Patent Application
20080060096
The DNA which codes for the ketolase NP60.79:BKT was amplified by PCR from Nostoc punctiforme SAG 60.79 (SAG: Sammiung von Algenkulturen Göttingen).
To prepare genomic DNA from a suspension culture of Nostoc punctiforme SAG 60.79, which was grown in BG 11 medium (1.5 g/l NaNO3, 0.04 g/l K2PO4×3H2O, 0.075 g/l MgSO4×H2O, 0.036 g/l CaCl2×2H2O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na2CO3,1 ml trace metal mix A5+Co (2.86 g/l H3BO3, 1.81 g/l MnCl2×4H2O, 0.222 gA ZnSO4×7H2o, 0.39 g/l NaMoO4×2H2o, 0.079 g/l CuSO4×5H2O, 0.0494 g/l Co(NO3)2×6H2O)) at 25° C. with continuous light and constant shaking (150 rpm) for 1 week, the cells were harvested by centrifugation, frozen in liquid nitrogen and powdered in a mortar.
Protocol for DNA Isolation from Nostoc punctiforme SAG 60.79:
The bacterial cells from a 10 ml liquid culture were pelleted by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 100 mM Tris HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After addition of 100 μl of proteinase K (concentration: 20 mg/ml), the cell suspension was incubated at 37° C. for 3 hours. The suspension was then extracted with 50 μl of phenol. After centrifugation at 13 000 rpm for 5 minutes, the upper, aqueous phase was transferred into a new 2 ml Eppendorf reaction vessel. The extraction with phenol was repeated 3 times. The DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved by heating to 65° C.
The nucleic acid coding for the ketolase NP60.79:BKT from Nostoc punctiforme SAG 60.79 was amplified by a polymerase chain reaction (PCR) from Nostoc punctiforme SAG 60.79 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NP196-2 SEQ ID No. 60).
The PCR conditions were as follows:
The PCR for amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence took place in a 50 ul reaction mixture which comprised:
The PCR was, carried out under the following cycle conditions:.
The PCR amplification with SEQ ID No. 59 and SEQ ID No. 60 resulted in a 792 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 61). The amplicon was cloned, using standard methods, into the PCR cloning vector pCR 2.1-TOPO (Invitrogen), and the clone pNP60.79 was obtained.
NP60.79:BKT from Nostoc punctiforme SAG 60.79 in Lycopersicon esculentum and Tagetes erecta Expression of the ketolase from Nostoc punctiforme SAG 60.79 in Lycopersicon esculentum and in Tagetes erecta took place under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase) from Arabidopsis thaliana. The expression took place with the pea transit peptide rbcS (Anderson et al-. 1986, Biochem J. 240:709-715).
The DNA fragment which comprises the FNR promoter region −635 to −1 from Arabidopsis thaliana (SEQ ID No. 65) was prepared by means of PCR using genomic DNA (isolated from Arabidopsis thaliana by standard methods) and the primers FNR-1 (SEQ ID No.63) and FNR-2 (SEQ ID No. 64).
The PCR conditions were as follows:
The PCR for amplifying the DNA which comprises the FNR promoter fragment (−635 to −1) took place in a 50 ul reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The 653 bp amplicon (SEQ ID No. 65) was cloned into the PCR-cloning vector pCR 2.1-TOPO (Invitrogen) using standard methods, and the plasmid pFNR was obtained.
Sequencing of the clone pFNR confirmed a sequence which agrees with a sequence segment on chromosome 5 of Arabidopsis thaliana (database entry ABOL 1474) from position 70127 to 69493. The gene starts at base pair 69492 and is annotated as “ferredoxin-NADP+reductase”.
The clone pFNR was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
The cloning took place by isolating the 637 bp KpnI-HindIII fragment from pFNR and ligating into the KpnI-HindIII cut vector pJIT117. The clone which the promoter FNR instead of the original promoter d35S is called pJFNR.
The clone pNP60.79 was used for cloning into the expression vector pJFNR (Example 2). The cloning took place by isolating the 790 Bp SphI fragment from pNP60.79 and ligating into the SphI cut vector pJFNR. The clone which comprises the ketolase from Nostoc punctiforme SAG 60.79 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is called pJFNRNP60.79.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the ketolase NP60.79:BKT from Nostoc punctiforme SAG 60.79 into Lycopersicon esculentum took place using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector pS3FNRNP60.79, the 2.4 Kb KpnI fragment from pJFNRNP60.79 was ligated to the KpnI cut vector pSUN3. This clone is called MSP1.
Preparation of an Expression Cassette for Agrobacterium-Mediated Transformation of the expression vector with the ketolase NP60.79:BKT from Nostoc punctiforme SAG 60.79 in Tagetes erecta took place using the binary vector pSUN5 (WO02/00900).
To prepare the expression vector pS5FNRNP60.69, the 2.4 Kb KpnI fragment from pJFNRNP60.79 was ligated to the KpnI cut vector pSUN5. This clone is called MSP2.
NP60.79:BKT from Nostoc punctiforme SAG 71.79 The DNA which codes for the ketolase NP71.79:BKT was amplified by PCR from Nostoc punctiforme SAG 71.79 (SAG: Sammiung von Algenkulturen Göttingen).
To prepare genomic DNA from a suspension culture of Nostoc punctiforme SAG 71.79, which was grown in BG 11 medium (1.5 g/l NaNO3, 0.04 g/l K2PO4×3H2O, 0.075 g/l MgSO4×H2O, 0.036 g/l CaCl2×2H2O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na2CO3, 1 ml trace metal mix A5+Co (2.86 g/l H3BO3, 1.81 g/l MnCl2×4H2o, 0.222 g/l ZnSO4×7H2o, 0.39 g/l NaMoO4×2H2o, 0.079 g/l CuSO4×5H2O, 0.0494 g/l Co(NO3)2×6H2O)) at 25° C. with continuous light and constant shaking (150 rpm) for 1 week, the cells were harvested by centrifugation, frozen in liquid nitrogen and powdered in a mortar.
Protocol for DNA isolation from Nostoc punctiforme SAG 71.79:
The bacterial cells from a 10 ml, liquid culture were pelleted by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After addition of 100 μl of proteinase K (concentration: 20 mg/ml), the cell suspension was incubated at 37° C. for 3 hours. The suspension was then extracted with 500 μl of phenol. After centrifugation at 13 000 rpm for 5 minutes, the upper, aqueous phase was transferred into a new 2 ml Eppendorf reaction vessel. The extraction with phenol was repeated. 3 times. The DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 700% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved by heating to 65° C.
The nucleic acid coding for the ketolase NP71.79:BKT from Nostoc punctiforme SAG 71.79 was amplified by a polymerase chain reaction (PCR) from Nostoc punctiforme SAG 71.79 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NP196-2 SEQ ID No. 60).
The PCR conditions were as follows:
The PCR for amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence took place in a 50 ul reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The PCR amplification with SEQ ID No. 59 and SEQ ID No. 60 resulted in a 792 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 66). The amplificate was cloned, using standard methods, into the PCR cloning vector pCR 2.1-TOPO (Invitrogen), and the clone pNP71.79 was obtained.
Expression of the ketolase from Nostoc punctiforme SAG 71.79 in Lycopersicon esculentum and in Tagetes erecta took place under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase) from Arabidopsis thaliana. The expression took place with the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240:709-715).
The clone pNP71.79 was used for cloning into the expression vector pJFNR (Example 2). The cloning took place by isolating the 790 Bp SphI fragment from pNP71.79 and ligating into the SphI cut vector pJFNR. The clone which comprises the ketolase from Nostoc punctiforme SAG 71.79 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is called pJFNRNP71.79.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the ketolase NP71.79:BKT from Nostoc punctiforme SAG 71.79 into Lycopersicon esculentum took place using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector pS3FNRNP71.79, the 2.4 Kb KpnI fragment from pJFNRNP71.79 was ligated to the KpnI cut vector pSUN3. This clone is called MSP3.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the expression vector with the ketolase NP71.79:BKT from Nostoc punctiforme SAG 71.79 in Tagetes erecta took place using the binary vector pSUN5 (WO02/00900).
To prepare the expression vector pS5FNRNP71.69, the 2.4 Kb KpnI fragment from pJFNRNP71.79 was ligated to the KpnI cut vector pSUN5. This clone is called MSP4.
The DNA which codes for the ketolase NS037:BKT was amplified by PCR from Nodularia spumigena CCAUV 01-037 (CCAUV: Culture Collection of Algae at the University of Vienna).
To prepare genomic DNA from a suspension culture of Nodularia spumigena CCAUV 01-037, which was grown in BG 11 medium (1.5 g/l NaNO3, 0.04 g/l K2PO4×3H2O, 0.075 g/l MgSO4×H2O, 0.036 g/l CaCl2×2H2O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na2CO3, 1 ml trace metal mix A5+Co (2.86 g/l H3BO3, 1.81 g/l MnCl2×4H2o, 0.222 g/l ZnSO4×7H2o, 0.39 g/l NaMoO4×2H2o, 0.079 g/l CuSO4×5H2O, 0.0494 g/l Co(NO3)2×6H2O)) at 25° C. with continuous light and constant shaking (150 rpm) for 1 week, the cells were harvested by centrifugation, frozen in liquid nitrogen and powdered in a mortar.
Protocol for DNA isolation from Nodularia spumigena CCAUV 01-037:
The bacterial cells from a 10 ml liquid culture were pelleted by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After addition of 100 μl of proteinase K (concentration: 20 mg/ml), the cell suspension was incubated at 37° C. for 3 hours. The suspension was then extracted with 500 μl of phenol. After centrifugation at 13 000 rpm for 5 minutes, the upper, aqueous phase was transferred into a new 2 ml Eppendorf reaction vessel. The extraction with phenol was repeated 3 times. The DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved by heating to 65° C.
The nucleic acid coding for the ketolase NS037:BKT from Nodularia spumigena CCAUV 01-037 was amplified by a polymerase chain reaction (PCR) from Nodularia spumigena CCAUV 01-037 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NSK-2 SEQ ID No. 68).
The PCR conditions were as follows:
The PCR for amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence took place in a 50 ul reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The PCR amplification with SEQ ID No. 59 and SEQ ID No. 68 resulted in an 807 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 69). The amplicon was cloned, using standard methods, into the PCR cloning vector pCR 2.1-TOPO (Invitrogen), and the clone pNS037 was obtained.
Expression of the ketolase from Nodularia spumigena CCAUV 01-037 in Lycopersicon esculentum and in Tagetes erecta took place under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase) from Arabidopsis thaliana. The expression took place with the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240:709-715).
The clone pNS037 was used for cloning into the expression vector PJFNR (Example 2). The cloning took place by isolating the 797 Bp SphI fragment from pNS037 and ligating into the SphI cut vector pJFNR. The clone which comprises the ketolase from Nodularia spumigena CCAUV 01-037 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is called pJFNRNS037.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the ketolase NS037:BKT from Nodularia spumigena CCAUVO1-037 into Lycopersicon esculentum took place using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector pS3FNRNS037, the 2.4 Kb KpnI fragment from pJFNRS037 was ligated to the KpnI cut vector pSUN3. This clone is called MSP5.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the expression vector with the ketolase NS037:BKT from Nodularia spumigena CCAUV 01-037 in Tagetes erecta took place using the binary vector pSUN5 (WO02/00900).
To prepare the expression vector pS5FNRNS037, the 2.4 Kb KpnI fragment from pJFNRNS037 was ligated to the KpnI cut vector pSUN5. This clone is called MSP6.
The DNA which codes for the ketolase NS053:BKT was amplified by PCR from Nodularia spumigena CCAUV 01-053 (CCAUV: Culture Collection of Algae at the University of Vienna).
To prepare genomic DNA from a suspension culture of Nodularia spumigena CCAUV 01-053, which was grown in BG 11 medium (1.5 g/l NaNO3, 0.04 g/l K2PO4×3H2O, 0.075 g/l MgSO4×H2O, 0.036 g/l CaCl2×2H2O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na2CO3, 1 ml trace metal mix A5+Co (2.86 g/l H3BO3, 1.81 g/l MnCl2×4H2o, 0.222 g/l ZnSO4×7H2o, 0.39 g/l NaMoO4×2H2o, 0.079 g/l CuSO4×5H2O, 0.0494 g/l Co(NO3)2×6H2O)) at 25° C. with continuous light and constant shaking (150 rpm) for 1 week, the cells were harvested by centrifugation, frozen in liquid nitrogen and powdered in a mortar.
Protocol for DNA isolation from Nodularia spumigena CCAUV 01-053:
The bacterial cells from a 10 ml liquid culture were pelleted by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After addition of 100 μl of proteinase K (concentration: 20 mg/ml), the cell suspension was incubated at 37° C. for 3 hours. The suspension was then extracted with 500 μl of phenol. After centrifugation at 13 000 rpm for 5 minutes, the upper, aqueous phase was transferred into a new 2 ml Eppendorf reaction vessel. The extraction with phenol was repeated 3 times. The DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved by heating to 65° C.
The nucleic acid coding for the ketolase NS053:BKT from Nodularia spumigena CCAUV 01-053 was amplified by a polymerase chain reaction (PCR) from Nodularia spumigena CCAUV 01-053 using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NSK-2 SEQ ID No. 68).
The PCR conditions were as follows:
The PCR for amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence took place in a 50 ul reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The PCR amplification with SEQ ID No. 59 and SEQ ID No. 68 resulted in an 807 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 71). The amplicon was cloned, using standard methods, into the PCR cloning vector pCR 2.1-TOPO (Invitrogen), and the clone pNS053 was obtained.
Expression of the ketolase from Nodularia spumigena CCAUV 01-053 in Lycopersicon esculentum and in Tagetes erecta took place under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase) from Arabidopsis thaliana. The expression took place with the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240:709-715).
The clone pNS053 was used for cloning into the expression vector pJFNR (Example 2). The cloning took place by isolating the 797 Bp SphI fragment from pNS053 and ligating into the SphI cut vector pJFNR. The clone which comprises the ketolase from Nodularia spumigena CCAUV 01-053 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is called pJFNRNS053.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the ketolase NS053:BKT from Nodularia spumigena CCAUV 01-053 into Lycopersicon esculentum took place using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector pS3FNRNS053, the 2.4 Kb KpnI fragment from pJFNRS053 was ligated to the KpnI cut vector pSUN3. This clone is called MSP7.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the expression vector with the ketolase NS053:BKT from Nodularia spumigena CCAUV 01-053 in Tagetes erecta took place using the binary vector pSUN5 (WO02/00900).
To prepare the expression vector pS5FNRNS053, the 2.4 Kb KpnI fragment from pJFNRNS053 was ligated to the KpnI cut vector pSUN5. This clone is called MSP8.
The DNA which codes for the ketolase GV35.87:BKT was amplified by PCR from Gloeobacter violaceus SAG 35.87 (SAG: Sammlung von Algenkulturen Göttingen).
To prepare genomic DNA from a suspension culture of Gloeobacter violaceus SAG 35.87, which was grown in BG 11 medium (1.5 g/l NaNO3, 0.04 g/l K2PO4×3H2O, 0.075 g/l MgSO4×H2O, 0.036 g/l CaCl2×2H2O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na2CO3, 1 ml trace metal mix A5+Co (2.86 g/l H3BO3, 1.81 g/l MnCl2×4H2o, 0.222 g/l ZnSO4×7H2o, 0.39 g/l NaMoO4×2H2o, 0.079 g/l CuSO4×5H2O, 0.0494 g/l Co(NO3)2×6H2O)) at 25° C. with continuous light and constant shaking (150 rpm) for 1 week, the cells were harvested by centrifugation, frozen in liquid nitrogen and powdered in a mortar.
Protocol for DNA isolation from Gloeobacter violaceus SAG 35.87:
The bacterial cells from a 10 ml liquid culture were pelleted by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were then crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris HCl (pH 7.5) and transferred into an Eppendorf reaction vessel (2 ml volume). After addition of 100 μl of proteinase K (concentration: 20 mg/ml), the cell suspension was incubated at 37° C. for 3 hours. The suspension was then extracted with 500 μl of phenol. After centrifugation at 13 000 rpm for 5 minutes, the upper, aqueous phase was transferred into a new 2 ml Eppendorf reaction vessel. The extraction with phenol was repeated 3 times. The DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.6 volume of isopropanol and then washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved by heating to 65° C.
The nucleic acid coding for the ketolase GV35.87:BKT from Gloeobacter violaceus SAG 35.87 was amplified by a polymerase chain reaction (PCR) from Gloeobacter violaceus SAG 35.87 using a sense-specific primer (GVK-F1, SEQ ID No. 73) and an antisense-specific primer (GVK-R1SEQ ID No. 74).
The PCR conditions were as follows:
The PCR for amplification of the DNA which codes for a ketolase protein consisting of the entire primary sequence took place in a 50 ul reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The PCR amplification with SEQ ID No. 73 and SEQ ID No. 74 resulted in a 785 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 75). The amplicon was cloned, using standard methods, into the PCR cloning vector pCR 2.1-TOPO (invitrogen), and the clone pGV35.87 was obtained.
Expression of the ketolase from Gloeobacter violaceus SAG 35.87 in Lycopersicon esculentum and in Tagetes erecta took place under the control of the constitutive promoter FNR (ferredoxin NADPH oxidoreductase) from Arabidopsis thaliana. The expression took place with the pea transit peptide rbcS (Anderson et al. 1986, Biochem J. 240:709-715).
The clone pGV35.87 was used for cloning into the expression vector pJFNR (Example 2). The cloning took place by isolating the 797 Bp SphI fragment from pGV35.87 and ligating into the SphI cut vector pJFNR. The clone which comprises the ketolase from Gloeobacter violaceus SAG 35.87 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is called pJFNRGV35.87.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the ketolase GV35.87:BKT from Gloeobacter violaceus SAG 35.87 into Lycopersicon esculentum took place using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector pS3FNRGV35.87, the 2.4 Kb KpnI fragment (partial KpnI hydrolysis) from pJFNRGV35.87 was ligated to the KpnI cut vector pSUN3. This clone is called MSP9.
Preparation of an expression cassette for Agrobacterium-mediated transformation of the expression vector with the ketolase GV35.87:BKT from Gloeobacter violaceus SAG 35.87 in Tagetes erecta took place using the binary vector pSUN5 (WO02/00900).
To prepare the expression vector pS5FNRGV35.87, the 22.4 Kb KpnI fragment (partial KpnI hydrolysis) from pJFNRGV35.87 was ligated to the KpnI cut vector pSUN5. This clone is called MSP10.
Construction of pMCL-CrtYIBZ/idi/gps took place in three steps via the intermediate stages of pMCL-CrtYIBZ and pMCL-CrtYIBZ/idi. The vector used was the plasmid pMCL200 which is compatible with high copy number vectors (Nakano, Y., Yoshida, Y., Yamashita, Y. and Koga, T.; Construction of a series of pACYC-derived plasmid vectors; Gene 162 (1995), 157-158).
The biosynthesis genes crtY, crtB, crtI and crtZ are derived from the bacterium Erwinia uredovora and were amplified by PCR. Genomic DNA from Erwinia uredovora (DSM 30080) was provided by the preparation service of the Deutsche Sammlung von Microorganismen und Zellkuturen (DSMZ, Brunswick). The PCR reaction was carried out in accordance with the manufacturer's information (Roche, Long Template PCR: Procedure for amplification of 5-20 kb targets with the expand long template PCR system). The PCR conditions for amplifying the Erwinia uredovora biosynthesis cluster were as follows:
Master Mix 1:
Master Mix 2:
The two mixtures “Master Mix 1” and “Master Mix 2” were pipetted together. The PCR was carried out in a total volume of 50 ul under the following cycle conditions:
The PCR amplification with SEQ ID No. 77 and SEQ ID No. 78 resulted in a fragment (SEQ ID NO. 79) which codes for the genes CrtY (protein: SEQ ID NO. 80), CrtI (protein: SEQ ID NO. 81), crtB (protein: SEQ ID NO. 82) and CrtZ (iDNA). The amplicon was cloned into the PCR cloning vector pCR2.1 (Invitrogen) by using standard methods, and the clone pCR2.1-CrtYIBZ was obtained.
The plasmid pCR2.1-CrtYIBZ was SalI and HindIII cut, and the resulting SalI/HindIII fragment was isolated and transferred by ligation into the SalI/HindIII cut vector pMCL200. The SalI/HindIII fragment from pCR2.1-CrtYIBZ cloned into pMCL 200 is 4624 Bp long, codes for the CrtY, CrtI, crtB and CrtZ genes and corresponds to the sequence from position 2295 to 6918 in D90087 (SEQ ID No. 79). The CrtZ gene is transcribed by means of its endogenous promoter contrary to the direction of reading of the CrtY, CrtI and CrtB genes. The resulting clone is called pMCL-CrtYIBZ.
The gene idi (isopentenyl-diphosphate isomerase; IPP isomerase) was amplified from E. coli by PCR. The nucleic acid encoding the entire idi gene with idi promoter and ribosome binding site was amplified from E. coli by a polymerase chain reaction (PCR) using a sense-specific primer (5′-idi SEQ ID No. 81) and an antisense-specific primer (3′-idi SEQ ID No. 82).
The PCR conditions were as follows:
The PCR for amplifying the DNA took place in a 50 μl reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The PCR amplification with SEQ ID No. 81 and SEQ ID No. 82 resulted in a 679 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 83). The amplicon was cloned into the PCR cloning vector pCR2.1 (Invitrogen) using standard methods, and the clone pCR2.1-idi was obtained.
Sequencing of the clone pCR2.1-idi confirmed a sequence which does not differ from the published sequence AE000372 in position 8774 to position 9440. This region comprises the promoter region, the potential ribosome binding site and the entire open reading frame for the IPP isomerase. The fragment cloned into pCR2.1-idi has, owing to the insertion of an XhoI cleavage site at the 5′ end and of a SalI cleavage site at the 3′ end of the idi gene, a total length of 679 Bp.
This clone was therefore used to clone the idi gene into the vector pMCL-CrtYIBZ. The cloning took place by isolating the XhoI/SalI fragmente from pCR2.1-idi. and ligating into the XhoI/SalI cut vector pMCL-CrtYIBZ. The resulting clone is called pMCL-CrtYIBZ/idi.
The gene gps (geranylgeranyl-pyrophosphate synthase; GGPP synthase) was amplified from Archaeoglobus fulgidus by PCR. The nucleic acid encoding gps from Archaeoglobus fulgidus was amplified by a polymerase chain reaction (PCR) using a sense-specific primer (5′-gps SEQ ID No. 85) and an antisense-specific primer (3′-gps SEQ ID No. 86).
The DNA from Archaeoglobus fulgidus was provided by the preparation service of the Deutsche Sammiung von Microorganismen und Zellkulturen (DSMZ, Brunswick). The PCR conditions were as follows:
The PCR for amplifying the DNA which codes for a GGPP synthase protein consisting of the entire primary sequence took place in a 50 μl reaction mixture which comprised:
The PCR was carried out under the following cycle conditions:
The DNA fragment amplified by PCR and the primers SEQ ID No. 85 and SEQ ID No. 86 was eluted by methods known per se from the agarose gel and cut with the restriction enzymes NcoI and HindIII. This resulted in a 962 Bp fragment which codes for a protein consisting of the entire primary sequence (SEQ ID No. 87). The NcoI/HindIII cut amplicon was cloned by using standard methods into the vector pCB97-30, and the clone pCB-gps was obtained.
Sequencing the clone pCB-gps confirmed a sequence for the GGPP synthase from A. fulgidus which differs from the published sequence AF120272 in one nucleotide. The second codon of the GGPP synthase was altered by the insertion of an NcoI cleavage site in the gps gene. In the published sequence AF120272, CTG (position 4-6) codes for leucine. The amplification with the two primers SEQ ID No. 85 and SEQ ID No. 86 changed this second codon to GTG, which codes for valine.
The clone pCB-gps was therefore used for cloning the gps gene into the vector pMCL-CrtYIBZ/idi. The cloning took place by isolating the KpnI/XhoI fragment from pCB-gps and ligating into the KpnI and XhoI cut vector pMCL-CrtYIBZ/idi. The cloned KpnI/XhoI fragment harbors the Prrn16 promoter together with a minimal 5′-UTR sequence of rbcL, the first 6 codons of rbcL which extend the GGPP synthase N-terminally, and 3′ of the gps gene the psbA sequence. The N terminus of the GGPP synthase thus has instead of the natural amino acid sequence with Met-Leu-Lys-Glu (amino acid 1 to 4 from AF120272) the altered amino acid sequence Met-Thr-Pro-Gln-Thr-Ala-Met-Val-Lys-Glu. The result of this is that the recombinant GGPP synthase is identical starting with Lys in position 3 (in AF120272) and shows no further alterations in the amino acid sequence. The rbcL and psbA sequences were used in accordance with a reference by Eibl et al. (Plant J. 19. (1999), 1-13). The resulting clone is called pMCL-CrtYI BZ/idi/gps.
Biotransformation of Zeaxanthin in Recombinant E. coli Strains
For the zeaxanthin biotransformation, recombinant E. coli strains which are able, through heterologous complementation, to produce zeaxanthin were prepared. Strains of E. coli TOP10 were used as host cells for the complementation experiments with the plasmids i) pNP60.79:BKT or ii) pNP71.79:BKT or iii) pNS037:BKT and pMCL-CrtYIBZ/idi/gps as second plasmid.
In order to prepare E. coli strains which make it possible to synthesize zeaxanthin in high concentration, the plasmid pMCL-CrtYIBZ/idi/gps was constructed. The plasmid harbors the biosynthesis genes crtY, crtB, crtI and crtY from Erwinia uredovora, the gene gps (for geranylgeranyl-pyrophoshate synthastase) from Archaeoglobus fulgidus and the gene idi (isopentenyl-diphosphate isomerase) from E. coli. This construct was used to eliminate limiting steps for high accumulation of carotenoids and their biosynthetic precursors. This has been described previously by Wang et al. in a similar manner with a plurality of plasmids (Wang, C.-W., Oh, M.-K. and Liao, J. C.; Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli, Biotechnology and Bioengineering 62 (1999), 235-241).
Cultures of E. coli TOP10 were transformed in a manner known per se with the plasmids pMCL-CrtYIBZ/idi/gps and i) pNP60.79:BKT, or ii) pNP71.79:BKT or iii) pNS037:BKT and cultivated in LB medium at 30° C. or 37° C. overnight. Ampicillin (50 μg/ml), chloramphenicol (50 μg/ml) and isopropyl α-thiogalactoside (1 mmol) were added in a manner which is usual per se, likewise overnight The E. coli cultures thus harbored in each case a low copy number and a high copy number plasmid.
Carotenoids were isolated from the recombinant strains by extracting the cells with acetone, evaporating the organic solvent to dryness and fractionating the carotenoids by HPLC on a C30 column. The following process conditions were set.
Separating column: Prontosil C30 column, 250×4,6 mm, (Bischoff, Leonberg)
Flow rate: 1.0 ml/min
The spectra were determined directly from the eluted peaks using a photodiode array detector. The isolated substances were identified from their absorption spectra and their retention times by comparison with standard samples.
The ketolase from Nostoc punctiforme 71.79 was expressed using the plasmid pNP71.79:BKT, the ketolase from Nostoc punctiforme 60.79 was expressed using the plasmid pNP60.79:BKT and the ketolase from Nodularia spumigena was expressed using pNS037:BKT. Determinations at the wavelength 600 nm before extraction of the carotenoids showed a total cell count of 6.1×109 for E. coli/pNP71.79:BKT, a total cell count of 6.3×109 for E. coli/pNP60.79:BKT and a total cell count of 6.2×109 for E. coli/pNS037:BKT.
Table 1 shows a comparison of the amounts of bacterially produced carotenoids:
E: coli
The total carotenoid concentration after incubation for about 18 hours was about ⅓ higher in E. coli/pNP60.79:BKT than in E. coli/pNP71.79:BKT. The amount of ketocarotenoids (canthaxanthin, adonirubin, adonixanthin and astaxanthin) was 1966 ng in E. coli/pNP60.79:BKT, which was distinctly higher than the 284 ng in E. coli/pNP71.79:BKT, the cell count being the same. The proportion of ketocarotenoids is 58% in E. coli/pNP60.79:BKT and only 13% in E. coli/pNP71.79:BKT, in each case based on the total carotenoid content. The proportion of the ketocarotenoids is 56% in E. coli/pNsO37:BKT based on the total carotenoid content.
Tomato plants were transformed and regenerated by the published method of Ling and coworkers (Plant Cell Reports (1998), 17:843-847). A higher kanamycin concentration was used to select the Microtom variety (100 mg/L).
Cotyledons and hypocotyls of seven- to ten-day old seedlings of the Microtom line were used as initial explant for the transformation. The culture medium of Murashige and Skoog (1962: Murashige and Skoog, 19,62, Physiol. Plant 15, 473-) with 2% sucrose, pH 6.1, was used for germination. The germination took place at 21° C. with a low light level (20-100 μE). After seven to ten days, the cotyledons were divided transversely, and the hypocotyls were cut into segments about 5-10 mm long and placed on the MSBN medium (MS, pH 6.1, 3% sucrose+1 mg/l BAP, 0.1 mg/l NM) which were charged the preceding day with suspension-cultured tomato cells. The tomato cells were covered, free of air bubbles, with sterile filter paper. The preculture of the explants on the described medium took place for three to five days. Cells of the Agrobacterium tumefaciens LBA4404 strain were transformed singly with the plasmids pS3FNRNP60.79, pS3FNRNP71.79, pS3FNRNS037, pS3FNRNS053, pS3FNRGV35.87. Each of the individual agrobacterium strains transformed with the binary vectors pS3FNRNP60.79, pS3FNRNP71.79, pS3FNRNS037, pS3FNRNS053, pS3FNRGV35.87 was cultivated in an overnight culture in YEB medium with kanamycin (20 mg/l) at 28° C., and the cells were centrifuged. The bacterial pellet was resuspended with liquid MS medium (3% sucrose, pH 6,1) and adjusted to an optical density of 0.3 (at 600 nm). The precultured explants were transferred into the suspension and incubated at room temperature with gentle shaking for 30 minutes. The explants were then dried with sterile filter paper and returned to their preculture medium for the three-day coculture (21° C.).
After the coculture, the explants were transferred to MSZ2 medium (MS pH 6.1+3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160 mg/l Timentin) and stored under low light conditions (20-100 gE, 16 h/8 h light rhythm) at 21° C. for the selective regeneration. The explants were transferred every two to three weeks until shoots formed. Small shoots could be detached from the explant and rooted on MS (pH 6.1+3% sucrose) 160 mg/l Timentin, 30 mg/l kanamycin, 0.1 mg/l IAA. Rooted plants were transferred into a glasshouse.
The following lines were obtained by the transformation method described above using the following expression constructs:
pS3FNRNP60.79 resulted in: MSP1-1, MSP1-2, MSP1-3
pS3FNRNP71.79 resulted in: MSP3-1, MSP3-2, MSP3-3
pS3FNRNS037 resulted in: MSP5-1, MSP5-2, MSP5-3
pS3FNRNS053 resulted in: MSP7-1, MSP7-2, MSP7-3
pS3FNRGV35.87 resulted in: MSP9-1, MSP9-2, MSP9-3
Production of Transgenic Tagetes Plants
Tagetes seeds are sterilized and placed on germination medium (MS medium; Murashige and Skoog, Physiol. Plant. 15 (1962), 473-497) pH 5.8, 2% sucrose). The germination takes place in a temperature/light/time interval of 18-28° C./20-200 μE/3-16 weeks, but preferably at 21° C., 20-70 μE, for 4-8 weeks.
All the leaves of the in vitro plants which have developed by then are harvested and cut transverse to the midrib. The leaf explants produced thereby with a size of 10-60 mm2 are stored during the preparation in liquid MS medium at room temperature for a maximum of 2 h.
Any Agrobacterium tumefaciens strain, but preferably a supervirulent strain, such as, for example, EHA105 with an appropriate binary plasmid which may harbor a selection marker gene (preferably bar or pat) and one or more trait genes or reporter genes is (pS5FNRNP60.79, pS5FNRNP71.79, pS5FNRNS037, pS5FNRNS053, pS5FNRGV35.87) cultured overnight and used for coculturing with the leaf material. The culturing of the bacterial strain can take place as follows: a single colony of the appropriate strain is inoculated in YEB (0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.5% magnesium sulfate×7H2O) with 25 mg/l kanamycin and cultured at 28° C. for 16 to 20 h. The bacterial suspension is then harvested by centrifugation at 6000 g for 10 min and resuspended in liquid MS medium in such a way that an OD600 of about 0.1 to 0.8 result. This suspension is used for the coculturing with the leaf material.
Immediately before the cocultivation, the MS medium in which the leaves have been stored is replaced by the suspension of bacteria. Incubation of the leaflets in the suspension of agrobacteria took place at room temperature with gentle shaking for 30 min. The infected explants are then placed on MS medium which has been solidified with agar e.g. 0.8% plant agar (Duchefa, N L) and comprises growth regulators such as, for example, 3 mg/l benzylaminopurine (BAP) and 1 mg/l indolylacetic acid (IAA). The orientation of the leaves on the medium has no significance. The explants are cultivated for 1 to 8 days, but preferably for 6 days, using the following conditions in this case: light intensity: 30-80 μMol/m2×sec, temperature: 22-24° C., 16/8 hours light/dark alternation. The cocultivated explants are then transferred to fresh MS medium, preferably comprising the same growth regulators, this second medium additionally comprising an antibiotic to suppress bacterial growth. Timentin in a concentration of from 200 to 500 mg/l is very suitable for this purpose. The second selective component employed is one to select for successful transformation. Phosphinothricin in a concentration of from 1 to 5 mg/l selects very efficiently, but other selective components are conceivable according to the process used.
After from one to three weeks in each case, the explants are transferred to fresh medium until shoot buds and small shoots develop, which are then transferred to the same basal medium including timentin and PPT or alternative components with growth regulators, namely, for example, 0.5 mg/l indolylbutyric acid (IBA) and 0.5 mg/l gibberillic acid GA3, for rooting. Rooted shoots can be transferred to a glasshouse.
In addition to the method described, the following advantageous modifications are possible:
Before the explants are infected with the bacteria, they can be preincubated for from 1 to 12 days, preferably 3-4, on the medium described above for the coculture. This is followed by infection, coculture and selective regeneration as described above.
The pH for the regeneration (normally 5.8) can be reduced to pH 5.2. This improves control of the growth of agrobacteria.
Addition of AgNO3 (3-10 mg/l) to the regeneration medium improves the condition of the culture, including the regeneration itself.
Components which reduce phenol formation and are known to the skilled worker, such as, for example, citric acid, ascorbic acid, PVP and many others, have a positive effect on the culture.
Liquid culture medium can also be used for the overall process. The culture can also be incubated on commercially available supports which are positioned on the liquid medium.
The following lines were obtained by the transformation method described above using the following expression constructs:
pS5FNRNP60.79 resulted in: MSP2-1, MSP2-2, MSP2-3
pS5FNRNP71.79 resulted in: MSP4-1, MSP4-2, MSP4-3
pS5FNRNS037 resulted in: MSP6-1, MSP6-2, MSP6-3
pS5FNRNS053 resulted in: MSP8-1, MSP8-2, MSP8-3
pS5FNRGV35.87 resulted in: MSP10-1, MSP10-2, MSP10-3
a) Ground plant material (e.g. petal material) (30-100 mg fresh weight) is extracted with 100% acetone (three times 500 μl; shake for about 15 minutes each time). The solvent is evaporated. Carotenoids are then taken up in 495 μl of acetone, 4.95 ml of potassium phosphate buffer (100 mM, pH7.4) are added and thoroughly mixed. This is followed by addition of about 17 mg of bile salts (Sigma) and 149 μl of an NaCl/CaCl2 solution (3M NaCl and 75 mM CaCl2). The suspension is incubated at 37° C. for 30 minutes. For the enzymatic hydrolysis of the carotenoid esters, 595 μl of a lipase solution (50 mg/ml lipase type 7 from Candida rugosa (Sigma)) are added and incubated at 37 C with shaking. After about 21 hours, 595 μl of lipase are again added, and incubation is continued at 37° C. for at least 5 hours. Approximately about 700 mg of Na2SO4 are then dissolved in the solution. After addition of 1800 μl of petroleum ether, the carotenoids are extracted into the organic phase by vigorous mixing. This extraction is repeated until the organic phase remains colorless. The petroleum ether fractions are combined, and the petroleum ether is evaporated. Free carotenoids are taken up in 100-120 μl of acetone. Free carotenoids can be identified by means of HPLC and C30 reverse phase column on the basis of the retention time and UV-VIS spectra.
The bile salts or bile acid salts used are 1:1 mixtures of cholate and deoxycholate.
b) Procedure for workup if only small amounts of carotenoid esters are present in the plant material
Alternatively, the carotenoid esters can be hydrolyzed by Candida rugosa lipase after separation by means of thin-layer chromatography. For this purpose, 50-100 mg of plant material are extracted three times with about 750 μl of acetone. The solvent extract is concentrated in rotary evaporator in vacuo (raised temperatures of 40-50° C. are tolerable). This is followed by addition of 300 μl of petroleum ether:acetone (ratio 5:1) and thorough mixing. Suspended materials are sedimented by centrifugation (1-2 minutes). The upper phase is transferred into a new reaction vessel. The remaining residue is again extracted with 200 μl of petroleum ether:acetone (ratio 5:1), and suspended materials are removed by centrifugation. The two extracts are put together (volume 500 μl) and the solvents are evaporated. The residue is resuspended in 30 μl of petroleum ether:acetone (ratio 5:1) and loaded onto a thin-layer plate (silica gel 60, Merck). If more than loading is necessary for preparative-analytical purposes, several aliquots each with a fresh weight of 50-100 mg should be prepared in the described manner for the separation by thin-layer chromatography. The thin-layer plate is developed in petroleum ether:acetone (ratio 5:1). Carotenoid bands can be identified visually on the basis of their color. Individual carotenoid bands are scraped off and can be pooled for preparative-analytical purposes. The carotenoids are eluted from the silica material with acetone; the solvent is evaporated in vacuo. To hydrolyze the carotenoid esters, the residue is dissolved in 495 μl of acetone, and 17 mg of bile salts (Sigma), 4.95 ml of 0.1M potassium phosphate buffer (pH 7.4) and 149 μl (3M NaCl, 75 mM CaCl2) are added. After thorough mixing, the mixture is equilibrated at 37° C. for 30 min. This is followed by addition of 595 μl of Candida rugosa lipase (Sigma, stock solution of 50 mg/ml in 5 mM CaCl2). Incubation with lipase takes place overnight with shaking at 37° C. After about 21 hours, the same amount of lipase is again added; incubation is continued with shaking at 37° C. for at least 5 hours. This is followed by addition of 700 mg of Na2SO4 (anhydrous); extraction is carried out with 1800 μl petroleum ether for about 1 minute, and the mixture is centrifuged at 3500 revolutions/minute for 5 minutes. The upper phase is transferred into a new reaction vessel, and the extraction is continued until the upper phase is colorless. The combined petroleum ether phase is concentrated in vacuo (temperatures of 40-50° C. are possible). The residue is dissolved in 120 μl of acetone, possibly using ultrasound. The dissolved carotenoids can be separated by HPLC using a C30 column and quantified by means of reference substances.
The samples obtained by the procedures in Example 15 are analyzed under the following conditions:
The following HPLC conditions were set.
Separating column: Prontosil C30 column, 250×4,6 mm, (Bischoff, Leonberg, Germany)
Flow rate: 1.0 ml/min
Gradient profile:
Some typical retention times for carotenoids produced according to the invention are, for example:
violaxanthin 11.7 min, astaxanthin 17.7 min, adonixanthin 19 min, adonirubin 19.9 min, zeaxanthin 21 min.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP03/09101 | Aug 2003 | EP | regional |
| PCT/EP03/09102 | Aug 2003 | EP | regional |
| PCT/EP03/09105 | Aug 2003 | EP | regional |
| PCT/EP03/09106 | Aug 2003 | EP | regional |
| PCT/EP03/09107 | Aug 2003 | EP | regional |
| PCT/EP03/09109 | Aug 2003 | EP | regional |
| 102004007624.3 | Feb 2004 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/08625 | 7/31/2004 | WO | 00 | 2/17/2006 |
