Patent Application: US-85326804-A

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 that is produced .

Description:
the invention now having been 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 . fig1 . 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 ). fig2 . 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 ) synthesised 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 on a tlc plate by electronic autoradiography ( instant imager , packard , us ). 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 . triacylglycerol accumulation is reduced in yeast cells that lack the are1 gene 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 :: his3 ade2 can 1 - 100 leu2 - 3 , 112 trp1 - 1 ura3 - 1 , was generated by crossing the two strains scy60 ( matα are1 - δ :: his3 ade2 - 1 can 1 - 100 leu2 - 3 , 112 trp1 - 1 ura3 - 1 ) and scy61 ( mata 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 ( mata 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 . primers 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 stui sites within the yor245c open reading frame . a linear fragment containing the resulting yor245c :: kanmx4 disruption cassette was finally obtained through cleavage by bamhi / 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 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 his3 - 11 , 15 trp1 - 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 :: his3 pdat - δ :: trp1 ade2 can 1 - 100 leu2 - 3 , 112 ura3 - 1 trp1 - 1 , was generated by transforming h1111 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 trp1 - 1 , was constructed by transforming h1224 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 . 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 triple 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 and 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 . 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 pjn92 ( ronne et al ., 1991 ), thus generating pus5 . the wild type yeast strain scy62 ( mata 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 . 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 promotor ( 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 . method a : a wild type yeast ( strain scy62 ) was transformed with a plasmid ( pus5 ) containing the are1 gene under the control of a gal1 promotor ( 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 induced 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 tris - 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 beads ( 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 ( h1236 ), 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 promotor 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 ammnonium 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 ). 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 ( fig1 ). 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 ( fig2 ) 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 . triacylglycerol accumulation is increased in the seeds of arabidopsis thaliana that express the are1 gene 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 methyl 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 . 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 . becker , d ., kemper , e ., schell , j ., and masterson , r . 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