Patent Application: US-52609800-A

Abstract:
the present biotechnological approach for the production of polyhydroxyalkanotes uses microbial systems . the high production costs makes them substantially more expensive than synthetic plastics . engineering a novel pathway in eucaryotic cell systems is a beneficial alternative to the production of phas in bacteria . this pathway will initially produce free fatty acids from the fatty acid synthetic pathway through the action of thioesterase , that will then add a coa moiety to the free fatty acid through the action of an acyl - coa synthetase , that will produce 3 - ketoacyl - coas from the acyl - coa through the action of a thiolase , that will produce r -- oh - acyl - coas from the 3 - keto acid coas through the action of a dehydrogenase isoform from yeasts . these r -- 3 - oh - acyl - coas will finally be used as substrate for the pha synthase reaction .

Description:
the present invention is directed at the production of polyhydroxyalkanoates in recombinant organisms , through the engineering of a new metabolic pathway which produces r -(−)- 3 - oh - acyl - coas monomeric subunits of adequate length to serve as substrates for the activity of pha synthases . more specifically , the present invention is directed to a methodology that is used to produce transgenic organisms with a new metabolic pathway that partially deviates fatty acids from their normal synthetic pathways , towards the formation of r -(−)- 3 - oh - acyl - coas that serves as substrates for the synthesis of hydroxyalkanoate polymers in chloroplasts . the new synthetic pathway of the present invention is depicted in fig3 . in one embodiment of the present invention the transgenic organism is a plant or any organ of a plant where there is active plastid activity . according to the present invention examples of suitable plants include but are not limited to arabidopsis , tobacco , alfalfa and tuber plants such as potato , sweet potato , beet and cassava . prior to the present invention , there was no demonstration of a metabolic pathway that would supply monomeric subunits to the polymerization reaction in pseudomonades , nor in any other organisms . known degradation pathways starting with acyl - coas produce s -(−) 3 - oh - acyl - coas and synthetic pathways produce r -(−)- acyl - acps , none of which can serve as substrate for the pha synthesis reaction . thus , the present invention is directed to a synthetic pathway that will produce r -(−)- oh - acyl - coas from 3 - keto acid - coas through the action of a dehydrogenase isoform from yeast . these r -(−)- oh - acyl - coas will then serve as a substrate for the pha synthase reaction . according to the present invention the term polyhydroxyalkanoate , is intended to include a polymer of r -(−)- 3 - hydoxyalkanoic acid monomers from about 3 to about 14 carbons in length . in one embodiment of the present invention the pha synthase from pseudamonas aeruginosa is used in the last step of the synthetic pathway . this enzyme prefers r -(−)- 3 - oh - acyl - coas of c6 to c 14 as a substrate . the biosynthetic pathway of the present invention involves five enzymes : a thioesterase , a acyl - coa synthetase , a thiolase , a d - 3 - hydoxyacyl - coa dehydrogenase and a pha synthase . the first reaction is catalyzed by a thioesterase . in one embodiment of the present invention the enzyme has been cloned from a cdna library from cuphea hookeriana , a mexican bush plant which accumulates up to 75 % of c8 : 0 - and c10 : 0 - fatty acids in seeds . this clone cl fatb2 ( genbank accession # u39834 ) has been expressed in e . coli where it exhibited a high specificity for c8 : 0 and c10 : 0 - acps as substrates . in the chloroplasts , this enzyme removes c8 - and c10 - acyl - acps from the fatty acid synthetic pathway and releases free medium - chain length fatty acids in the stroma much as endogenous thioesterases do with c 16 - and c18 - acyls - acps in the fatty acid synthetic pathway ( dehesh , k . et al ., 1996 , the plant journal 9 ( 2 ): 167 - 172 ). the second reaction is catalysed by an acyl - coa synthetase . in one embodiment of the invention , the enzyme was isolated from pseudomonas oleovorans , a bacteria which accumulates phas mcl ( medium - chain length polyhydroxy alkanoates ). the enzyme is encoded by gene k of operon alkbfghjkl which is responsible for alkanoate synthesis . this acyl - coa synthetase ( genbank accession # x65936 ) is specific to medium - chain length fatty acids ( van beilen , j . b . et al . ( 1992 ) dna sequence determination and functional characterization of the oct - plasmid - encoded alkjkl genes of pseudomonas oleovorans . molecular microbiology 6 ( 21 ): 3121 - 3136 ). the third reaction is catalysed by a keto thiolase . this reaction is a condensation reaction which will add one acetyl - coa moiety to the acyl - coa , thus releasing one coa molecule . this condensation reaction is reversible , and creates a 3 - keto acyl coa with two extra carbon . the products following this reaction will be therefore c10 - and c12 - 3 - oh - acyl coas and free coa . the enzyme described in one example of the present invention has been isolated from brassica napus , in which it is part of the oxidation pathway ( olesen , c . j . et al . ( 1997 ) the glyoxysomal 3 - ketoacyl - coa thiolase precursor from brassica napus has enzymatic activity when synthesized in e . coli ( febs letters 6 ( 21 ): 138 - 140 ; genbank accession # x93015 ). the fourth reaction , according to the present invention , is catalysed by a yeast 3 - hydroxyacyl - coa dehydrogenase which produces r -(−)- 3 - oh - acyl - coas ( hiltunen , j . k . et al . ( 1992 ) peroxisomal multi functional β - oxidation protein of saccharomyces cerevisiae . j . biol . chem . 267 : 6646 - 6653 ; genbank accession # m86456 ). homologs of this 3 - keto - acyl - coa dehydrogenases usually produce s -(−)- 3 - oh - acyl - coas in the β - oxidation pathway . the 3 - hydroxyacyl - coa dehydrogenase domain of the multi functional protein ( mfp ) of yeasts exhibits this unique catalytic property . thus the product of this reaction will be r -(−)- 3 - oh - decanoyl - coa and r -(−)- 3 - oh - dodecanoyl - coa . both molecules can serve as substrate for the polymerization reaction catalyzed by pha synthases . the last reaction of this embodiment is catalysed by a pha synthase from pseudomonas aeruginosa , which accumulates large amounts of pha granules in nutrient stress conditions ( timm , a . and steinbüchel , a ., 1992 ) cloning and molecular analysis of the poly ( 3 - hydroxyalkanoic acid ) gene locus of pseudomonas aeruginosa pao1 . eur . j . appl . microbiol . 209 : 15 - 30 ; genbank accession # x66592 ). analysis of depolymerization products shows that this enzyme uses r -(−)- 3 - oh - acyl - coas of c6 to c14 in length as substrates , with an apparent preference for c10 and c12 r -(−)- 3 - oh - acyl - coas . in one embodiment of the present invention , each of these genes is sub - cloned in an appropriate expression vector . in one embodiment of this invention the host is a plant cell and any know plant expression vector can be used according to the present invention . said plant expression vector can contain a promoter sequence , a 5 ′ utr sequence , a chloroplast transit peptide sequence , the complete coding sequence of the gene , a stop codon , an a 3 ′ utr region containing a eukaryotic polyadenylation signal and a polyadenylation site . the polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3 ′ end of the mrna precursor . polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5 ′ aataaa - 3 ′ although variations are not uncommon . examples of suitable 3 ′ regions are the 3 ′ transcribed non - translated regions containing a polyadenylation signal of agrobacterium tumor inducing ( ti ) plasmid genes , such as the nopaline synthase ( nos gene ) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose - 1 , 5 - bisphosphate carboxylase ( ssrubisco ) gene . the 3 ′ untranslated region from the structural gene of the present construct can therefore be used to construct chimeric genes for expression in plants . the chimeric gene construct of the present invention can also include further enhancers , either translation or transcription enhancers , as may be required . these enhancer regions are well known to persons skilled in the art , and can include the atg initiation codon and adjacent sequences . the initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence . the translation control signals and initiation codons can be from a variety of origins , both natural and synthetic . translational initiation regions may be provided from the source of the transcriptional initiation region , or from the structural gene . the sequence can also be derived from the promoter selected to express the gene , and can be specifically modified so as to increase translation of the mrna . to aid in identification of transformed plant cells , the constructs of this invention may be further manipulated to include plant selectable markers . useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin , hygromycin , kanamycin , and the like . similarly , enzymes providing for production of a compound identifiable by colour change such as gus ( β - glucuronidase ), or luminescence , such as luciferase are useful . this invention is directed at any means by which the genes of interest can be transfected in a plant providing it results in stable integration and expression . preferred means are , agrobacterium mediated dna transfer which requires t - dna borders , and selectable markers ; dna bombardment , which requires selectable markers , and electroporation which can in some cases be used without screenable markers . these various cloning and plant transformation methods are well know in the art . for reviews of such techniques see for example weissbach and weissbach , methods for plant molecular biology , academy press , new york viii , pp . 421 - 463 ( 1988 ); and geierson and corey , plant molecular biology , 2d ed . ( 1988 ). also considered part of this invention are transgenic organisms containing the chimeric gene construct of the present invention . in one embodiment of this invention the transgenic organism is a plant . methods of regenerating whole plants from plant cells are known in the art , and the method of obtaining transformed and regenerated plants is not critical to this invention . in general , transformed plant cells are cultured in an appropriate medium , which may contain selective agents such as antibiotics , where selectable markers are used to facilitate identification of transformed plant cells . once callus forms , shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants . the plants may then be used to establish repetitive generations , either from seeds or using vegetative propagation techniques . when specific sequences are referred to in the present invention , it is understood that these sequences include within their scope sequences that are “ substantially similar ” to said specific sequences . sequences are “ substantially similar ” when at least about 80 %, preferably at least about 90 % and most preferably at least about 95 % of the nucleotides match over a defined length of the molecule . sequences that are “ substantially similar ” include any substitution , deletion , or addition within the sequence . dna sequences that are substantially similar can be identified in southern hybridization experiments , for example under stringent hybridization conditions ( see maniatis et al ., in molecular cloning ( a laboratory manual ), cold spring harbor laboratory ( 1982 ) p 387 to 389 ). the specific sequences , referred to in the present invention , also include sequences which are “ functionally equivalent ” to said specific sequences . in the present invention functionally equivalent sequences refer to sequences which although not identical to the specific sequences provide the same or substantially the same function . dna sequences that are functionally equivalent include any substitution , deletion or addition within the sequence . since the creation of the novel metabolic pathway requires the simultaneous expression of 5 different transgenes , 5 independent transformants can be produced , and genotypes containing the 5 transgenes are produced by repeated crossing of mono - transgenics and selection . alternately , series of genes can be co - transfected on single constructs , thus reducing the need for extensive crossing . other means of integrating the novel genes in chloroplasts also include the direct transfection of dna on chloroplastic dna by recombination using homologous or heterologous border sequences . using this methodology , polycistronic constructs ( multiple gene construct under the control of a single promoter ) could be used to bring the 5 modifications . these methods are well known to persons of skill in the art . while this invention is described in detail with particular reference to preferred embodiments thereof , said embodiments are offered to illustrate but not limit the invention , as shown in the following example . cloning of the pha synthase gene phac1 ( x66592 , timm , 1992 ) was performed by pcr amplification of genomic dna from pseudomonas aeruginosa strain pao1 with the “ expand ” system from boehringer mannheim . template dna was extracted and purified . the primer at the 5 ′ terminus was 5 ′ gatc gcatgc gaaggatttc t atg agtcag3 ′ ( seq id no : 4 ); it contains the sphi and xmni restriction sites upstream from the atg . the primer at the 3 ′ terminus was 5 ′ gatc gaattctca tcgttcatgcacgtagg3 ′ ( seq id no : 5 ); a ecori site had been introduced downstream of the stop codon . conditions for the pcr were : 94 ° c .- 2 ′ the pcr products formed were separated on agarose gels and the band corresponding to the expected size was removed from the gel . purified dna was then digested with sphi / ecori , and the 1692 pb fragments were cloned in puc1 8 . complete homology of the selected clone was verified by sequencing . the synthase gene was then cloned at the sphi / ecori sites of pgem - 7zf ( promega ) containing the 3 ′ non coding region ( 3 ′ nc ) of ssu - rubisco gene rbcsk ( khoudi et al 1997 ) previously cloned at the ecori / smai site . the sphi / saci fragment ( 2138 pb ) containing phac1 + 3 ′ nc was then sub - cloned into litmus28 ( ne biolabs ) containing the complete 5 ′ region of rbcsk with the promoter and transit peptide ( 5 ′ rbcsk ), previously cloned at the sali / sphi site . finally , a fragment of 4112 bp containing the 5 ′ rbcsk + phac1 + 3 ′ nc was cloned into the sali / saci site of pbi 101 . 2 ( clontech ), removing then the gus gene . this construct was then transfected to agrobacterium tumefaciens strain lba4404 , and incorporated in the genome of selected plant cells cells through co - cultivation with transgenic a . tumefaciens , as described by desgagnés et al ( 1995 ) plants were regenerated from transgenic cells , and leaf tissue is used for the selection of the best transgenic lines by northern analysis . pha synthase activity of the selected lines was then measured in clarified leaf extracts as described in the prior art . the domain of the multi functional b - oxidation protein ( mfp ) ( m86456 ) ( hiltunen , 1992 ) which encodes for r - 3 - hydroxyacyl - coa dehydrogenase was amplified by pcr from saccharomyces cerevisiae . it has been shown that the protein contains two activities : a 2 - enoyl - coa hydratase 2 , converting trans - 2 - enoyl - coa to r - 3 - hydroxyacyl - coa , and a r - 3 - hydroxyacyl - coa dehydrogenase , converting r - 3 - hydroxyacyl - coa to 3 - ketoacyl - coa . a truncated version of mfp lacking 271 carboxyl - terminal amino acids was also overexpressed and purified and it was shown that it has only the r - 3 - hydroxyacyl - coa dehydrogenase activity . these results clearly demonstrate that the b - oxidation of fatty acids in the yeast follows a previously unknown stereochemical course , namely it occurs via a r - 3 - hydroxyacyl - coa intermediates . the expression of the truncated version in chloroplasts of plants with a medium - chain length - specific thioesterase , an acyl - coa synthetase and a thiolase will allow the production of r - 3 - hydrozyacyl - coas , the substrate of the pha synthase . template genomic dna was extracted and purified from s . cerevisiae as described in current protocols in molecular biology , 1997 , section 13 . 11 . 1 - 13 . 11 . 4 . the 5 ′ primer used contains the sphi site upstream of the atg ( gatc gcatgc ta atg cctggaaatttatccttc ) ( seq id no : 6 ) and the 3 ′ primer has an apai site downstream a newly introduced stop codon ( gatc gggccctta cgggttgatagtgttgcgact ) ( seq id no : 7 ). the pcr conditions are described above , except that the annealing temperature used was 50 ° c . the pcr fragment was purified , digested sphi / apai and the 1799 bp fragment was cloned in pkitmus / rbsck - 3 ′ nc . this vector is a plitmus28 derivative that contains a cassette for chloroplasts expression . this cassette has in 5 ′ the promoter of the small subunit of the ribulose 1 , 5 - biphosphate carboxylase ( rubisco ) of alfalfa , its 5 ′ non - translated region and the targeting signal to chloroplasts , followed by a multiple cloning site ( mcs ), and in 3 ′, the 3 ′ non coding region of rubisco ( khoudi , et al ., 1997 ). the region in 5 ′ is a 1978 bp sali / sphi fragment that has at its 3 ′ end the sequence that codes for the 58 amino acids of the transit peptide , followed by the atg of the mature protein . this atg is found to be the one in the sphi site . the dehydrogenase clone in the mcs is then in frame with the targeting signal , having a leucine in between the atg of the mature rubisco protein and the one of the gene . the 3 ′ non coding region is a 441 bp ecori / smai fragment that has the two polya signals of the small subunit of rubisco . and a wild type clone , pkitmus / rbsck - dh - 3 ′ nc # 9 , has been conserved for further manipulations ( fig5 a - 5 c ). the gene was put in a cassette for transient expression . plasmid 35sc4ppdk - sgfp - tyg - nos was obtained from jen sheen at the department of molecular biology at massachusetts general hospital and contains the following : the 35s - c4ppdk promoter flanked by xhoi and bamhi sites ; a gene encoding gfp flanked by bamhi and psti sites that contains an amino acid change at position 65 for increased fluorescence and whose codon usage is optimized for plant expression ; and a polyadenylation sequence flanked by psti and ecori sites . the bamhi site after the promoter was changed for a sphi site by a treatment of the vector digested bamhi with the klenow and its ligation with a sphi linker . the vector produced was then digested ecori , treated with the klenow and digested sphi to yield a sphi / blunt vector fragment of 3290 bp that has lost the gene gfp and the nos . pkitmus / rbsck - dh - 3 ′ nc was digested sphi - smai and the 2198 bp fragment containing the dehydrogenase and the 3 ′ non coding region of alfalfa rubisco was ligated in the vector to produce puc / 35s . c4ppdk . dh . 3 ′ nc , a 5488 bp clone used in the transient expression experiment ( fig5 d - 5 e ). for plant expression , the cassette containing the alfalfa rubisco promoter , the dehydrogenase and the 3 ′ non coding region of alfalfa rubisco was obtained by a sali / smai digestion of pkitmus / rbsck - dh - 3 ′ nc # 9 and cloned in pcambia 2300 . the clone formed is called pcambia / rbsck - dh - 3 ′ nc ( fig5 f - 5 j ). diagrams of the constructs are shown in fig4 and the sequences files are found in fig5 a - 5j . for the enzymatic activity of the ( d )- 3 - hydroxyacyl - coa dehydrogenase in e . coli , a 2xyt . ap liquid culture ( 5 ml ) is innoculated with a single colony of dh5α : ptrcn / fox2 and the culture is placed under agitation at 30 ° c . for 16 hours . this overnight culture is used to innoculate ( 1 %) 50 ml of 2xyt . ap media . the cultures are put at 30 ° c . under agitation until the od600 nm reach 0 . 6 . iptg ( 0 . 4 mm ) is added and the culture is incubated for another 4 hours . the cells are collected by centrifugation ( 15 min ./ 5000 g / 4 ° c .) and stored at − 80 ° c . the cells are resuspended in 20 mm kh 2 po 4 buffer ( ph 7 . 0 ), 0 . 5 mm dtt , 0 . 1 mm pmsf and are disrupted by sonication with pulses of 0 . 2 sec for a total period of 20 sec . the cells are returned to ice for cooling purposes and the sonication procedure is repeated two more times to ensure lysis . the extract is clarified by centrifugation in a microfuge ( 12000 g / 15 min ./ 4 ° c .) prior to activity measurements . the dehydrogenase reaction measured the oxidation of a 3 - hydroxyacyl - coa in a 3 - ketoacyl - coa and is followed by monitoring the formation of the mg 2 + complex of 3 - ketoacyl - coa at 303 nm . the incubation mixture consisted of 50 μmol tris - ci ( ph 9 . 0 ), 50 μg bovine serum albumin , 50 μmol kcl , 1 μmol nad + , 25 μmol mgcl 2 , 1 μmol pyruvate and 10 μg lactate dehydrogenase in a total volume of 1 ml . the lactate dehydrogenase allows the regeneration of nad + using the pyruvate as substrate . the reaction is monitored at room temperature with all the components of the incubation mixture and then 10 μg of l - 3 - hydroxyacyl - coa dehydrogenase is added . after about 1 minute , 50 nmol of the substrate dl - 3 - hydroxyoctanoyl - coa is added . when the od 303nm is stabilized , meaning that the l - 3 - hydroxyoctanoyl - coa is completely oxidized , then the extract ( 1 %) is added . the reaction is monitored for another 5 minutes . the r - 3 - hydroxyacyl - coa dehydrogenase activity was measured in dh5α overexpression the mfp fox2 gene of saccharomyces cerevisiae . the extinction coefficient used for the 3 - ketooctanoyl - coa is 14 . 5 × 10 3 cm − 1 m − 1 . the monitoring activity is shown on fig6 a and the linear part of the graph that represents the r - 3 - hydroxyacyl - coa dehydrogenase activity is shown in fig6 b . the specific activity is 0 . 81 units / mg protein . transient expression of the 3 - hydroxyacyl - coa dehydrogenase in plant cells was done by transformation of arabidopsis thaliana protoplasts with puc / 35s . c4ppdk . dh . 3 ′ nc ( sheen , j ., et al ., 1995 ). for the enzymatic activity of the ( d )- 3 - hydroxyacyl - coa dehydrogenase in plant cells , arabidopsis thaliana protoplasts are harvested by centrifugation ( 115 g ) and the supernatant is removed . an aliquot ( 14 μl ) of 7 × stock of protease inhibitor stock is added to the sample and the sample is brought to a final volume of 100 μl with a solution containing 20 mm kh 2 po 4 buffer ( ph 7 . 0 ), 0 . 5 mm dtt , 0 . 1 mm pmsf . the 7 × stock of protease inhibitors is prepared by dissolving one “ complete mini protease inhibitor tablet ” ( boehringer manneheim ) in 1 . 5 ml 20 mm kh 2 po 4 buffer ( ph 7 . 0 ), 0 . 5 mm dtt , 0 . 1 mm pmsf . the protoplasts are disrupted in a 1 . 5 ml centrifuge tube using a pellet pestle mixer ( kontes ) for 30 seconds . soluble proteins are separated from insoluble proteins by centrifugation at maximum speed in a microcentrifuge ( 10 min , 4 ° c .). dehydrogenase activity was measured as described above . the r - 3 - hydroxyacyl - coa dehydrogenase activity was measured in protoplasts of arabidopsis thaliana transformed with puc / 35s . c4ppdk . dh . 3 ′ nc . the extinction coefficient used for the 3 - ketooctanoyl - coa is 14 . 5 × 10 3 cm − 1 m − 1 . arabidopsis thaliana plants were transformed with pcambia / rbsck - dh - 3 ′ nc following a floral dip protocol ( clough , s . j ., et al . 1998 ) and using the agrobacterium tumefasciens strain gv3101 / pmp90 ( koncz , c ., et al ., 1986 ). for the enzymatic activity of the ( d )- 3 - hydroxyacyl - coa dehydrogenase in plants , leaves of arabidopsis thaliana expressing the enzyme are collected and grinded in 5 volumes of extraction buffer containing 50 mm tris - cl buffer ( ph 8 . 0 ), 0 . 4 % β - mercaptoethanol , 2 mm pmsf . the extract is clarified on miracloth and centrifuged ( 12 000g / 15 min ./ 4 ° c .). the supernatant is desalted on a sephadex g - 25 pd - 10 column ( pharmacia ) eluting in a buffer containing 20 mm kh 2 po 4 buffer ( ph 7 . 0 ), 0 . 5 mm dtt , 0 . 1 mm pmsf . dehydrogenase activity was determined as described above . the r - 3 - hydroxyacyl - coa dehydrogenase activity was measured in plants of arabidopsis thaliana transformed with pcambia / rbsck - dh - 3 ′ nc . the extinction coefficient used for the 3 - ketooctanoyl - coa is 14 . 5 × 10 3 cm − 1 m − 1 . cloning of the thiolase gene ( x93015 ) ( olesen , 1997 ) was performed by pcr amplification of genomic dna from brassica napus as described above , except that the annealing temperature used was 50 ° c . template dna was prepared from b . napus leaves as described by rogers & amp ; bendich , plant mol . biol ., 1988 , a6 : 1 - 10 . the primer at the 5 ′ end was 5 ′ gatc gcatgc tagctggggacagtgctgcgtatc - 3 ′ ( seq id no : 12 ) with an added sphi site , and at the 3 ′ end 5 ′- gatc gaattccta acgagcgtccttggacaaaag - 3 ′ ( seq id no : 13 ) with an ecori site downstream of the stop codon . primers were selected so that amplification would be initiated at position 106 of the cdna and therefore eliminate the n - terminal targeting signal for glyoxysomes . the gel - purified pcr products were digested with sphi / ecori and cloned in a derivative of plitmus 28 modified as described above for cloning of yeast dehydrogenase . suitable clones are fully sequenced . sequence was compared with the published cdna ( 1389 pb ), although amplicons were produced from genomic dna template . homologous amplicons with introns and without the targeting signal are 2568 - bp in size . the whole construct with the homologous gene was sub - cloned at the sali / smai site in pbi 101 . 2 . transformation of a . tumefaciens , transformation of selected plants , and regeneration of transgenic lines was performed as described above . selection of best transgenic lines was performed with northern hybridization . a gene encoding for a thioesterase with specificity for 8 : 0 and 10 : 0 - acp substrates ( u39834 ) ( dehesh , 1996 ) was amplified by pcr from cuphea hookeriana as described above , except that the annealing temperature used was 60 ° c . template genomic dna was prepared with the qiagen genomic tip protocol as described by the manufacturer . the primer at the 5 ′ end was 5 ′- gatc tctagaatg gtggctgctgcagcaagttccg - 3 ′ ( seq id no : 14 ) with a xbai site upstream of the atg ; the primer at the 3 ′ end was 5 ′- gatc gggccccta agagaccgagtttccatttgaag - 3 ′ ( seq id no : 15 ) with an apai site downstream of the stop codon . the pcr product was cloned at the xbai / apai site in the plant vector pcambia 2300 , modified to harbor the rbsck promotor ( sali / sphi ) with the transit peptide , a multiple cloning site between sphi and ecori and the 3 ′ non coding region of rbsck ( ecori / smai ). transformation of a . tumefaciens , transformation of selected plants , and regeneration of transgenic lines was performed as described above . selection of best transgenic lines was performed with northern hybridization . cloning of the acyl - coa synthetase gene ( x65936 ) ( van beilen , 1992 ) was performed by pcr amplification of genomic dna from pseudomonas oleovorans . template dna was prepared as described in current protocols in molecular biology , 1997 , 2 . 4 . 1 - 2 . 4 . 2 . the primer at the 5 ′- end was gatc ggatccatg ttaggtcagatgatgcgt - 3 ′ ( seq id no : 16 ) with a bamhi site upstream of the atg ; the primer at the 3 ′ end was 5 ′- gatc gaattctta ttcacagacagaagaact - 3 ′ ( seq id no : 17 ) with an ecori site downstream of the stop codon . the pcr product was cloned in the bamhi / ecori site of plitmus 28 modified as described above for cloning of yeast dehydrogenase . suitable clones were fully sequenced . wjole constructs were then sub - cloned into pbi . 101 . 2 . transformation of a . tumefaciens , transformation of alfalfa and tobacco plants , and regeneration of transgenic lines was performed as described above . selection of best transgenic lines was performed with northern hybridization . all scientific references and patent documents are incorporated herein by reference . the invention as herein described can be varied in many ways . such variations are not to be regarded as a departure from the spirit and scope of the present invention . all modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims . anderson , a . j . & amp ; dawes , e . a . 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( 1992 ) chemical regulation of bacillus thuringiensis d - endotoxin expression in transgenic plants . bio / technology 10 : 540 - 543 . atg agg gtc ccc gct cag ctc ctg ggg ctc ctg ctg ctc tgg ctc cca 48 ggt gca cga tgt gcc tat gaa ctg act cag cca ccc tcg gtg tca gtg 96 gly ala arg cys ala tyr glu leu thr gln pro pro ser val ser val tcc cca gga cag acg gcc agg atc acc tgt ggg gga gac aac agt aga 144 ser pro gly gln thr ala arg ile thr cys gly gly asp asn ser arg aat gaa tat gtc cac tgg tac cag cag aag cca gcg cgg gcc cct ata 192 asn glu tyr val his trp tyr gln gln lys pro ala arg ala pro ile ctg gtc atc tat gat gat agt gac cgg ccc tca ggg atc cct gag cga 240 ttc tct ggc tcc aaa tca ggg aac acc gcc acc ctg acc atc aac ggg 288 gtc gag gcc ggg gat gag gct gac tat tac tgt cag gtg tgg gac agg 336 gct agt gat cat ccg gtc ttc gga gga ggg acc cgg gtg acc gtc cta 384 ala ser asp his pro val phe gly gly gly thr arg val thr val leu ggt cag ccc aag gct gcc ccc tcg gtc act ctg ttc ccg ccc tcc tct 432 gag gag ctt caa gcc aac aag gcc aca ctg gtg tgt ctc ata agt gac 480 glu glu leu gln ala asn lys ala thr leu val cys leu ile ser asp ttc tac ccg gga gcc gtg aca gtg gcc tgg aag gca gat agc agc ccc 528 phe tyr pro gly ala val thr val ala trp lys ala asp ser ser pro gtc aag gcg gga gtg gag acc acc aca ccc tcc aaa caa agc aac aac 576 aag tac gcg gcc agc agc tac ctg agc ctg acg cct gag cag tgg aag 624 tcc cac aga agc tac agc tgc cag gtc acg cat gaa ggg agc acc gtg 672 gag aag aca gtg gcc cct aca gaa tgt tca tga 705 gly ala arg cys ala tyr glu leu thr gln pro pro ser val ser val ser pro gly gln thr ala arg ile thr cys gly gly asp asn ser arg asn glu tyr val his trp tyr gln gln lys pro ala arg ala pro ile ala ser asp his pro val phe gly gly gly thr arg val thr val leu glu glu leu gln ala asn lys ala thr leu val cys leu ile ser asp phe tyr pro gly ala val thr val ala trp lys ala asp ser ser pro atg aaa cac ctg tgg ttc ttc ctc ctc ctg gtg gca gct ccc aga tgg 48 gtc ctg tcc cag gtg aag ctg cag cag tgg ggc gaa gga ctt ctg cag 96 cct tcg gag acc ctg tcc cgc acc tgc gtt gtc tct ggt ggc tcc atc 144 agc ggt tac tac tac tgg acc tgg atc cgc cag acc cca ggg agg gga 192 ctg gag tgg att ggc cat att tat ggt aat ggt gcg acc acc aac tac 240 aat ccc tcc ctc aag agt cga gtc acc att tca aaa gac acg tcc aag 288 aac cag ttc ttc ctg aac ttg aat tct gtg acc gac gcg gac acg gcc 336 gtc tat tac tgt gcg aga ggc cct cgc cct gat tgc aca acc att tgt 384 tat ggc ggc tgg gtc gat gtc tgg ggc ccg gga gac ctg gtc acc gtc 432 tcc tca gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc 480 tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag 528 gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg 576 asp tyr phe pro glu pro val thr val ser trp asn ser gly ala leu acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc 624 tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc 672 cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg 720 gln thr tyr ile cys asn val asn his lys pro ser asn thr lys val gac aag aaa gca gag ccc aaa tct tgt gac aaa act cac aca tgc cca 768 ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc 816 ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc 864 pro pro lys pro lys asp thr leu met ile ser arg thr pro glu val aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc 912 aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg 960 asn trp tyr val asp gly val glu val his asn ala lys thr lys pro cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc 1008 gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc 1056 val leu his gln asp trp leu asn gly lys glu tyr lys cys lys val tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc 1104 aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg 1152 lys gly gln pro arg glu pro gln val tyr thr leu pro pro ser arg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc 1200 asp glu leu thr lys asn gln val ser leu thr cys leu val lys gly ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg 1248 phe tyr pro ser asp ile ala val glu trp glu ser asn gly gln pro gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc 1296 glu asn asn tyr lys thr thr pro pro val leu asp ser asp gly ser ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag 1344 phe phe leu tyr ser lys leu thr val asp lys ser arg trp gln gln ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac 1392 gly asn val phe ser cys ser val met his glu ala leu his asn his tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1431 asp tyr phe pro glu pro val thr val ser trp asn ser gly ala leu gln thr tyr ile cys asn val asn his lys pro ser asn thr lys val pro pro lys pro lys asp thr leu met ile ser arg thr pro glu val asn trp tyr val asp gly val glu val his asn ala lys thr lys pro val leu his gln asp trp leu asn gly lys glu tyr lys cys lys val lys gly gln pro arg glu pro gln val tyr thr leu pro pro ser arg asp glu leu thr lys asn gln val ser leu thr cys leu val lys gly phe tyr pro ser asp ile ala val glu trp glu ser asn gly gln pro glu asn asn tyr lys thr thr pro pro val leu asp ser asp gly ser phe phe leu tyr ser lys leu thr val asp lys ser arg trp gln gln gly asn val phe ser cys ser val met his glu ala leu his asn his atg agc ctc cct gct cag ctc ctc ggg ctg cta ttg ctc tgc gtc ccc 48 ggg tcc agt ggg gaa gtt gtg atg act cag tct cca ctg tcc ctt ccc 96 atc aca cct gga gag ccg gcc tcc atc tcc tgt agg tct agt caa agc 144 ctt aaa cac agt aat gga gac acc ttc ctg agt tgg tat cag cag aag 192 leu lys his ser asn gly asp thr phe leu ser trp tyr gln gln lys cca ggc caa cct cca agg ctc ctg att tat aag gtt tct aac cgg gac 240 pro gly gln pro pro arg leu leu ile tyr lys val ser asn arg asp tct ggg gtc cca gac aga ttc agc ggc agt ggg gca ggg aca gat ttc 288 aca ctg aaa atc agc gca gtg gag gct gaa gat gtt ggg gtt tat ttc 336 thr leu lys ile ser ala val glu ala glu asp val gly val tyr phe tgc ggg caa ggt aca agg act cct ccc act ttc ggc gga ggg acc aag 384 gtg gaa atc aaa cgt acg gtg gct gca cca tct gtc ttc atc ttc ccg 432 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 pro ser asp glu gln leu lys ser gly thr ala ser val val cys leu ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 leu asn asn phe tyr pro arg glu ala lys val gln trp lys val asp aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tga 720 gly leu ser ser pro val thr lys ser phe asn arg gly glu cys leu lys his ser asn gly asp thr phe leu ser trp tyr gln gln lys pro gly gln pro pro arg leu leu ile tyr lys val ser asn arg asp thr leu lys ile ser ala val glu ala glu asp val gly val tyr phe pro ser asp glu gln leu lys ser gly thr ala ser val val cys leu leu asn asn phe tyr pro arg glu ala lys val gln trp lys val asp gly leu ser ser pro val thr lys ser phe asn arg gly glu cys atg ggt tgg agc ctc atc ttg ctc ttc ctt gtc gct gtt gct acg cgt 48 met gly trp ser leu ile leu leu phe leu val ala val ala thr arg gtc cag tgt gag gtg caa ctg gtg gag tct ggg gga ggc ttg gtc cag 96 cct ggc ggg tcc ctg aga gtc tcc tgt gca gtc tct gga ttc acc ttc 144 agt gac cac tac atg tat tgg ttc cgc cag gct cca ggg aag ggg ccg 192 ser asp his tyr met tyr trp phe arg gln ala pro gly lys gly pro gaa tgg gta ggt ttc att aga aac aaa ccg aac ggt ggg aca aca gaa 240 glu trp val gly phe ile arg asn lys pro asn gly gly thr thr glu tac gcc gcg tct gtg aaa gac aga ttc acc atc tcc aga gat gat tcc 288 aaa agc atc gcc tat ctg caa atg agc agc ctg aaa atc gag gac acg 336 lys ser ile ala tyr leu gln met ser ser leu lys ile glu asp thr gcc gtc tat tac tgt act aca tcc tac att tca cat tgt cgg ggt ggt 384 gtc tgc tat gga ggt tac ttc gaa ttc tgg ggc cag ggc gcc ctg gtc 432 acc gtc tcc tca gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca 480 ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg 528 gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc 576 val lys asp tyr phe pro glu pro val thr val ser trp asn ser gly gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca 624 gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg 672 ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc 720 gly thr gln thr tyr ile cys asn val asn his lys pro ser asn thr aag gtg gac aag aaa gca gag ccc aaa tct tgt gac aaa act cac aca 768 tgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc 816 ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 864 gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 912 aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 960 lys phe asn trp tyr val asp gly val glu val his asn ala lys thr aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 1008 ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 1056 leu thr val leu his gln asp trp leu asn gly lys glu tyr lys cys aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 1104 aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 1152 lys ala lys gly gln pro arg glu pro gln val tyr thr leu pro pro tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 1200 ser arg asp glu leu thr lys asn gln val ser leu thr cys leu val aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1248 lys gly phe tyr pro ser asp ile ala val glu trp glu ser asn gly cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 1296 gln pro glu asn asn tyr lys thr thr pro pro val leu asp ser asp ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 1344 gly ser phe phe leu tyr ser lys leu thr val asp lys ser arg trp cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 1392 gln gln gly asn val phe ser cys ser val met his glu ala leu his aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1437 met gly trp ser leu ile leu leu phe leu val ala val ala thr arg ser asp his tyr met tyr trp phe arg gln ala pro gly lys gly pro glu trp val gly phe ile arg asn lys pro asn gly gly thr thr glu lys ser ile ala tyr leu gln met ser ser leu lys ile glu asp thr val lys asp tyr phe pro glu pro val thr val ser trp asn ser gly gly thr gln thr tyr ile cys asn val asn his lys pro ser asn thr lys phe asn trp tyr val asp gly val glu val his asn ala lys thr leu thr val leu his gln asp trp leu asn gly lys glu tyr lys cys lys ala lys gly gln pro arg glu pro gln val tyr thr leu pro pro ser arg asp glu leu thr lys asn gln val ser leu thr cys leu val lys gly phe tyr pro ser asp ile ala val glu trp glu ser asn gly gln pro glu asn asn tyr lys thr thr pro pro val leu asp ser asp gly ser phe phe leu tyr ser lys leu thr val asp lys ser arg trp gln gln gly asn val phe ser cys ser val met his glu ala leu his atg agg gtc ccc gct cag ctc ctg ggg ctc ctg ctg ctc tgg ctc cca 48 ggt gca cga tgt gag tct gtc ctg aca cag ccg ccc tca gtg tct ggg 96 gly ala arg cys glu ser val leu thr gln pro pro ser val ser gly gcc cca ggg cag aag gtc acc atc tcg tgc act ggg agc acc tcc aac 144 ala pro gly gln lys val thr ile ser cys thr gly ser thr ser asn att gga ggt tat gat cta cat tgg tac cag cag ctc cca gga acg gcc 192 ile gly gly tyr asp leu his trp tyr gln gln leu pro gly thr ala ccc aaa ctc ctc atc tat gac att aac aag cga ccc tca gga att tct 240 gac cga ttc tct ggc tcc aag tct ggt acc gcg gcc tcc ctg gcc atc 288 act ggg ctc cag act gag gat gag gct gat tat tac tgc cag tcc tat 336 gac agc agc ctg aat gct cag gta ttc gga gga ggg acc cgg ctg acc 384 asp ser ser leu asn ala gln val phe gly gly gly thr arg leu thr gtc cta ggt cag ccc aag gct gcc ccc tcg gtc act ctg ttc ccg ccc 432 tcc tct gag gag ctt caa gcc aac aag gcc aca ctg gtg tgt ctc ata 480 ser ser glu glu leu gln ala asn lys ala thr leu val cys leu ile agt gac ttc tac ccg gga gcc gtg aca gtg gcc tgg aag gca gat agc 528 ser asp phe tyr pro gly ala val thr val ala trp lys ala asp ser agc ccc gtc aag gcg gga gtg gag acc acc aca ccc tcc aaa caa agc 576 aac aac aag tac gcg gcc agc agc tac ctg agc ctg acg cct gag cag 624 tgg aag tcc cac aga agc tac agc tgc cag gtc acg cat gaa ggg agc 672 trp lys ser his arg ser tyr ser cys gln val thr his glu gly ser acc gtg gag aag aca gtg gcc cct aca gaa tgt tca tga 711 gly ala arg cys glu ser val leu thr gln pro pro ser val ser gly ala pro gly gln lys val thr ile ser cys thr gly ser thr ser asn ile gly gly tyr asp leu his trp tyr gln gln leu pro gly thr ala asp ser ser leu asn ala gln val phe gly gly gly thr arg leu thr ser ser glu glu leu gln ala asn lys ala thr leu val cys leu ile ser asp phe tyr pro gly ala val thr val ala trp lys ala asp ser trp lys ser his arg ser tyr ser cys gln val thr his glu gly ser atg aaa cac ctg tgg ttc ttc ctc ctc ctg gtg gca gct ccc aga tgg 48 gtc ctg tcc cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag 96 cct tcg gag acc ctg tcc ctc acc tgc gct gtc tct ggt ggc tcc atc 144 agc ggt ggt tat ggc tgg ggc tgg atc cgc cag ccc cca ggg aag ggg 192 ctg gag tgg att ggg agt ttc tat agt agt agt ggg aac acc tac tac 240 aac ccc tcc ctc aag agt caa gtc acc att tca aca gac acg tcc aag 288 aac cag ttc tcc ctg aag ctg aac tct atg acc gcc gcg gac acg gcc 336 gtg tat tac tgt gtg aga gat cgt ctt ttt tca gtt gtt gga atg gtt 384 tac aac aac tgg ttc gat gtc tgg ggc ccg gga gtc ctg gtc acc gtc 432 tcc tca gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc 480 tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag 528 gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg 576 asp tyr phe pro glu pro val thr val ser trp asn ser gly ala leu acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc 624 tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc 672 cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg 720 gln thr tyr ile cys asn val asn his lys pro ser 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