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IMPROVED METHOD FOR THE PRODUCTION OF ISOPRENOIDS

20230383321 · 2023-11-30

    Inventors

    Cpc classification

    International classification

    Abstract

    Disclosed herein is a method for the production of isopentenyl diphosphate, dimethylallyl diphosphate and/or isoprenoids derived therefrom.

    Claims

    1. An isolated kinase 1 capable of catalysing a reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate or a salt thereof in an aqueous medium comprising water, kinase 1 and isoprenol and/or prenol, wherein after incubation at least 10% of the isoprenol and/or prenol have been converted to isopentenylphosphate or a salt thereof and/or dimethylallyl phosphate or a salt thereof

    2. The isolated kinase 1 of claim 1 wherein after incubation at least 20% of the isoprenol and/or prenol have been converted to isopentenylphosphate or a salt thereof and/or dimethylallyl phosphate or a salt thereof.

    3. The isolated kinase 1 of claim 1 comprising a sequence selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium.

    4. (canceled)

    5. A process for producing isopentenylphosphate and/or dimethylallyl phosphate or salt thereof comprising the steps of i. Providing an aqueous medium comprising water, one or more kinase 1 and prenol and/or isoprenol, ii. Incubating the aqueous medium and iii. Optionally isolating the isopentenylphosphate and/or dimethylallyl phosphate or salt thereof from the reaction mixture, wherein the one or more kinase 1 is capable of catalyzing the reaction from prenol and/or isoprenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium comprising water, kinase 1 and prenol and/or isoprenol, wherein after incubation at least 10% of the isoprenol and/or prenol have been converted to isopentenylphosphate or a salt thereof and/or dimethylallyl phosphate or a salt thereof

    6. The process according to claim 5 wherein after incubation at least 20% of the isoprenol and/or prenol have been converted to isopentenylphosphate or a salt thereof and/or dimethylallyl phosphate or a salt thereof

    7. The process according to claim 5 wherein the kinase 1 a sequence selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393 or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium.

    8. (canceled)

    9. A recombinant construct comprising a kinase 1 wherein the kinase 1 comprises a sequence encoding an amino acid molecule selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium.

    10. The recombinant construct of claim 9, wherein the kinase 1 is functionally linked to a heterologous promoter.

    11. A recombinant vector comprising the recombinant construct of claim 9.

    12. A recombinant microorganism comprising the recombinant construct of claim 9.

    13. The recombinant microorganism of claim 12 wherein the microorganism is Rhodococcus rhodochrous, Aerococcus sp., Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, Escherichia coli, Haloferax volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis, Streptomyces cacaoi, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum, Vibrio natrigens or Yarrowia lipolytica.

    14. A composition comprising water, a kinase 1, isopentenylphosphate and/or dimethylallyl phosphate wherein the kinase 1 comprises a sequence selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium.

    15. A process for producing isopentenylpyrophosphate and/or dimethylallyl pyrophosphate or salt thereof comprising the steps of i. Providing an aqueous medium comprising water, one or more kinase 1, one or more kinase 2 and prenol and/or isoprenol, ii. Incubating the aqueous medium and iii. Optionally isolating the isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate or salt thereof from the reaction mixture, wherein the one or more kinase 1 is capable of catalysing the reaction from prenol and/or isoprenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium comprising water, kinase 1 and prenol and/or isoprenol, and wherein the one or more kinase 2 is capable of catalysing the reaction from isopentenylphosphate and/or dimethylallyl phosphate to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate in an aqueous medium comprising water, kinase 2 and isopentenylphosphate and/or dimethylallyl phosphate, wherein after incubation at least 10% of the isoprenol and/or prenol have been converted to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate.

    16. The process according to claim 15 wherein after incubation at least 20% of the isoprenol and/or prenol have been converted to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate.

    17. The process according to claim 15 wherein the kinase comprises a sequence selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl phosphate in an aqueous medium.

    18. The process according to claim 17 wherein the kinase 2 comprises a sequence selected from the group consisting of f. The amino acid molecule of SEQ ID NO: 76, 85, 88,103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483, g. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 76, 85, 88, 103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483 or a functional fragment thereof, h. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, i. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, and j. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, wherein the amino acid molecule as defined in g., h., i. and j. is capable of catalysing the reaction from isopentenylphosphate and/or dimethylallyl phosphate to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate in an aqueous medium.

    19. (canceled)

    20. (canceled)

    21. (canceled)

    22. (canceled)

    23. (canceled)

    24. (canceled)

    25. (canceled)

    26. (canceled)

    27. (canceled)

    28. A recombinant microorganism comprising an introduced, increased or enhanced activity and/or expression of one or more kinase 1, one or more kinase 2 and optionally one or more pathways capable of producing one or more isoprenoids, wherein the one or more kinase 1 is capable of catalysing the reaction from prenol and/or isoprenol to isopentenylphosphate and/or dimethylallyl phosphate, and wherein the one or more kinase 2 is capable of catalysing the reaction from isopentenylphosphate and/or dimethylallyl phosphate to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate, and wherein the kinase 1 comprises a sequence selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393 b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393 or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl.

    29. The composition of claim 28 wherein the kinase 2 comprises a sequence selected from the group consisting of f. The amino acid molecule of SEQ ID NO: 76, 85, 88, 103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483, g. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 76, 85, 88, 103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483 or a functional fragment thereof, h. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, i. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, and j. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, and wherein the amino acid molecule as defined in g., h., i. and j. is capable of catalysing the reaction from isopentenylphosphate and/or dimethylallyl phosphate to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate.

    30. A method for fermentative production of one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate or salt thereof comprising the steps of i. Providing a recombinant microorganism of claim 28, and ii. Culturing said microorganism in a medium comprising prenol and/or isoprenol under conditions that allow for the production of said one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate or salt thereof and optionally isolating said one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate or salt thereof from the medium.

    31. (canceled)

    32. (canceled)

    33. A method for producing a recombinant microorganism of claim 28 comprising the steps of: (I) introducing, increasing or enhancing the activity and/or expression of a kinase 1 gene encoding a kinase 1 enzyme having a prenol and/or isoprenol phosphorylating activity in said microorganism; (II) introducing, increasing or enhancing the activity and/or expression of a kinase 2 gene encoding a kinase 2 enzyme having an isopentenylphosphate and/or dimethylallyl phosphate phosphorylating activity in said microorganism; and optionally (III) further introducing, increasing or enhancing the activity and/or expression of an isoprenoid producing pathway in said microorganism.

    34. The microorganism of claim 28, wherein the microorganism is Rhodococcus rhodochrous, Aerococcus sp., Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, Escherichia coli, Haloferax volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis, Streptomyces cacaoi, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum, Vibrio natrigens or Yarrowia lipolytica.

    35. A recombinant expression construct comprising i. a promoter functional in a microorganism functionally linked to a nucleic acid molecule encoding a kinase 1 and ii. a promoter functional in a microorganism functionally linked to a nucleic acid molecule encoding kinase 2, wherein at least one of the promoters functionally linked to the kinase 1 or kinase 2 is heterologous to the kinase 1 or kinase 2, wherein the kinase 1 comprises a sequence encoding an amino acid molecule selected from the group consisting of a. The amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393, b. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 43, 46, 49, 52, 123, 126, 135, 138, 144, 150, 153, 324, 351, 357, 375, 378, 390, or 393 or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 44, 45, 47, 48, 50, 51, 53, 54, 124, 127, 136, 139, 145, 151, 154, 325, 358, 379, 394, 125, 128, 137, 140, 146, 152, 155, 326, 353, 359, 377, 380, 392, or 395 or a functional fragment thereof, and wherein the amino acid molecule as defined in b., c., d. and e. is capable of catalysing the reaction from isoprenol and/or prenol to isopentenylphosphate and/or dimethylallyl.

    36. The recombinant expression construct of claim 35 wherein the kinase 2 comprises comprising a sequence selected from the group consisting of f. The amino acid molecule of SEQ ID NO: 76, 85, 88, 103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483, g. An amino acid molecule having at least 50% identity to the amino acid molecule of SEQ ID NO: 76, 85, 88, 103, 138, 171, 174, 177, 180, 183, 189, 192, 195, 198, 201, 204, 207, 210, 216, 219, 222, 225, 228, 231, 234, 237, 240, 246, 249, 252, 255, 258, 261, 270, 273, 279, 282, 288, 426, 429, 435, 447, or 483 or a functional fragment thereof, h. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, i. An amino acid molecule encoded by a nucleic acid molecule having at least 50% identity to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, and j. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 77, 78, 86, 87, 89, 90, 104, 105, 139, 172, 175, 178, 181, 184, 190, 193, 196, 199, 202, 205, 208, 211, 217, 220, 223, 226, 229, 232, 235, 238, 241, 247, 250, 253, 256, 259, 262, 271, 274, 280, 283, 289, 430, 448, 484, 140, 173, 176, 179, 182, 185, 191, 194, 197, 200, 203, 206, 209, 212, 218, 221, 224, 227, 230, 233, 236, 239, 242, 248, 251, 254, 257, 260, 263, 272, 275, 281, 284, 290, 428, 431, 437, 449, or 485 or a functional fragment thereof, and wherein the amino acid molecule as defined in g., h., i. and j. is capable of catalysing the reaction from isopentenylphosphate and/or dimethylallyl phosphate to isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate.

    37. A recombinant vector comprising the recombinant expression construct of claim 35.

    38. A recombinant microorganism comprising the recombinant expression construct of claim 35.

    39. A method of culturing or growing the recombinant microorganism of claim 28 comprising inoculating a culture medium with one or more of said recombinant microorganisms and culturing or growing said recombinant microorganism in culture medium comprising prenol and/or isoprenol.

    40. (canceled)

    41. A process for whole cell bio-conversion of prenol and/or isoprenol to one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate comprising the steps of I) growing the recombinant microorganism according to claim 28 in a fermenter comprising isoprenol and/or prenol, a medium suitable for growing said recombinant microorganism and a C-source and II) recovering the one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate thereof from the fermentation broth obtained in I).

    42. A process for whole cell bio-conversion of prenol and/or isoprenol to one or more isoprenoid or salt thereof or isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate comprising the steps of i) growing the recombinant microorganism according to claim 28 in a fermenter comprising a medium suitable for growing said recombinant microorganism and a C-source, ii) recovering the recombinant microorganism from the fermenter, iii) performing a whole cell bio-conversion in an aqueous medium by supplementing isoprenol/prenol, and iv) recovering the one or more isoprenoid or salt thereof or IPP and/or DMAPP from the aqueous medium obtained in iii).

    Description

    FIGURES

    [0334] FIG. 1 shows reactions catalysed by kinase 1. (A) Synthesis of isopentenyl phosphate (IP) from isoprenol and (B) Synthesis of Dimethylallyl phosphate (DMAP) from prenol.

    [0335] FIG. 2 shows the screening results for kinase 1. Enzyme activity was monitored by measuring IP formation in cleared E. coli cell lysates supplemented with isoprenol in the presence of ATP and MgC12.

    [0336] FIG. 3 shows reactions catalysed by kinase 2. (A) Synthesis of isopentenyl diphosphate (IPP) from IP and (B) Synthesis of Dimethylallyl diphosphate (DMAPP) from DMAP.

    [0337] FIG. 4 shows the screening results for kinase 2. Enzyme activity was monitored by measuring IPP formation in cleared E. coli cell lysates supplemented with IP in the presence of ATP and MgCl.sub.2.

    [0338] FIG. 5 shows the cascade reaction of kinase 1 and 2. (A) Synthesis of IPP from isoprenol and (B) Synthesis of DMAPP from prenol.

    [0339] FIG. 6 shows an example of the cascade reaction of kinase 1 (SEQ ID NO 43) and kinase 2 converting isoprenol to IPP. Enzyme activity was monitored by measuring IPP formation in cleared E. coli cell lysates supplemented with isoprenol in the presence of ATP and MgC12.

    [0340] FIG. 7 shows the screening results for kinase 1. Enzyme activity was monitored by measuring IP formation in cleared E. coli cell lysates supplemented with isoprenol in the presence of ATP and MgCl.sub.2.

    [0341] FIG. 8 shows the screening results for kinase 2. Enzyme activity was monitored by measuring IPP formation in cleared E. coli cell lysates supplemented with IP in the presence of ATP and MgCl.sub.2.

    [0342] FIG. 9 shows the cellular (in vivo) production of IP over time. Depicted are the concentrations of IP normalized to the OD600 of the culture. Values are the result of three independent measurements and error bars indicate the standard deviation.

    [0343] FIG. 10 shows the cellular (in vivo) production of IPP over time. Depicted are the concentrations of IPP normalized to the OD600 of the culture. Values are the result of three independent measurements and error bars indicate the standard deviation.

    EXAMPLES

    Chemicals and Common Methods

    [0344] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, Ligation of nucleic acids, transformation, selection and cultivation of bacterial cells were performed as described (Sambrook et al., 1989). Sequence analyses of recombinant DNA were performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, WI, USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, CA, USA). Restriction endonucleases were from New England Biolabs (Ipswich, MA, USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides were synthesized by Eurofins Eurofins Genomics (Ebersberg, Germany) or Integrated DNA Technologies (Coralville, IA, USA).

    Example 1

    Cloning of Kinases

    [0345] The amino acid sequences of the kinases were identified from public databases. The respective DNA sequences were derived thereof using standard codon usage of Escherichia coli. The DNA sequences were synthesized (BioCat GmbH) and cloned into the plasmid pDHE19.2 (Ress-Loeschke, M. et al., DE 19848129, 1998, (BASF AG)). The resulting plasmids were used to transform competent cells (Chung, C. T. et al., Proc Natl Acad Sci U S A, 1989, 86, 2172) of the E. coli strain TG10, pAgro, pHSG575 (E. coli TG10 (Kesseler, M. et al., WO2004050877A1, 2004, (BASF AG)):rhaA.sup.−-derivate of E. coli TG1(DSMZ 6056) transformed with pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397).

    Example 2

    Recombinant Production of Kinases

    [0346] E. coli TG10 carrying the recombinant plasmids of the kinases were used to inoculate 25 ml LB medium (Bertani, G., J Bacteriol, 1951, 62, 293) supplemented with 100 μg/ml ampicillin, 100 pg/ml spectinomycin, 20 pg/ml chloramphenicol, 0.1 mM isopropyl-β-D-thiogalactopyranosid, and 0.5 g/ml rhamnose in a 100 ml baffled Erlenmeyer-flask. The culture was incubated at 37° C. for 18 h under shaking conditions. Subsequently, the biomass was harvested by centrifugation at 5000×g for 10 min. Cells were washed in buffer (50 mM TRIS*HCl, 1 mM MgCl.sub.2 at pH 7.0) before being resuspended in 1 ml of the same buffer.

    [0347] The cell free cleared raw lysates were prepared by breaking 300 mg biomass with 0.7 ml quartz-beads (∅0.1 mm) in a homogenizer (Peqlab Precellys24, VWR) for two 30 second cycles. In between cycles samples were chilled on ice. The resulting cell free lysates were cleared by centrifugation 20817×g at 10° C. The supernatant (=cleared cell lysate) was isolated and it commonly contained 10 to 15 mg/ml of protein.

    Example 3

    Enzyme Screening

    [0348] Kinase 1, Conversion of Isoprenol to Isopentenyl Phosphate (IP)

    [0349] Enzyme activity was assessed in a screening assay. In that regard, buffer (50 mM NH.sub.4HCO.sub.3 at pH 7.5) was supplemented with 5 mM isoprenol (BASF production), 15 mM ATP, 20 mM MgCl.sub.2. The reactions were initiated by adding 20 vol-% (final) of the cleared cell lysate. Subsequently, reactions were incubated at 37° C., 300 rpm, for 24 hours in a thermomixer (Eppendorf) before being quenched by 1:5 dilution with acetonitrile and vigorous mixing. Quenched reactions were cleared by centrifugation at 14,100×g for 5 min at room temperature and the resulting supernatant was used to quantify the levels of IP and isopentenyl pyrophosphate (IPP) by liquid-chromatography coupled to mass spectrometry (LC-MS).

    [0350] Kinase 2, Conversion of IP to Isopentenyl Pyrophosphate (IPP)

    [0351] Here, 50 mM NH4HCO.sub.3 buffer at pH 7.5 was supplemented with 5 mM IP dilithium salt (Sigma-Aldrich), 15 mM ATP, 20 mM MgCl.sub.2. The reactions were initiated by adding 20 vol-% (final) of the cleared cell lysate. The reactions were treated and analysed as described in the previous paragraph.

    Example 4

    In Vitro Cascade Reaction Kinase 1 and Kinase 2, Conversion of Isoprenol to IPP

    [0352] To assess the potential of converting isoprenol to IPP with a combination of kinase 1 and kinase 2 a cascade reaction was carried out in vitro. In that regard, 50 mM NH.sub.4HCO.sub.3 buffer at pH 7.5 was supplemented with 5 mM isoprenol, 30 mM ATP, and 20 mM MgCl.sub.2. Reactions were initiated by adding 20 vol-% of the cleared cell lysate of kinase 1 and kinase 2 protein production (40 vol-% total). The reactions were treated and analysed as described above. LC-MS quantification indicated successful synthesis of IPP and/or DMAPP

    Example 5

    In Vivo Cascade Reaction Kinase 1 and Kinase 2, Conversion of Isoprenol to IPP

    [0353] Genes of kinases identified as hits in the in vitro screenings were recloned (BioCat GmbH) into the commercially available pCDFDuet1-plasmid system (Novagen) following the logic pCDFDuet1_Kinase2_Kinasel (e.g. SEQ ID NO 486). Resulting plasmids were used to transform E. coli BL21-Gold(DE3) (Agilent). A single colony of the transformants was transferred into a 12 ml reaction tube containing 4 ml LB medium supplemented with 100 pg/ml spectinomycin dihydrochloride (Sigma Aldrich) and incubated overnight at 37° C. at an agitation of 200 rpm. The overnight culture was used to inoculate 100 ml Neidhardt supplemented medium (Clomburg, J. M. et al., Proc Natl Acad Sci USA, 2019, 116(26)), 12810 containing 100 μg/ml spectinomycin dihydrochloride filled into a 500 ml baffled Erlenmeyer flask to a final optical density at 600 nm (0D600) of 0.1. Subsequently, the culture was incubated at 37° C. and 200 rpm for 3 hours before the temperature was lowered to 30° C. and gene expression was induced by the addition of 1 mM IPTG. Isoprenol was added at a final concentration of 26.9 mM and 5 ml samples were taken after 0 (prior to isoprenol addition), 1, 2.5, 5, 22, and 28 hours of incubation. The OD600 of the samples was measured and they were directly further processed by fast filtration (Castano-Cerezo, S. et al., Metabolomics, 2019, 15, 115). In brief, the sample was filtered under reduced pressure through a 0.45 μm polyamide filter membrane (Sartorius) with a diameter of 5 cm fitted on a 1 l filter apparatus (Sartorius). The filter cake and filter were transferred to a 15 ml conical tube and immediately frozen in liquid nitrogen. The filters were stored at −80° C. until being further processed. In that regard, the filter was submerged in a 1:1 mixture of isopropanol and a 50 mM aqueous NH.sub.4HCO.sub.3 solution at pH 7.5 prewarmed to 70° C. The suspension was subsequently incubated for 20 min at 70° C. and an agitation of 1000 rpm on a Thermomixer before being placed on ice and sonicated for 3 min (Branson Sonifier 250, 70%, Output 7). All debris was removed by centrifugation at 4° C. and 1 ml of the resulting supernatant transferred to a fresh 1.5 ml reaction tube. All of the solvent was completely removed from the sample using a SpeedVac vacuum concentrator (Thermo Fisher, Savant, SPD131DDA) at 45° C. overnight. The residue was taken up in 200 μl 1:1 mixture of methanol and 50 mM aqueous NH.sub.4HCO.sub.3 solution at pH 7.5. IP/DMAP and IPP/DMAPP were quantified via liquid chromatography coupled to mass spectrometry. Reported results of IP/DMAP and IPP/DMAPP concentrations are normalized to the respective OD600 of the sample. A culture in which gene expression was not induced and no isoprenol was added served as a negative control. Reported values show the average of three independent measurements

    Example 6

    LC-MS Quantification

    [0354] In Vitro Assay

    [0355] Reactants were quantified on an ultra-high-pressure liquid chromatography (UPLC) system (Vanquish, Thermo-Fisher) coupled to a single-quadrupole mass spectrometer with electron spray ionization (ISQ-EC, Thermo-Fisher). The UPLC system was run in ion pairing chromatography mode. It was equipped with a peptide C-18 column (Waters, ACQUITY peptide BEH, pore size 130 Å, particle size 1.7 μm, inner diameter×length 2.1×50 mm) that was eluted under isocratic conditions with 25 vol-% eluent A (buffer containing 10 mM tributylamine and 15 mM acetic acid) and 75 vol-% eluent B (acetonitrile). The method was run with a flow of 0.5 ml/min for 2.5 min and the column was heated to 40° C. Analytes were detected and quantified via mass spectrometry. In that regard, the instrument was set to negative ionization mode, collision-induced dissociation voltage was set to 40 V, the vaporizer temperature was 282° C. and the temperature of the ion transfer tube 300° C. The sweep, sheath, and aux gas pressures were set to 0.5 psig, 49.9 psig, and 57 psig, respectively. IP eluted at 0.6 min and its appearance could be followed at a mass-to-charge ratio (m/z) 165, while IPP eluted at 1.3 min and its appearance could be followed at m/z 245. The quantification was based on standard curves prepared by analysis of authentic standards of IP dilithium salt and IPP triammonium salt (Sigma-Aldrich). Reaction yields are reported relative to the amount of the respective substrate used in the reaction.

    [0356] In Vivo Assay

    [0357] All measurements were performed on a Thermo Vanquish Flex UHPLC system using an Acquity PREMIER BEH C18, 50×2.1 mm, 1.7 μm dp column (Waters, Germany). Separation of 2.5 μL sample was achieved by a multistep gradient from (A) 10 mM AmFo (Ammonium formate)+10 mM DBA (Dibutylamine) in H.sub.2O to (B) 10 mM AmFo+10 mM DBA in H.sub.2O/ACN (1:9) at a flow rate of 600 pl/min and 45° C. The gradient was initiated by a 0.5 min isocratic step at 2% B, followed by an increase to 60% B in 5.5 min, followed by an increase to 100% B in 0.5 min to end up with a 1 min step at 100% B before reequilibration at initial conditions. UV spectra were recorded by a DAD (Diode Array Detector) in the range from 200 to 600 nm. The LC flow was split to approx. 75 μL/min before entering the maXis II hr-ToF mass spectrometer (Bruker Daltonics, Germany) using the Apollo ESI source in negative mode. In the source region, the temperature was set to 200° C., the capillary voltage was 3200 V, the dry-gas flow was 5.0 L/min and the nebulizer was set to 1.0 bar. To transfer the generated ions funnel 1 RF was set to 400 Vpp and the multipole RF to 350 Vpp. Then ions passed the quadrupole with a low cutoff at 110 m/z and an ion energy of 3.0 eV, forwarded to the collision cell which was operated in stepping mode (collision energy=8.0 eV, pre pulse storage=5.0 μs; collision RF=200-800 Vpp; transfer time=75-120 μs; timing=30-70%) before entering the ToF tube. Mass spectra were acquired in the focus mode ranging from 50-650 m/z at a 2.5 Hz scan rate. The quantification was based on standard curves prepared by analysis of authentic standards of IP dilithium salt and IPP triammonium salt.