Fermentative carotenoid production

A process for the preparation of lycopene wherein the process comprises culturing a recombinant cell that has been transformed with an expression vector having a regulatory sequence and a polynucleotide comprising the following DNA sequences:

• a) a DNA sequence which encodes the gerany/granylpyrophosphate (GGPP) synthase of Flavobacterium sp. R1534 (CrtE) or a DNA sequence encoding GGPP synthase activity and which hybridizes to SEQ ID NO:2 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• b) a DNA sequence which encodes the prephycoene synthase of Flavobacterium sp. R1534 (crtB) or a DNA sequence encoding prephytoene synthase activity and which hybridizes to SEQ ID NO:4 under the following hybridization conditions:
• prehybridizaiion and hybridization in 7% SDS, 1% BSA, 0.5M NA2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 miniutes each, and
• c) a DNA sequence which encodes the phytoene desarurase of Flavobacterium sp. R1534 (crtI) or a DNA sequence encoding phytoene desaturase activity and which hybridizes to SEQ ID NO:6 under the following hybridizaton conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room tern-premature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each;
• the polynucleotide being substantially free of other polynucleotides of Flavobacterium sp R1534 in the presence of farnesyl pyrophosphate and isopenryl pyrophosphate under culture conditions sufficient for the expression of enzymes which catalyze the conversion of the farnesyl pyrophosphate and isopentyl pyrophosphate to lycopene, and isolating the lycopene from cells or the culture medium.

A process for the preparation of β-carotiene wherein the process comprises culturing a recombinant cell that has been transformed with an expression vector having a regulatory sequence and a polynucleotide comprising the following DNA sequences:

• a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534 (CrtE) or a DNA sequence encoding GGPP synthase activity and which hybridizes to SEQ ID NO:2 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• b) a DNA sequence which encodes the prephytoene synthase of Flavobacterium sp. R1534 (crtB) or a DNA sequence encoding phytoene synthase activity and which hybridizes to SEID NO:4 under the flowing hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 al 65° C. and washing twice in 2×SSC 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC 0.1% SDS at 65° C. for 15 minutes each,
• c) a DNA sequence which encodes the phytoene desaturase of Flavobacterium sp. R1534 (crtI) or a DNA sequence encoding phytoene desaturase activity and which hybridizes to SEQ ID NO:6 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each, and
• d) a DNA sequence which encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY) or a DNA sequence encoding lycopene cyclase activity and which hybridizes to SEQ ID NO:8 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each;
• the polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534, in the presence of farnesyl pyrophosphate and isopentyl pyrophosphate under culture conditions sufficient for the expression of enzymes which catalyze the conversion of farnesyl pyrophosphate and isopentyl pyrophosphate to β-carotene, and isolating he β-carotene from such cells or the culture medium.

A process for the preparation of echinenone wherein the process comprises culturing a recombinant cell that has been transformed with an expression vector having a regulatory sequence and a polynucleotide comprising the following DNA sequences;

• a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534 (CrtE) or a DNA sequence encoding GGPP synthase activity and which hybridizes to SEQ ID NO:2 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• b) a DNA sequence which encodes the prephytoene synthase of Flavobacterium sp. R1534 (crtB) or a DNA sequence encoding prephytoene synthase activity and which hybridizes to SEQ ID NO:4 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• c) a DNA sequence which encodes the phytoene desaturase of Flavobacterium sp R1534 (crdI) or a DNA sequence encoding phytoene desaturase activity and which hybridizes to SEQ ID NO:6 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.% SDS at 65° C. for 15 minutes each,
• d) a DNA sequence which encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY) or a DNA sequence encoding lycopene cyclase activity and which hybridizes to SEQ ID NO:8 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each, and
• e) a DNA sequence which encodes the β-carotene β4-oxygenase of Alcaligenes strain PC-1 (crtW);
• the polynucleotide being substantially free of other polynucleotides of Flavobacterium sp. R1534, in the presence of farnesyl pyrophosphate and isopentyl pyrophosphate under culture conditions sufficient for the expression of ones which catalyze the conversion of the famesyl pyrophosphate and isopentyl pyrophosphate to echinenone, and isolating the echinenone from such cells or the culture medium.

A process for the preparation of cataxanthin wherein the process comprises culturing a recombinant cell that has been transformed with an expression vector having a regulatory sequence and a polynucleotide comprnsing the following DNA sequences:

• a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534 (CrTE) or a DNA sequence encoding GGPP synthase activity and which hybridizes to SEQ ID NO:2 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing rwice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• b) a DNA sequence which encodes the prephytoene synthase of Flavobacterium sp. R1534 (crtB) or a DNA sequence encoding prephytoene synthase activity and which hybridizes to SEQ ID NO:4 under the following hybridization conditions:
• prehybridizartion and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each folowed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• c) a DNA sequence which encodes the phytoene desatuase of Flavobacteriun sp. R1534 (crtI) or a DNA sequence encoding phytoene desaturase activity and which hybridizes to SEQ ID NO:6 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• d) a DNA sequence which encodes the lycopene cyclase of Flavobacteriumn sp. R1534 (crtY) or a DNA sequence encoding lycolyne cyclase activity and which hybridizes to SEQ ID NO:8 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing, twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0 1% SSC, 0.1% SDS at 65° C. for 15 minutes each, and
• e) a DNA sequence which encodes the β-carotene β4-oxygenase of Alcaligenes srin PC-1 (crtW);
• the polynucleotide being substantially free of other polynucleotides of Flavobaterium sp. R1534, in the presence of farnesyl pyrophosphate and isopenyl pyrophosphate under culture conditions sufficient for the expression of enzymes which catalyze the conversion of the farnesyl pyrophosphate and isopentyl pyrophosphate to cataxanthin, and isolating the cataxanthin from such cells or the culture medium.

A process for the preparation of adonixathin wherein the process comprises culturing a recombinant cell that has been transformed with an expression vector having a regulatory sequence and a polynucleotide comprising the following DNA sequences:

• a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp. R1534 (CrtE) or a DNA sequence encoding GGPP synthase activity and which hybridizes to SEQ ID NO:2 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 mirnutes each,
• b) a DNA sequence which encodes the prephytoene syntase of Flavobacterium sp. R1534 (crtB) or a DNA sequence encoding prephytoene synthase activity and which hybridizes to SEQ ID NO:4 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• c) a DNA sequence which encodes the phytoene desaturase of Flavobacterium sp- R1534 (crtI) or a DNA sequence encoding phytoene desaturase activity and which hybridizs to SEQ ID NO:6 under the following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• d) a DNA sequence which encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY) or a DNA sequence encoding lycopene cyclase activity and which hybridizes to SEQ ID NO:8 under the following hybridization conditions:
• pehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five minutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each,
• e) a DNA sequence which encodes the β-carotene β4-oxygenase of Alcaligenes stain PC-1 (crtW), and
• f) a DNA sequence which encodes the β-carotene hydroxylase of Flavobacteriurn sp. R1534 (crtZ or a DNA seuence encoding β-carotene hydroxylase activity and which hybridizes to SEQ ID NO:10 under tihe following hybridization conditions:
• prehybridization and hybridization in 7% SDS, 1% BSA, 0.5M Na2HPO4, pH 7.2 at 65° C. and washing twice in 2×SSC, 1% SDS at room temperature for five mninutes each followed by two additional washes in 0.1% SSC, 0.1% SDS at 65° C. for 15 minutes each
• the polypeptide being substantially free of other polynaucleotides of Flavobactehum sp. R1534, in the presence of famesyl pyrophosphate and isopentyl pyrophosphate under culture conditions sufficient for the expression of enzymes which catalyze the conversion of the farnesyl pyrophosphate and isopentyl pyrophosphate to adonixanthin, and isolating the adonixanthin from such cells or the culture medium.