′ as primers . the resulting pcr product was purified by agarose gel electrophoresis and cloned into the ecorv site of pbluescript ( pbluescript - pdat ). for complementation experiments , the cloned fragment was released from pbluescript by hindiii - saci digestion and then cloned between the hindiii and saci sites of pfl39 ( 9 ), thus generating pus1 . for overexpression of the pdat gene , a 2202 bp ecori fragment from the pbluescript plasmid which contains only 24 bp of 5 ′ flanking dna was cloned into the bamhi site of the gal1 - tpk2 expression vector pjn92 ( 12 ), thus generating pus4 . microsomal preparations . microsomes from developing seeds of sunflower ( helianthus annuus ), ricinus communis and crepis palaestina were prepared using the procedure of stobart and stymne ( 11 ). to obtain yeast microsomes , 1 g of yeast cells ( fresh weight ) was re - suspended in 8 ml of ice - cold buffer ( 20 mm tris - cl , ph 7 . 9 , 10 mm mgcl 2 , 1 mm edta , 5 % ( v / v ) glycerol , 1 mm dtt , 0 . 3 m ammonium sulfate ) in a 12 ml glass tube . to this tube , 4 ml of glass beads ( diameter 0 . 45 - 0 . 5 mm ) were added , and the tube was then heavily shaken ( 3 × 60 s ) in an msk cell homogenizer ( b . braun melsungen ag , germany ). the homogenized suspension was centrifuged at 20 , 000 × g for 15 min at 6 ° c . and the resulting supernatant was again centrifuged at 100 , 000 × g for 2 h at 6 ° c . the 100 , 000 × g pellet was resuspended in 0 . 1 m potassium phosphate ( ph 7 . 2 ), and stored at − 80 ° c . it is subsequently referred to as the crude yeast microsomal fraction . lipid substrates . radio - labeled ricinoleic ( 12 - hydroxy - octadecenoic ) and vernolic ( 12 , 13 - epoxy - octadecenoic ) acids were synthesized enzymatically from [ 1 - 14 c ] oleic acid and [ 1 - 14 c ] linoleic acid , respectively , by incubation with microsomal preparations from seeds of ricinus communis and crepis palaestina , respectively ( 12 ). the synthesis of phosphatidylcholines ( pc ) or phosphatidylethanolamines ( pe ) with 14 c - labeled acyl groups in the sn - 2 position was performed using either enzymatic ( 13 ), or synthetic ( 14 ) acylation of [ 14 c ] oleic , [ 14 c ] ricinoleic , or [ 14 c ] vernolic acid . dioleoyl - pc that was labeled in the sn - 1 position was synthesized from sn - 1 -[ 14 c ] oleoyl - lyso - pc and unlabeled oleic acid as described in ( 14 ). sn - 1 - oleoyl - sn - 2 -[ 14 c ] ricinoleoyl - dag was synthesized from pc by the action of phospholipase c type xi from b . cereus ( sigma chemical co .) as described in ( 15 ). monovernoloyl - and divernoleoyl - dag were synthesized from tag extracted from seeds of euphorbia lagascae , using the tag - lipase ( rizhopus arrhizus , sigma chemical co .) as previously described ( 16 ). monoricinoleoyl - tag was synthesized according to the same method using tag extracted from castor bean . lipid analysis . total lipid composition of yeast were determined from cells harvested from a 40 ml liquid culture , broken in a glass - bead shaker and extracted into chloroform as described by bligh and dyer ( 17 ), and then separated by thin layer chromatography in hexane / diethylether / acetic acid ( 80 : 20 : 1 ) using pre - coated silica gel 60 plates ( merck ). the lipid areas were located by brief exposure to i 2 vapors and identified by means of appropriate standards . polar lipids , sterol - esters and triacylglycerols , as well as the remaining minor lipid classes , referred to as other lipids , were excised from the plates . fatty acid methylesters were prepared by heating the dry excised material at 85 ° c . for 60 min in 2 % ( v / v ) sulfuric acid in dry methanol . the methyl esters were extracted with hexane and analyzed by glc through a 50 m × 0 . 32 mm cp - wax58 - cb fused - silica column ( chrompack ), with methylheptadecanoic acid as an internal standard . the fatty acid content of each fraction was quantified and used to calculate the relative amount of each lipid class . in order to determine the total lipid content , 3 ml aliquots from yeast cultures were harvested by centrifugation and the resulting pellets were washed with distilled water and lyophilized . the weight of the dried cells was determined and the fatty acid content was quantified by glc - analyses after conversion to methylesters as described above . the lipid content was then calculated as nmol fatty acid ( fa ) per mg dry weight yeast . enzyme assays . aliquots of crude microsomal fractions ( corresponding to 10 nmol of microsomal pc ) from developing plant seeds or yeast cells were lyophilized over night . 14 c - labeled substrate lipids dissolved in benzene were then added to the dried microsomes . the benzene was evaporated under a stream of n 2 , leaving the lipids in direct contact with the membranes , and 0 . 1 ml of 50 mm potassium phosphate ( ph 7 . 2 ) was added . the suspension was thoroughly mixed and incubated at 30 ° c . for the time period indicated , up to 90 min . lipids were extracted from the reaction mixture using chloroform and separated by thin layer chromatography in hexane / diethylether / acetic acid ( 35 : 70 : 1 . 5 ) using silica gel 60 plates ( merck ). the radioactive lipids were visualized and quantified on the plates by electronic autoradiography ( instant imager , packard , us ). yeast cultivation . yeast cells were grown at 28 ° c . on a rotatory shaker in liquid ypd medium ( 1 % yeast extract , 2 % peptone , 2 % glucose ), synthetic medium ( 18 ) containing 2 % ( v / v ) glycerol and 2 % ( v / v ) ethanol , or minimal medium ( 19 ) containing 16 g / l of glycerol . the instant invention is further characterized by the following examples which are not limiting : acyl - coa - independent synthesis of tag by oil seed microsomes . a large number of unusual fatty acids can be found in oil seeds ( 20 ). many of these fatty acids , such as ricinoleic ( 21 ) and vernolic acids ( 22 ), are synthesized using phosphatidylcholin ( pc ) with oleoyl or linoleoyl groups esterified to the sn - 2 position , respectively , as the immediate precursor . however , even though pc can be a substrate for unusual fatty acid synthesis and is the major membrane lipids in seeds , unusually fatty acids are rarely found in the membranes . instead , they are mainly incorporated into the tag . a mechanism for efficient and selective transfer of these unusual acyl groups from pc into tag must therefore exist in oil seeds that accumulate such unusual fatty acids . this transfer reaction was biochemically characterized in seeds from castor bean ( ricinus communis ) and crepis palaestina , plants which accumulate high levels of ricinoleic and vernolic acid , respectively , and sunflower ( helianthus annuus ), a plant which has only common fatty acids in its seed oil . crude microsomal fractions from developing seeds were incubated with pc having 14 c - labeled oleoyl , ricinoleoyl or vernoloyl groups at the sn - 2 position . after the incubation , lipids were extracted and analyzed by thin layer chromatography . we found that the amount of radioactivity that was incorporated into the neutral lipid fraction increased linearly over a period of 4 hours ( data not shown ). the distribution of [ 14 c ] acyl groups within the neutral lipid fraction was analyzed after 80 min ( fig1 ). interestingly the amount and distribution of radioactivity between different neutral lipids were strongly dependent both on the plant species and on the type of [ 14 c ] acyl chain . thus , sunflower microsomes incorporated most of the label into dag , regardless of the type of [ 14 c ] acyl group . in contrast , r . communis microsomes preferentially incorporated [ 14 c ] ricinoleoyl and [ 14 c ] vernoloyl groups into tag , while [ 14 c ] oleyl groups mostly were found in dag . c . palaestina microsomes , finally , incorporated only [ 14 c ] vernolyol groups into tag , with [ 14 c ] ricinoleyl groups being found mostly as free fatty acids , and [ 14 c ] oleyl groups in dag . this shows that the high in vivo levels of ricinoleic acid and vernolic acid in the tag pool of r . communis and c . palaestina , respectively , can be explained by an efficient and selective transfer of the corresponding acyl groups from pc to tag in these organisms . the in - vitro synthesis of triacylglycerols in microsomal preparations of developing castor bean is summarized in table 1 . pdat : a novel enzyme that catalyzes acyl - coa - independent synthesis of tag . it was investigated if dag could serve both as an acyl donor as well as an acyl acceptor in the reactions catalyzed by the oil seed microsomes . therefore , unlabeled divernoloyl - dag was incubated with either sn - 1 - oleoyl - sn - 2 -[ 14 c ] ricinoleoyl - dag or sn - 1 - oleoyl - sn - 2 -[ 14 c ] ricinoleoyl - pc in the presence of r . communis microsomes . the synthesis of tag molecules containing both [ 14 c ] ricinoleoyl and vernoloyl groups was 5 fold higher when [ 14 c ] ricinoleoyl - pc served as acyl donor as compared to [ 14 c ] ricinoleoyl - dag ( fig1 b ). these data strongly suggests that pc is the immediate acyl donor and dag the acyl acceptor in the acyl - coa - independent formation of tag by oil seed microsomes . therefore , this reaction is catalyzed by a new enzyme which we called phospholipid : diacylglycerol acyltransferase ( pdat ). pdat activity in yeast microsomes . wild type yeast cells were cultivated under conditions where tag synthesis is induced . microsomal membranes were prepared from these cells and incubated with sn - 2 -[ 14 c ]- ricinoleoyl - pc and dag and the 14 c - labeled products formed were analyzed . the pc - derived [ 14 c ] ricinoleoyl groups within the neutral lipid fraction mainly were found in free fatty acids or tag , and also that the amount of tag synthesized was dependent on the amount of dag that was added to the reaction ( fig2 ). the in vitro synthesis of tag containing both ricinoleoyl and vernoloyl groups , a tag species not present in vivo , from exogenous added sn - 2 -[ 14 c ] ricinoleoyl - pc and unlabeled vernoloyl - dag ( fig2 , lane 3 ) clearly demonstrates the existence of an acyl - coa - independent synthesis of tag involving pc and dag as substrates in yeast microsomal membranes . consequently , tag synthesis in yeast can be catalyzed by an enzyme similar to the pdat found in plants . the pdat encoding gene in yeast . a gene in the yeast genome ( ynr008w ) is known , but nothing is known about the function of ynr008w , except that the gene is not essential for growth under normal circumstances . microsomal membranes were prepared from the yeast strain fvkt004 - 04c ( al ) ( 8 ) in which the gene with unknown function had been disrupted . pdat activity in the microsomes were assayed using pc with radiolabelled fatty acids at the sn - 2 position . the activity was found to be completely absent in the disruption strain ( fig2 lane 4 ). significantly , the activity could be partially restored by the presence of ynr008w on the single copy plasmid pus1 ( fig2 lane 5 ). moreover , acyl groups of phosphatidylethanolamine ( pe ) were efficiently incorporated into tag by microsomes from the wild type strain whereas no incorporation occurred from this substrate in the mutant strain . this shows that ynr008w encodes a yeast pdat which catalyzes the transfer of an acyl group from the sn - 2 position of phospholipids to dag , thus forming tag . it should be noted that no cholesterol esters were formed from radioactive pc even in incubations with added ergosterols , nor were the amount of radioactive free fatty acids formed from pc affected by disruption of the ynr008w gene . this demonstrates that yeast pdat do not have cholesterol ester synthesising or phospholipase activities . increased tag content in yeast cells that overexpress pdat . the effect of overexpressing the pdat - encoding gene was studied by transforming a wild type yeast strain with the pus4 plasmid in which the gene is expressed from the galactose - induced gal1 : tpk2 promoter . cells containing the empty expression vector were used as a control . the cells were grown in synthetic glycerol - ethanol medium , and expression of the gene was induced after either 2 hours ( early log phase ) or 25 hours ( stationary phase ) by the addition of galactose . the cells were then incubated for another 21 hours , after which they were harvested and assays were performed . we found that overexpression of pdat had no significant effect on the growth rate as determined by the optical density . however , the total lipid content , measured as total μmol fatty acids per mg yeast dry weight , was 47 % ( log phase ) or 29 % ( stationary phase ) higher in the pdat overexpressing strain than in the control . furthermore , the polar lipid and sterolester content was unaffected by overexpression of pdat . instead , the elevated lipid content in these cells is entirely due to an increased tag content ( fig3 a , b ). thus , the amount of tag was increased by 2 - fold in pdat overexpressing early log phase cells and by 40 % in stationary phase cells . it is interesting to note that a significant increase in the tag content was achieved by overexpressing pdat even under conditions ( i . e . in stationary phase ) where dagat is induced and thus contributes significantly to tag synthesis . in vitro pdat activity assayed in microsomes from the pdat overexpressing strain was 7 - fold higher than in the control strain , a finding which is consistent with the increased levels of tag that we observed in vivo ( fig3 c ). these results clearly demonstrate the potential use of the pdat gene in increasing the oil content in transgenic organisms . substrate specificity of yeast pdat . the substrate specificity of yeast pdat was analyzed using microsomes prepared from the pdat overexpressing strain ( see fig4 ). the rate of tag synthesis , under conditions given in fig4 with di - oleoyl - pc as the acyl - donor , was 0 . 15 nmol per min and mg protein . with both oleoyl groups of pc labeled it was possible , under the given assay conditions , to detect the transfer of 11 pmol / min of [ 14 c ] oleoyl chain into tag and the formation of 15 pmol / min of lyso - pc . in microsomes from the pdat - deficient strain , no tag at all and only trace amounts of lyso - pc was detected , strongly suggesting that yeast pdat catalyses the formation of equilmolar amounts of tag and lyso - pc when supplied with pc and dag as substrates . the fact that somewhat more lyso - pc than tag is formed can be explained by the presence of a phospholipase in yeast microsomes , which produces lyso - pc and unesterified fatty acids from pc ( data not shown ). the specificity of yeast pdat for different acyl group positions was investigated by incubating the microsomes with di - oleoyl - pc carrying a [ 14 c ] acyl group either at the sn - 1 position ( fig4 a bar 2 ) or the sn - 2 position ( fig4 a bar 3 ). we found that the major 14 c - labeled product formed in the former case was lyso - pc , and in the latter case tag . we conclude that yeast pdat has a specificity for the transfer of acyl groups from the sn - 2 position of the phospholipid to dag , thus forming sn - 1 - lyso - pc and tag . under the given assay conditions , trace amounts of 14 c - labelled dag is formed from the sn - 1 labeled pc by the reversible action of a cdp - choline : choline phosphotransferase ( data not shown ). this labeled dag can then be further converted into tag by the pdat activity . it is therefore not possible to distinguish whether the minor amounts of labeled tag that is formed in the presence of di - oleoyl - pc carrying a [ 14 c ] acyl group in the sn - 1 position , is synthesized directly from the sn - 1 - labeled pc by a pdat that also can act on the sn - 1 position , or if it is first converted to sn - 1 - labeled dag and then acylated by a pdat with strict selectivity for the transfer of acyl groups at the sn - 2 position of pc . taken together , this shows that the pdat encoded by ynr008w catalyses an acyl transfer from the sn - 2 position of pc to dag , thus causing the formation of tag and lyso - pc . the substrate specificity of yeast pdat was further analyzed with respect to the headgroup of the acyl donor , the acyl group transferred and the acyl chains of the acceptor dag molecule . the two major membrane lipids of s . cerevisiae are pc and pe , and as shown in fig4 b ( bars 1 and 2 ), dioleoyl - pe is nearly 4 - fold more efficient than dioleoyl - pc as acyl donor in the pdat - catalyzed reaction . moreover , the rate of acyl transfer is strongly dependent on the type of acyl group that is transferred . thus , a ricinoleoyl group at the sn - 2 position of pc is 2 . 5 times more efficiently transferred into tag than an oleoyl group in the same position ( fig4 b bars 1 and 3 ). in contrast , yeast pdat has no preference for the transfer of vernoloyl groups over oleoyl groups ( fig4 b bars 1 and 4 ). the acyl chain of the acceptor dag molecule also affects the efficiency of the reaction . thus , dag with a ricinoleoyl or a vernoloyl group is a more efficient acyl acceptor than dioleoyl - dag ( fig4 b bars 1 , 5 and 6 ). taken together , these results clearly show that the efficiency of the pdat - catalyzed acyl transfer is strongly dependent on the properties of the substrate lipids . pdat genes . nucleotide and amino acid sequences of several pdat genes are given as seq id no . 1 through 15 . further provisional and / or partial sequences are given as seq id no . 16 through 19 , respectively . one of the arabidopsis genomic sequences ( seq id no . 4 ) identified an arabidopsis est cdna clone ; t04806 . this cdna clone was fully characterized and the nucleotide sequence is given as seq id no . 5 . based on the sequence homology of the t04806 cdna and the arabidopsis thaliana genomic dna sequence ( seq id no . 4 ) it is apparent that an additional a is present at position 417 in the cdna clone ( data not shown ). excluding this nucleotide would give the amino acid represented by the nucleotide sequence depicted in seq id no . 12 . increased tag content in seeds of arabidopsis thaliana that express the yeast pdat . for the expression of the yeast pdat gene in arabidopsis thaliana an ecori fragment from the pbluescript - pdat was cloned together with napin promoter ( 26 ) into the vector pgptv - kan ( 27 ). a plasmid ( pgnappdat ) having the yeast pdat gene in the correct orientation was identified and transformed into agrobacterium tumefaciens . these bacteria were used to transform arabidopsis thaliana columbia ( c - 24 ) plants using the root transformation method ( 28 ). plants transformed with an empty vector were used as controls . first generation seeds ( t1 ) were harvested and germinated on kanamycin containing medium . second generation seeds ( t2 ) were pooled from individual plants and their fatty acid contents analysed by quantification of their methyl esthers by gas liquid chromatography after methylation of the seeds with 2 % sulphuric acid in methanol at 85 ° c . for 1 . 5 hours . quantification was done with heptadecanoic acid methyl esters as internal standard . from the transformation with pgnappdat one t1 plant ( 26 - 14 ) gave raise to seven t2 plants of which 3 plants yielded seeds with statistically ( in a mean difference two - 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