WO2011021717A2 - Procédé de production d'acides aminés hydroxylés - Google Patents

Procédé de production d'acides aminés hydroxylés Download PDF

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WO2011021717A2
WO2011021717A2 PCT/JP2010/064323 JP2010064323W WO2011021717A2 WO 2011021717 A2 WO2011021717 A2 WO 2011021717A2 JP 2010064323 W JP2010064323 W JP 2010064323W WO 2011021717 A2 WO2011021717 A2 WO 2011021717A2
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seq
dioxygenase
hydroxylated
amino acid
dna
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PCT/JP2010/064323
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WO2011021717A3 (fr
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Sergey Vasilievich Smirnov
Natalia Nikolaevna Samsonova
Veronika Aleksandrovna Kotliarova
Natalia Yurievna Rushkevich
Ekaterina Aleksandrovna Fedorina
Pavel Mikhailovich Sokolov
Aleksandra Viktorovna Kolokolova
Jun Ogawa
Makoto Hibi
Sakayu Shimizu
Yuki Imabayashi
Shunichi Suzuki
Masakazu Sugiyama
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Ajinomoto Co.,Inc.
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Priority claimed from RU2010107900/10A external-priority patent/RU2460779C2/ru
Application filed by Ajinomoto Co.,Inc. filed Critical Ajinomoto Co.,Inc.
Publication of WO2011021717A2 publication Critical patent/WO2011021717A2/fr
Publication of WO2011021717A3 publication Critical patent/WO2011021717A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for manufacturing hydroxylated amino acids using a bacterium containing DNA encoding dioxygenase.
  • Hydroxylated amino acids may have practical importance.
  • 4-hydroxy-L- isoleucine displays an insulinotropic activity, which is of great interest because of its stimulating effect which is clearly dependent on the plasma glucose concentration in the medium.
  • This effect has been demonstrated both in isolated perfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al, Diabetes, 47: 206—210, (1998)).
  • Such a glucose dependency has not been confirmed for sulfonylurea (Drucker, D.
  • 4-hydroxy-L-isoleucine is only found in plants, and due to its particular insulinotropic action, might be considered a novel secretagogue with potential applications for the treatment of type II diabetes, since this is a disease characterized by defective insulin secretion associated with various degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623, (1999)).
  • Oxidizing iron, ascorbic acid, ⁇ -ketoglutaric acid, and oxygen-dependent isoleucine by utilizing dioxygenase activity in fenugreek extract has been reported as a method for manufacturing 4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997).
  • this method is unsatisfactory for the manufacturing of 4-hydroxy-L-isoleucine because the activity of the enzyme is inhibited by the substrate at isoleucine concentrations of 20 mM and above.
  • the enzyme has not been identified, is derived from plant extracts, is not readily obtained in large quantities, and is unstable.
  • An aspect of present invention is to provide a method for manufacturing hydroxy- L- amino acids by enzymatic conversion using a dioxygenase.
  • microorganisms can produce hydroxylated L-amino acid.
  • dioxygenase is selected from the group consisting of dioxygenases isolated from a bacterium belonging to the genera Escherichia, Pantoea, Agrobacterium, Bordetella,
  • Burkholderia Bacillus, Methylobacillus, Gluconobacter, Pseudomonas, Nostoc.
  • dioxygenase is selected from the group consisting of dioxygenases SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No: 58, SEQ ID No: 60, SEQ ID No: 62, SEQ ID No: 64 or variant thereof.
  • dioxygenase is selected from the group consisting of dioxygenases from a bacterium belonging to the genera Escherichia, Pantoea, Agrobacterium, Bordetella,
  • Burkholderia Bacillus, Methylobacillus, Gluconobacter, Pseudomonas, Nostoc.
  • dioxygenase is selected from the group consisting of dioxygenases SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 14, SEQ ID No: 16, SEQ ID No: 18, SEQ ID No: 20, SEQ ID No: 22, SEQ ID No: 24, SEQ ID No: 26, SEQ ID No: 28, SEQ ID No: 30, SEQ ID No: 54, SEQ ID No: 56, SEQ ID No: 58, SEQ ID No: 60, SEQ ID No: 62, SEQ ID No: 64 or variant thereof.
  • a DNA encoding dioxygenase selected from the group consisting of dioxygenases SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, S
  • microorganism as described above, wherein said microorganism is belonging to genus selected from the group consisting of Escherichia, Pantoea, Pseudomonas, Corynebacterium, Arthrobacter, Aspergillus, Bacillus.
  • Figure 1 shows detection of the X (4'-HIL) and Y(4',4-DHIL) compounds (photograph).
  • Figure 2 shows biosynthesis and purification of the X (4'-HIL) (photograph).
  • Figure 3 shows biosynthesis and purification of 4', 4-DHIL (photograph).
  • Figure 4 shows the isoleucine hydroxy lation cascade catalyzed by HiIA and HiIB.
  • Figure 5 shows purification of HiI A and his6-tag-HilB (photograph).
  • a - SDS-PAGE of protein fractions obtained during purification process 1- Mw marker, 2 - crude cell lysate of the MG1655[pETAC-HilA] strain., 3 - unbounded with DEAE column fraction, 4 - ammonium precipitation step, 5 - active fraction eluted from SEC-column.
  • Figure 6 shows assay of the substrate specificity of the his6-tag-HilB (photograph).
  • Figure 7 shows alignment of homologues of the 4-IDO from Bacillus subtilis.
  • mor-DO-B - hypothetical protein PE36_03361 from Moritella sp. PE36 [gi
  • mme- DO-B - hypothetical protein GOS 1822161 [marine metagenome, gi
  • plu-DO-B - hypothetical protein plul881 from Photorhabdus luminescens subsp. laumondii TTOl
  • Gluconobacter oxydans 621H [gi
  • Figure 8 shows alignment of members of the HilA-dioxygenase family.
  • psyHilA hypothetical protein PSPPH 3987 from Pseudomonas syringae (GI:20804088); paaHilA - ; mme-DO-A - hypothetical protein GOS 9268270 from marine metagenome (GI:135403471); bce-DO-A - putative signal transduction protein from Bacillus cereus subsp. cytotoxis NVH 391-98 (GI: 152974310); bha-DO-A - hypothetical protein BH2933 from
  • Figure 9 shows the structures of the pSlo and pET plasmids harboring synthesized dioxygenase gene (e.g. HT-avi-DO-B).
  • Figure 10 shows activity assay of of the HT-bpe-DO- A (photograph). Enzyme hydroxylated methionine (lanes 1-5) and isoleucine (lanes 6-10). Lanes 3 and 8 - control reactions without Fe 2+ . Reactions 1, 2 and 6, 7 were carried out in HEPES buffer but reactions 4,5 and 9, 10 in 5OmM Tris buffer pH 8. Each reaction was duplicated.
  • Figure 11 shows the novel threonine-4-dioxygenase activity of dioxygenase from Bordetella petrii DSM 12804 strain (photograph).
  • A The novel threonine-4-dioxygenase activity of dioxygenase from Bordetella petrii DSM 12804 strain.
  • B TLC-analysis of the reaction mixture containing 100 mM Hepes pH7, 5mM threonine, 5mM ⁇ -ketoglutarate, purified HT-bpe-DO-B, and 5mM Fe 2+ (track 1). As a control reaction we used the same mixture without Fe 2+ .
  • Figure 12 shows investigation of substrate specificity of gvi-DO-B to several amino acids including Leu and Met (photograph). Full reaction mixtures are designated as +. Control reaction mixtures (without Fe 2+ ) are designated as -. Hydroxylated leucine and methionine are marked by red circle.
  • Figure 13 shows the structure of the pELAC-HT-mfl-DO-B plasmid.
  • Figure 14 shows the structure of the pELAC-IDO(Lys, 23) plasmid.
  • Figure 15 shows scheme of the He ⁇ 4',4-DHIL biotransformation.
  • Figure 16 shows results concerning He— » ⁇ 4',4-DHIL biotransformation process (photograph).
  • TLC was performed using alkali (A) and acidic (B) developer. Lanes: Al, A4, A7, Bl, B2, B7 and BlO - initial reaction mixtures containing 100 mM of He; A2(B2), A3(B3) and A5(B5),
  • A6(B6) - duplicated reactions mixtures A and C after 16h cultivation respectively for reference see Fig.l
  • Figure 17 shows results of purification of the 4',4-DHIL using DOWEX 50 W-X2 (photograph).
  • Figure 18 shows results of partially purification of hLeu (photograph).
  • a - Chromatography 1 fractions 10-12 were pooled;
  • B,C-Chromatography 2 fractions 10-14 were pooled, start - applied preparation.
  • Figure 19 shows results of purification of hVal (photograph). Start - culture broth after biotransformation process; 1 - 6 - chromatographic fractions.
  • Figure 20 shows results of purification of methionine sulfoxide (photograph). Start - culture broth after biotransformation process; 1 - 12 - chromatographic fractions.
  • Figure 21 shows results concerning hydroxylation of threonine using crude cell lysate of
  • Figure 22 shows results of purification of hThr, fractions 9-14 were pooled (photograph).
  • Figure 23 shows a typical HPLC chromatogram of racemic L-methionine sulfoxide derivatized by Marfey's reagent.
  • Figure 24 shows HPLC chromatograms of IDO reaction products after Marfey's reagent treatment.
  • dioxygenases refers to group of enzymes that catalyze the insertion of an oxygen molecule into an organic substrate.
  • Dioxygenase according to the present invention has ⁇ -ketoglutarate dependent and Fe + dependent activity.
  • ⁇ -ketoglutarate-dependent enzyme activity refers to an enzymatic activity to catalyze a reaction coupled with the formation of succinate from ⁇ -ketoglutarate.
  • Fe 2+ -dependent enzyme activity means that catalytic center of the enzyme contains Fe 2+ ion.
  • dioxygenase according to the present invention contains Fe 2+ ion in the catalytic center thereof and catalyze insertion one atom of oxygen into substrate molecule, particularly into molecule of L-amino acid, coupled with the formation of succinate from ⁇ -ketoglutarate employing another atom of oxygen.
  • hydroxylated L-amino acid refers to an L-amino acid additionally containing one or more hydroxyl groups as a result of
  • ti can be any hydroxylated L-amino acid, but hydroxylated isoleucine, hydroxylated methionine, hydroxylated threonine, hydroxylated leucine or hydroxylated valine are preferable.
  • isoleucine can be converted during hydroxylation reaction to 4-hydroxyisoleucine, 4'-hydroxyisoleucine, 3- hydroxyisoleucine, 5-hydroxyisoleucine, 4,4'-dihydroxyisoleucine, 3,4-dihydroxyisoleucine etc; valine can be converted to 3-hydroxyvaline, 4-hydroxyvaline, 4'-hydroxyvaline, 3,4- dihydroxyvaline or 3,4'-dihydroxyvaline; threonine can be converted to 4-hydroxythreonine; leucine can be can be converted to 3 -hydroxy leucine, 4-hydroxyleucine, 5 -hydroxy leucine, 5'- hydroxyleucine, 3,4-dihydroxyisoleucine, 5,5'-dihydroxyisoleucine etc; methionine can be converted to 3-hydroxymethionine, 2-amino-4-(methylsulfoxy)butyric acid.
  • HiIA catalyzes conversion of He (2S, 3R) into 4'-HIL (2S, 3R)
  • HiIB catalyzes conversion of 4'-HIL (2S, 3R) into 4',4 -DHIL (2S, ' 3R, 4S) and 4',4 -DHIL (2S, 3R, 4R) (Fig.4).
  • Pantoea ananatis HT-paa-HilA and HT-paa-HilB
  • Methylobacillus flagellars KT HT-mfl-DO-B
  • Gluconobacter oxydans 62 IH (HT-gox-DO-B), Agrobacterium vitis (HT-avi-DO-B); Bordetella petrii DSM 12804 (HT-bpe-DO-A and HT-bpe-DO-B); Streptomyces fradiae (HT-sfr-DO-B); marine metagenome (HT-mme-DO-B); Photorhabdus luminescens subsp. laumondii TTOl (HT-plu-DO-B); Gloeobacter violaceus PCC7421 (HT-gvi-DO-B); Psychromonas sp.
  • CNPT3 (HT-psh-DO-A); Mesorhizobium loti (HT-mlo-DO-A); Anabaena variabilis ATCC 29413 (HT- ava-DO-A); Burkholderia s/?.(HT-bur-DO-A). It has been constructed library of 15
  • the DNA encoding dioxygenase HT-paa-HilA (from Pantoea ananatis) is shown in SEQ ID No: 1. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID No: 2.
  • the DNA encoding dioxygenase HT-paa-HilB (from Pantoea ananatis) is shown in SEQ ID No: 3. Furthermore, the amino acid sequence of HT-paa-HilB encoded by the nucleotide sequence of SEQ ID NO: 3 is shown in SEQ ID No: 4.
  • the DNA encoding dioxygenase KT HT-mfl-DO-B (from Methylobacillus flagellatus) is shown in SEQ ID No: 5. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 5 is shown in SEQ ID No: 6.
  • the DNA encoding dioxygenase HT-gox-DO-B (from Gluconobacter oxydans 621H) is shown in SEQ ID No: 7. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 7 is shown in SEQ ID No: 8.
  • the DNA encoding dioxygenase HT-avi-DO-B (from Agrobacterium vitis) is shown in SEQ ID No: 9. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 9 is shown in SEQ ID No: 10.
  • the DNA encoding dioxygenase HT-bpe-DO-A (from Bordetella petrii DSM 12804) is shown in SEQ ID No: 11. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 11 is shown in SEQ ID No: 12.
  • the DNA encoding dioxygenase HT-bpe-DO-B (from Bordetella petrii DSM 12804) is shown in SEQ ID No: 13. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 13 is shown in SEQ ID No: 14.
  • the DNA encoding dioxygenase HT-sfr-DO-B (from Streptomyces fradiae) is shown in SEQ ID No: 15. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 15 is shown in SEQ ID No: 16.
  • the DNA encoding dioxygenase HT-mme-DO-B (from marine metagenome) is shown in SEQ ID No: 17. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 17 is shown in SEQ ID No: 18.
  • the DNA encoding dioxygenase HT-plu-DO-B (from Photorhabdus luminescens subsp. laumondii TTOl) is shown in SEQ ID No: 19. Furthermore, the amino acid sequence of HT- paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 19 is shown in SEQ ID No: 20.
  • T-gvi-DO-B from Gloeobacter violaceus PCC7421
  • SEQ ID No: 22 The DNA encoding dioxygenase (T-gvi-DO-B (from Gloeobacter violaceus PCC7421) is shown in SEQ ID No: 21. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 21 is shown in SEQ ID No: 22.
  • the DNA encoding dioxygenase HT-psh-DO-A (from Psychromonas sp. CNPT3) is shown in SEQ ID No: 23. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 23 is shown in SEQ ID No: 24.
  • the DNA encoding dioxygenase HT-mlo-DO-A (from Mesorhizobium loti) is shown in SEQ ID No: 25. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 25 is shown in SEQ ID No: 26.
  • the DNA encoding dioxygenase T-ava-DO-A (from Anabaena variabilis ATCC 29413) is shown in SEQ ID No: 27. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 27 is shown in SEQ ID No: 28.
  • the DNA encoding dioxygenase HT-bur-DO-A (from Burkholderia sp.) is shown in SEQ ID No: 29. Furthermore, the amino acid sequence of HT-paa-HilA encoded by the nucleotide sequence of SEQ ID NO: 29 is shown in SEQ ID No: 30.
  • the DNA encoding dioxygenase Ido (from Bacillus thuringiensis 2e2) is shown in SEQ ID No: 53. Furthermore, the amino acid sequence Ido encoded by the nucleotide sequence of SEQ ID NO: 53 is shown in SEQ ID No: 54.
  • the DNA encoding dioxygenase Doxl (from Pseudomonas syringae pv. phaseolicola 1448A) is shown in SEQ ID No: 55. Furthermore, the amino acid sequence Doxl encoded by the nucleotide sequence of SEQ ID NO: 55 is shown in SEQ ID No: 56.
  • the DNA encoding dioxygenase Dox2 (from Pseudomonas syringae pv. phaseolicola 1448A) is shown in SEQ ID No: 57. Furthermore, the amino acid sequence Dox2 encoded by the nucleotide sequence of SEQ ID NO: 57 is shown in SEQ ID No: 58.
  • the DNA encoding dioxygenase Dox (from Burkholderia phytofirmans PsJN) is shown in SEQ ID No: 59. Furthermore, the amino acid sequence Dox encoded by the nucleotide sequence of SEQ ID NO: 59 is shown in SEQ ID No: 60.
  • the DNA encoding dioxygenase Dox (from Nostoc punctiforme PCC 73102) is shown in SEQ ID No: 61. Furthermore, the amino acid sequence Dox encoded by the nucleotide sequence of SEQ ID NO: 61 is shown in SEQ ID No: 62.
  • the DNA encoding dioxygenase Dox (from Burkholderia ambifaria AMMD) is shown in SEQ ID No: 63. Furthermore, the amino acid sequence Dox encoded by the nucleotide sequence of SEQ ID NO: 63 is shown in SEQ ID No: 64.
  • a DNA that encodes dioxygenase belonging to the HiIA or HiIB dioxygenase families is not only the DNA shown in SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61 or SEQ ID No: 63. This is because there may be differences in nucleotide sequences from each species and strains among bacteria that form dioxygenase belonging to the HiIA or HiIB dioxygenase families.
  • the DNA of the present invention not only includes the isolated DNA encoding dioxygenase belonging to the HiIA or HiIB dioxygenase families, but also a DNA in which mutations have been artificially added to the DNA that encodes dioxygenase belonging to the HiIA or HiIB dioxygenase families.
  • This DNA may be isolated from the chromosome of an microorganism producing dioxygenase belonging to the HiIA or HiIB dioxygenase families.
  • the DNA of the present invention must encode dioxygenase that is able to catalyze the hydroxylation reaction of amino acid. Methods for artificially adding mutations include typically used methods introducing site-specific mutations described in Method, in Enzymol., 154 (1987).
  • the "stringent conditions” refer to those conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to numerically express these conditions explicitly, by way of example, those conditions under which DNA molecules having higher homology e.g. preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more homology, hybridize with each other, while DNA molecules having lower homology do not hybridize with each other, or those conditions under which hybridization occurs under typical washing conditions in
  • Southern hybridization that is, at a salt concentration corresponding to 0. IxSSC and 0.1% SDS at 37°C, preferably 0.IxSSC and 0.1% SDS at 60 0 C, and more preferably 0.IxSSC and 0.1% SDS at 65°C.
  • the length of the probe may be suitably selected, depending on the hybridization conditions, and usually varies from 100 bp to 1 kbp.
  • "L-amino acid dioxygenase activity” may be described as the activity that synthesizes hydroxylated L-amino acid from the L- amino acid.
  • nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61 or SEQ ID No: 63, it preferably retains L-amino acid dioxygenase activity of 10% or more, preferably 30% or more, more preferably 50% or more, and still more preferably 70% or more, of protein having the amino acid sequence of SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No
  • a DNA encoding a protein which is substantially identical to the L-amino acid dioxygenase encoded by the DNA of SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61 or SEQ ID No: 63 is also included in the DNA of the present invention. Namely, the following DNAs are also included in the DNA of the present invention:
  • one or several refers to a number of changes which do not result in significant changes to the 3D structure of the protein, or signficant reduction of the L- amino acid dioxygenase activity, and more specifically, is in the range of 1 to 78, preferably 1 to 52, more preferably 1 to 26, and still more preferably 1 to 13.
  • substitution, deletion, insertion, addition, or inversion of one or several amino acid residues should be conservative mutation(s) so that the activity is maintained.
  • conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with GIn, His or Lys, substitution of Asn with GIu, GIn, Lys, His or Asp, substitution of Asp with Asn, GIu or GIn, substitution of Cys with Ser or Ala, substitution of GIn with Asn, GIu, Lys, His, Asp or Arg, substitution of GIu with Asn, GIn, Lys or Asp, substitution of GIy with Pro , substitution of His with Asn, Lys, GIn, Arg or Tyr, substitution of He with Leu, Met, VaI or Phe, substitution of Leu with He, Met, VaI or Phe , substitution of Lys with Asn, GIu, GIn, His or Arg, substitution of Met with He, Leu, VaI or Phe , substitution of Phe with Trp, Tyr, Met, He or Leu, substitution of Ser with Thr or Ala , substitution of Thr with Ser or Ala
  • L- amino acid dioxygenase activity refers to catalyzing the
  • a homologue DNA of SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61, SEQ ID No: 63 can be used as the gene encoding L-amino acid dioxygenase of present invention.
  • homologue DNA encodes L- amino acid dioxygenase or not can be confirmed by measuring L- amino acid dioxygenase activity of the cell lysate, or cell lysate of the
  • the homologue DNA of SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID No: 17, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 27, SEQ ID No: 29, SEQ ID No: 53, SEQ ID No: 55, SEQ ID No: 57, SEQ ID No: 59, SEQ ID No: 61, SEQ ID No: 63 can also be prepared from the genome of another species of bacteria belonging to the genera Pantoea, Methylobacillus, Gluconobacter, Agrobacterium, Bordetella, Streptomyces,
  • bacteria / "microorganism” as employed in the present specification includes an enzyme-producing bacterium/ microorganism, a mutant, and a genetic recombinant of such bacteria/ microorganism in which the targeted enzymatic activity is present or has been enhanced, and the like.
  • ⁇ -ketoglutarate-dependent dioxygenases A number of proteins having ⁇ -ketoglutarate-dependent enzyme activity, such as ⁇ - ketoglutarate-dependent dioxygenases have been reported.
  • the examples thereof include dioxygenases usable for productions of useful products, such as a Pro dioxygenase convering L- Pro to hydroxy-Pro (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1999, p. 4028-4031), a ⁇ -butyrobetaine dioxygenase converting ⁇ -butyrobetaine to L-carnitine
  • the bacterium has been modified to attenuate the expression of a gene coding for oxoglutarate dehydrogenase (such as AsucAB, ⁇ sucA, AsucB), preferably modified to attenuate the expression of genes coding for oxoglutarate dehdyrogenase and isocitrate lyase (such as (AsucAB, AsucA, or AsucB) plus AaceA), more preferably modified to attenuate the expression of genes coding for oxoglutarate dehdyrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase (such as (AsucAB, AsucA, or AsucB) plus AaceAK), which is represented by E.
  • oxoglutarate dehydrogenase such as AsucAB, ⁇ sucA, AsucB
  • coli strain MGl 655 (AsucAB, AaceAK) which is described in the below-mentioned Examples, is considered to be a general host for efficiently using a -ketoglutarate produced from a carbon source such as D-glucose, in a -ketoglutarate-dependent enzyme reactions.
  • the present invention will be described by referring to, as an example, an embodiment in which the protein having ⁇ -ketoglutarate-dependent enzyme activity is a protein having L- amino acid dioxygenase activity and the product of the reaction catalyzed by the protein is hydroxylated L-amino acid.
  • the present invention is not limited to this embodiment.
  • a bacterium belonging to the genus Escherichia means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).
  • the bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited; however, e.g., bacteria described by Neidhardt, RC. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology,
  • a bacterium belonging to the genus Pantoe ⁇ means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology.
  • Some species of Enterobacter agglomerans have been recently re- classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on nucleotide sequence analysis of 16S rRNA, etc (International Journal of Systematic
  • Pantoea ananatis Bacteriology, July 1989, 39(3),p.337-345. Furthermore, some bacteria belonging to the genus Erwinia were re-classified as Pantoea ananatis or Pantoea stewartii (International Journal of Systematic Bacteriology, Jan. 1993, 43(1), pp.162-173).
  • Typical strains of the Pantoea bacteria include, but are not limited to, Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples include the following strains: Pantoea ananatis AJ 13355 (FERM BP-6614, European Patent Publication No.
  • Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Publication No. 0952221), Pantoea ananatis AJ 13601 (FERM BP-7207, European Patent Publication No. 0952221).
  • a preferable ⁇ -Red resistant to be used for present invention is Pantoea ananatis SC 17(0) (VKPM B-9246, RU application 2006134574).
  • a bacterium belonging to the genus Bacillus means that the bacterium is classified into the genus Bacillus according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Bacillus as used in the present invention include, but are not limited to, Bacillus subtilis and Bacillus thuringiensis.
  • a bacterium belonging to the genus Pseudomonas means that the bacterium is classified into the genus Pseudomonas according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Pseudomonas as used in the present invention include, but are not limited to, Pseudomonas syringae.
  • a bacterium belonging to the genus Agrobacterium means that the bacterium is classified into the genus Agrobacterium according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Bacillus as used in the present invention include, but are not limited to, Agrobacterium vitis.
  • a bacterium belonging to the genus Bordetell ⁇ means that the bacterium is classified into the genus Bordetella according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Bordetella as used in the present invention include, but are not limited to, Bordetella petrii.
  • a bacterium belonging to the genus Streptomyces means that the bacterium is classified into the genus Streptomyces according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Streptomyces as used in the present invention include, but are not limited to, Streptomyces fradiae.
  • a bacterium belonging to the genus Photorhabdus means that the bacterium is classified into the genus Photorhabdus according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Photorhabdus as used in the present invention include, but are not limited to, Photorhabdus luminescens.
  • a bacterium belonging to the genus Gloeobacter means that the bacterium is classified into the genus Gloeobacter according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Gloeobacter as used in the present invention include, but are not limited to, Gloeobacter violaceus.
  • a bacterium belonging to the genus Psychromonas means that the bacterium is classified into the genus Psychromonas according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Mesorhizobium means that the bacterium is classified into the genus Mesorhizobium according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Mesorhizobium as used in the present invention include, but are not limited to, Mesorhizobium loti.
  • a bacterium belonging to the genus Anabaen ⁇ means that the bacterium is classified into the genus Anabaena according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Anabaena as used in the present invention include, but are not limited to, Anabaena variabilis.
  • a bacterium belonging to the genus Burkholderia means that the bacterium is classified into the genus Burkholderia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus
  • Burkholderia as used in the present invention include, but are not limited to, Burkholderia phytofirmans and Burkholderia ambifaria.
  • a bacterium belonging to the genus Methylobacillus means that the bacterium is classified into the genus Methylobacillus according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Methylobacillus as used in the present invention include, but are not limited to, Methylobacillus flagellatus.
  • a bacterium belonging to the genus Gluconobacter means that the bacterium is classified into the genus Gluconobacter according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Gluconobacter as used in the present invention include, but are not limited to, Gluconobacter oxydans.
  • a bacterium belonging to the genus Nostoc means that the bacterium is classified into the genus Nostoc according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Nostoc as used in the present invention include, but are not limited to, Nostoc punctiforme.
  • a microorganism belonging to the genus Corynebacterium means that the microorganism is classified into the genus Corynebacterium according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Corynebacterium as used in the present invention include, but are not limited to, Corynebacterium glutamicum.
  • a microorganism belonging to the genus Arthrobacter means that the microorganism is classified into the genus Arthrobacter according to the classification known to a person skilled in the art of microbiology.
  • a bacterium belonging to the genus Arthrobacter as used in the present invention include, but are not limited to, Arthrobacter simplex, Arthrobacter globiformis, Arthrobacter sulfureus, and Arthrobacter viscosus.
  • a microorganism belonging to the genus Aspergillus means that the microorganism is classified into the genus Aspergillus according to the classification known to a person skilled in the art of microbiology.
  • Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
  • Transformation of a bacterium with DNA encoding a protein means introduction of the DNA into a bacterium, for example, by conventional methods. Transformation of this DNA will result in an increase in expression of the gene encoding the protein(s) of present invention, and will enhance the activity of the protein in the bacterial cell. Methods of transformation include any known methods that have hitherto been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. MoI. Biol, 53, 159 (1970)) may be used.
  • transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).
  • the method of the present invention is a method for producing a product of a reaction catalyzed by a protein having ⁇ -ketoglutarate-dependent and Fe 2+ dependent enzyme activity by cultivating the bacterium of the present invention in a culture medium containing a substrate of the reaction, and isolating the produced product from the medium.
  • the product includes compounds in both the free form and a salt form thereof.
  • the substrate is suitably selected.
  • the substrate may be He (2S, 3R).
  • the method of the present invention may be a method for producing hydroxylated L-amino acid, including both the free form and a salt form thereof, by cultivating the bacterium of the present invention in a culture medium containing the L-amino acid, and isolating produced hydroxylated L-amino acid from the medium.
  • the medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • the carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the used microorganism, alcohol, including ethanol and glycerol, may be used.
  • As the nitrogen source various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used.
  • the medium of present invention contained corresponding L- amino acid (20-40 g/1).
  • the cultivation is preferably performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40 °C, preferably 30 to 38 °C.
  • the pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers.
  • Examples of separation and purification methods may include a method in which the hydroxylated L-amino acid is contacted with an ion exchange resin to adsorb basic amino acids followed by elution and crystallization, and a method in which the product obtained by elution is discolored and filtrated with activated charcoal followed by crystallization to obtain the hydroxylated L-amino acid.
  • the culture condition, the separation and pufification methods and the line are selected depending on the nature of the used bacterium and the target product.
  • pET-HilA plasmid a 935-bp DNA fragment containing the hilA gene was amplified using primers Pl(SEQ ID No:31) and P2(SEQ ID No:32), and chromosomal DNA of the P. ananatis SC 17 strain as a template. Resulted fragment was digested by Xba ⁇ and Sad and ligated with pET-ilvA plasmid (Ru patent application 2006131211) previously digested with the same restriction enzymes.
  • pET-Hil A-HiIB plasmid a 1,732-bp DNA fragment containing hilA and MB genes was amplified using primers Pl (SEQ ID No:31) and P3 (SEQ ID No:33), and chromosomal DNA of the SC 17 strain as a template. Resulted fragment was digested by Xbal and Sad and ligated with pET-ilvA plasmid previously digested with the same restriction enzymes.
  • pET-HilB plasmid a 791-bp DNA fragment containing hilB gene was amplified using primers P4 (SEQ ID No:34) and P3 (SEQ ID No:33), and chromosomal DNA of the SC 17 strain as a template. Resulted fragment was digested by Xbal and Sad and ligated with pET-ilvA plasmid previously digested with the same restriction enzymes.
  • pET-HT-HilB In a similar manner any plasmid pET-HT-xxx-DOA(B) can be constructed) plasmid, a 796-bp DNA fragment was amplified using primers P5 (SEQ ID No:35) and P6 (SEQ ID No:36), and chromosomal DNA of the SC 17 strain. Resulting fragment was digested with Ncol and BamHl and ligated with pET-15 (b+) vector (Novagen, Germany) previously digested with the same restriction enzymes.
  • E. coli MG1655 ( ⁇ attB-PL- brnQ) ⁇ sucAB::( ⁇ attB-Ptac) ⁇ aceAK::( ⁇ attB)( ⁇ amca 141) strain which is MGl 655 ( ⁇ sucAB, ⁇ aceAK, PL-brnQ) strain disclosed in WO 2009/082029, was transformed with plasmids pETAC-HilA and pETAC-Hil A-HiIB, thus AG7030 and
  • AG7029 strains were obtained.
  • the strains AG7030 and AG7029 were cultivated in 2 ml of TI medium [ 50 mM KH 2 PO 4 (pH 7 adjusted by KOH); 20 mM NH 4 Cl; 2 mM
  • HPLC analysis was performed as follows. The amino acids were determined using the Accq-Tag method (Waters). Synthesis of amino acid derivatives and their separation were performed according to manufacturer's recommendations. Separation was carried out at 20 °C using an Agilent 1100 series chromatograph equipped with XBridge Cl 8 (5) column (150*2.1 mm; Waters).
  • He under expression of the HiIA and HiIB is 4',4-dihydroxyisoleucine (4',4- DHIL) (Fig. 4).
  • DHIL 4,4'-dihydroxyisoleucine
  • Obtained suspensions were cultivated at 32 °C for about 16 hours. Synthesis of the 4',4-DHIL was detected by using TLC analysis (scheme and results of the experiment are shown on the Fig. 15, Fig. 16) .
  • Duplicated reaction mixtures B and D were pooled to obtained about 10 ml of the 4',4-DHIL solution. Two milliliters of obtained solution were applied to the DOWEX 50 W-X2 column (16 X 0.8 cm) equilibrated by 30 ⁇ M ammonia solution. An isocratic elution was down using the same ammonia solution at 1.5 ml/min elution rate. Twenty fractions were pooled in total. 4',4-DHIL was eluted in fractions 7 - 11 (Fig. 17). Eluted DHIL was lyophilization and strored at -20 0 C until used. Described procedure was repeated 5 times to purified all DHIL from 10 ml of starting solution, hi total, we obtained approximately 100 mg of the lyophilized powder of the 4'4-DHIL.
  • Induced biomass was harvested by centrifugation and re-suspended in 24 ml of reaction buffer containing: 100 mM HEPES pH 8, 10 mM Fe 2+ , L- valine 100 mM, ⁇ -ketoglutarate 100 mM. Reaction mixture was incubated at 37°C for 15 hours at vigorously shaking. Partially purification of hVal was conducted as was described previously for hLeu (Fig. 19). Furthur purification and
  • Reaction mixture (50 ⁇ l) contained 100 mM HEPES (pH 7 ajusted by KOH), 5 mM
  • Substrate specificities of the HiIA and HiIB were investigated as follows.
  • Reaction mixture 50 ⁇ l
  • Reaction mixture contained: 100 mM Hepes pH7.0, 5 mM of amino acid (He, VaI 5 Leu, Ala, GIy, Asp, Asn, GIu, GIn, Pro, Phe, Trp, Met, Cys, Lys, Arg, Thr, Ser, His were tested), 5mM 2-oxoglutarate, 5mM ascorbate, 5 mM FeSO 4 and protein preparation.
  • Reaction was incubated at 34 0 C fro 1 hour at vigorously shaking. Amino acids hydroxylation was monitored by TLC analysis.
  • Step 1 Cells were thawed and resuspended in 40 ml of buffer A (50 mM Tris pH 7, 50 mM NaCl, ImM DTT, ImM EDTA). Cells were disrupted by 2 passages through a French pressure cell (maximum pressure 2000 Psi) followed by centrifugation (14 000 g, 4°C, 20 min) to remove cell debris.
  • buffer A 50 mM Tris pH 7, 50 mM NaCl, ImM DTT, ImM EDTA
  • Step2 Forty milliliters of protein preparation obtained from Step 1 were applied to a DEAE-Sepharose column (1.6 x 25 cm) equilibrated with the buffer A. Unbounded (flow- through) fraction was collected.
  • Step 3 The protein preparation obtained from Step 2 was precipitated by adding (NH 4 ) 2 SO 4 up to 40% of saturation and then resuspended in 2 ml of the buffer A.
  • Step 4 One milliliter of protein preparation obtained from Step 3 was applied to a Superdex 200 HR 10/3 OA column equilibrated with the buffer A . Isocratic elution was performed at a flow rate of 0.5mL min-1. Each 1-mL fraction was collected. Active fractions were pooled.
  • the protein paaHilA have been purified (Fig. 5) and its basic properties investigated were investigated.
  • Obtained protein preparation (protein's concentration was about 4 mg/ml) was applied to a 1 ml His-trap column (Amersham) equilibrated with buffer His-tagl. The elution was carried out at flow rate 1 ml/min by liner gradient 0.02 - 0.5 M imidazole in buffer His-tag 1 (15 column volumes). Each 1 ml fraction was collected. Active fractions were pooled.
  • the protein HiIB has been purified as a his 6 -tag derivative (Fig.5) and its basic properties investigated. Purified protein is homodimer and exhibits substrate preference to 4'- He, methionine and He (side activity) (Fig. 6).
  • pET-HT-xxx-DOA(B) plasmid synthesized using SloningTM technology and received as a set of pSlo.X plasmids (Fig. 9).
  • Xbal-BamHI fragment of corresponding pSlo.X plasmid was re-cloned into pET15(b+)/XbaI-BamHI vector.
  • Obtained plasmid pET-HT- xxx-DOA(B) was introduced into BL21(DE3) strain.
  • Cells of the BL21 (DE3) [pET-HT HT- xxx-DOA(B)] strain were grown in LB broth at 37 °C up to A 540 ⁇ 1. Then IPTG was added up to final concentration of 1 mM and cultivation was prolonged within 2 hours. Induced cells were harvested and stored at -70 °C until used.
  • buffer HT-I (20 mM NaH 2 PO 4 , 0.5M NaCl, 20 mM imidazole, pH 7.4 adjusted by NaOH) and destroyed by using French-press. Cell debris was removed by centrifugation. Obtained protein preparation was applied to a 1 ml His-trap column (Amersham) equilibrated with buffer HT-I. The elution was carried out at flow rate 1 ml/min with liner gradient, 0.02 - 0.5 M imidazole in buffer HT-I (15 column volumes). Each 1 ml fraction was collected. Active fractions were pooled.
  • Obtained active protein preparation was desalted using PDlO column (Amersham) equilibrated with buffer IDO (50 mM HEPES, 50 mM NaCl, ImM EDTA, glycerol 10% v/v). The 0.2 ml aliquots of final protein preparation were stored at - 70 °C until used.
  • buffer IDO 50 mM HEPES, 50 mM NaCl, ImM EDTA, glycerol 10% v/v.
  • Reaction mixture (50 ⁇ l) contained: 50 mM HEPES pH8.0, 5 mM of amino acid (He, VaI, Leu, Ala, GIy, Asp, Asn, GIu, GIn, Pro, Phe, Trp, Met, Cys, Lys, Arg, Thr, Ser, His, R(S)-beta-phenylalanine were tested), 5 mM 2-oxoglutarate, 5 mM FeSO 4 , and protein preparation. It was incubated at 34 0 C for 0.5-1 hour with vigorously shaking. Amino acid hydroxylation was monitored by TLC analysis.
  • a 805-bp DNA fragment was amplified using primers P7 (SEQ ID NO: 37), P8 (SEQ ID NO: 38) and chromosomal DNA from the strain Methylobacillus flagelatus KT. Resulting fragment was digested with Ncol and BamHl and ligated with pET-15 (b+) vector previously digested with the same restriction enzymes. Obtained plasmid pET-HT-mfl-DO-B was introduced into E. coli strain BL21 (DE3) (Novagen, Germany).
  • Putative dioxygenase from Gluconobacter oxydans 621H (HT-gox-DO-B 2.3.1. Cloning, expression, purification and activity assay of the putative dioxygenase from Gluconobacter oxydans 62 IH (HT-gox-DO-B).
  • a 802-bp DNA fragment was amplified using primers P9 (SEQ ID NO: 39), PlO (SEQ ID NO: 40) and chromosomal DNA of the strain Gluconobacter oxydans 62 IH as a template. Resulting fragment was digested with Nc ⁇ l and BamHl and ligated with pET-15 (b+) vector previously digested with the same restriction enzymes.
  • reaction buffer 100 mM Hepes pH 8, 100 mM ⁇ -ketoglutarate, 100 mM Leu.
  • test-tubes (5 x 2 ml) at 37 0 C for 15 hours.
  • Culture broth was pooled, passed through 0.22 ⁇ m filter and applied to the DOWEX 50 W-X2 column (32 X 1.6 cm)
  • Fractions containing hLeu were pooled , applied again to the DOWEX 50 W-X2 column collected and chromatography process was repeated. Fractions containing hLeu were lyophilized.
  • reaction buffer contained: 100 mM HEPES pH 7, 10 mM Fe 2+ , Met/ ⁇ -ketoglutarate in concentration of 100 mM. Reaction was incubated at 37 0 C for 15 hours at vigorously shaking. Purification of methionine methionine sulfoxide was carried out as was described above for hLeu (Fig. 20).
  • reaction mixtures total volume 2 ml containing: ImI of cell lysate, 10 mM Fe 2+ , Thr (and ⁇ -ketoglutarate) in concentration of 10 mM, 20 mM, 50 mM, 70 mM, 100 mM. Reactions were incubated at 37 0 C for 15 hours with vigorously shaking. We analyzed resulting reaction mixtures using TLC (Fig.21). As can be seen, only 10 and 20 mM of Thr were completely hydroxylated. At higher concentration of Thr (and ⁇ -ketoglutarate) we observed massive protein precipitation which precluded effective Thr hydroxylation. Thus, optimal reaction mixture contained 20-25 mM Thr
  • reaction mixture contained 25 mM Thr, 25 mM ⁇ -ketoglutarate, 100 mM Hepes pH7, 10 mM Fe 2+ . Final reaction mixtures were centrifuged and supernatant containing hydroxythreonine was frozen until used.
  • Example 3 Application of the E.coli strain to synthesis of the new hydroxylated amino acid(s) using direct biotransformation process.
  • the ⁇ sucAB deletion was performed into 2 ⁇ strain (MGl 655 ace AK:: ⁇ attL-Kn- ⁇ attR) as described in WO05/010175.
  • 2 ⁇ # strain MG1655 sucAB:: ⁇ attR-Cm- ⁇ attL- Ptac, ace AK:: ⁇ attL-Kn- ⁇ attR was constructed.
  • CM7 - [(NFLO 2 SO 4 - 5g/l; KH 2 PO 4 - 1.5 g/1; MgSO 4 x 7H 2 O - lg/1; FeSO 4 x7H 2 O - 0,01 g/1; isoleucine - 26 g/1; glucose - 54 g/1; pH 7 adjusted by KOH]
  • the E.coli strains 2 ⁇ and 2 ⁇ # were transformed with the plasmid pEL-IDO (pEL- IDO(LyS, 23) plasmid has been constructed as follows. Large part of the lad gene was deleted from pEL AC-IDO(LyS, 23) plasmid (Fig. 14) by excision of the Sphl-EcoRV DNA fragment using corresponding restrictases followed by ligation of the remaining part of the plasmid). Thus the strains 2 ⁇ [ pEL-IDO] and 2 ⁇ #[pEL-IDO], respectively, were obtained.
  • a piece of biomass of the E.coli strain 2 ⁇ [ pEL-IDO] and the E.coli strain 2 ⁇ #[pEL- IDO] each grown on fresh-made LB-agar plate were inoculated in 750 ml-flask containing 50 ml of LB broth supplemented with Ap (100 mg/1) and cultivated at 37 0 C for about 4 hours. Obtained cells' cultures were used as inoculums in large-scale biotransformation.
  • Example 4 Cloning of dioxygenase genes from Bacillus thurineiensis 2e2. Pseudomonas syringae py. phaseolicola 1448 A, Burkholderia phytofirmans PsJN, Nostoc punctiforme PCC 73102. and Burkholderia ambifa ⁇ a AMMD.
  • LB medium (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl) was used.
  • King's medium B (20 g/L Proteose Peptone No. 3, 1% (v/v) Glycerol, 1.5 g/L K 2 HPO 4 , 1.5 g/L MgSO 4 -7H 2 O, 50 mg/L Rifampicin, and pH 7.2) was used.
  • King's medium B (20 g/L Proteose Peptone No. 3, 1% (v/v) Glycerol, 1.5 g/L K 2 HPO 4 , 1.5 g/L MgSO 4 -7H 2 O, 50 mg/L Rifampicin, and pH 7.2
  • TGY medium (5 g/L Tryptone, 1 g/L Glucose and 3 g/L Yeast Extract) was used.
  • Blue-green nitrogen-fixing medium 1.5 g/L NaNO 3 , 0.04 g/L
  • B. thuringiensis 2e2 P syringae pv. phaseolicola 1448 A, B. phytofirmans PsJN, and B. ambifaria AMMD were each cultivated overnight at 28°C in 5 ml (pre-culture).
  • N. punctiforme PCC 73102 was cultivated for 1 week at 22°C in 5 ml under light intensity of 2,000-3,000 lux (pre-culture).
  • a main culture was carried out in 50 ml of each medium. After cultivation up to a logarithmic growth phase, cells were harvested from 50 ml of the culture broth by centrifugation (12000 x g, 4°C, 15 min). From these cells, chromosomal DNA was prepared according to an ordinary method.
  • Bacillus thuringiensis strain 2e2 was named Bacillus thuringiensis AJl 10584 and deposited at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba, Ibaraki 305- 8566, Japan) on September 27, 2006 and given an accession number of FERM BP- 10688 under the provisions of Budapest Treaty.
  • amplification by PCR was carried out with PrimeSTAR (TaKaRa) under the following condition: 30 cycles for 10 seconds at 98°C, 15 seconds at 52°C and 1 minute at 72°C.
  • the obtained PCR product was subjected to agarose gel electrophoresis and
  • P syringae pv. phaseolicola 1448 A As for P syringae pv. phaseolicola 1448 A, by the primers based on published genomic sequence information about P. syringae pv. phaseolicola 1448A (GenBank accession No.
  • B. phytofirmans PsJN As for B. phytofirmans PsJN, by the primers based on published genomic sequence information about B. phytofirmans PsJN (GenBank accession No. NC O 10676), the primers Pl 7 (SEQ ID NO: 47) and Pl 8 (SEQ ID NO: 48) were synthesized, a dioxygenase gene, dox, was cloned by PCR in a similar manner to the above.
  • N. punctiforme PCC 73102 As for N. punctiforme PCC 73102, by the primers based on published genomic sequence information about N. punctiforme PCC 73102 (GenBank accession No.
  • B. ambifaria AMMD As for B. ambifaria AMMD, by the primers based on published genomic sequence information about B. AMMD (GenBank accession No. NC 008392), the primers P21 (SEQ ID NO: 51) and P22 (SEQ ID NO: 52) were synthesized, a dioxygenase gene, dox, was cloned by PCR in a similar manner.
  • IPTG was added at a final concentration of 1 mM, and the cultivation was carried out for further 3 hours.
  • cells were harvested, washed, suspended in 1 ml of 20 mM Tris-HCl (pH 7.6) and disrupted with a sonicator (INSONATOR 20 IM, KUBOTA). The lysate was centrifuged at 15000 rpm for 10 minutes to obtain a supernatant which was used as a crude enzyme solution.
  • the protein preparation obtained was added to the 5 ml of HisTrap HP (GE Healthcare) equilibrated in Binding buffer (20 mM Sodium phosphate, 0.5 M NaCl, 30 mM Imidazole, and pH 7.4). The elution was carried out at flow rate 1 ml/min by liner gradient in 10 column volumes of Elution buffer (20 mM Sodium phosphate, 0.5 M NaCl, 500 mM Imidazole, and pH 7.4). Each 1 ml fraction was collected. Active fractions were pooled and dialysis against 20 mM Tris-HCl (pH 7.6).
  • the reaction mixture had a composition as described below.
  • the reaction mixture was reacted with shaking (300 rpm) at 25°C for 16 hours, and then the produced succinate was determined by F-kit (Roche). The determination results were shown in Table 4.
  • Amino acid products in dioxygenase reaction were determined by LC-MS analysis.
  • Amino acids were derivatized using the AccQ-Tag method (Waters).
  • the amino acid derivatives were analysed using a high-performance liquid chromatography (HPLC) system equipped with an electrospray ionization quadrupole mass spectrometer (ESI-Q-MS).
  • HPLC high-performance liquid chromatography
  • ESI-Q-MS electrospray ionization quadrupole mass spectrometer
  • the XBridge C 18 column (5 ⁇ m, 2.1 x 150 mm; Waters) was used for the separation at 40 0 C.
  • the mobile phases were 1 mM ammonium acetate buffer (eluent A) and methanol (eluent B), and the flow rate of the eluent was 0.3 ml/min.
  • the eluent gradients were 20-40% (v/v) B in 0-15 min and
  • Example 6 Preparation of hydroxylated L-leucine. hvdroxylated L-norleucine, and oxidized L- methionine by IDO.
  • E. coli AG6772 (2 ⁇ [ p ⁇ L-IDO]) was innoculated into Sakaguchi flask containing 50 ml terrific broth (TB) medium (Yeast extract 24 g/1, Peptone 12 g/1, K 2 HPO 4 9.4 g/1, KH 2 PO 4 2.2 g/1, glycerol 4 ml/1) supplemented with 100 ⁇ g/ml ampicilin and 0.1 mM IPTG., and cultivated for 16 h at 34 0 C. The culture broth was centrifuged at 8,000 rpm for 5 min, and thus obtained cells were used in the following resting cell reactions.
  • TB terrific broth
  • Yeast extract 24 g/1, Peptone 12 g/1, K 2 HPO 4 9.4 g/1, KH 2 PO 4 2.2 g/1, glycerol 4 ml/1 supplemented with 100 ⁇ g/ml ampicilin and 0.1 mM IPTG.
  • the culture broth was centrif
  • the E. coli AG6772 cells were collected form 10 ml culture broth as described above, and resuspended in 2 ml of reaction mixture containing 100 mM KPB (pH7.0), 100 mM L-Leu, 100 mM ⁇ -ketoglutarate, 1 mM sodium ascorbate, 10 mM FeSO 4 7H 2 O.
  • the reaction mixture was centrifused at 8,000 rpm for 10 min.
  • the eution fractions were assayed using TLC, and fractions containing hydroxylated L-Leu were collected, evaporated, and applied onto Amberlite 120 H (bed volume was approximately 1 ml) and eluated with IN NFL ⁇ aq.
  • the eluate was collected, evaporated, and analyzed by NMR and ⁇ SI-MS. The result indicated the purified compound was 4- hydroxylated-L-leucine.
  • the E. coli AG6772 cells were collected form 10 ml culture broth as described above, and resuspended in 2 ml of reaction mixture containing 100 mM KPB (pH7.0), 100 mM L- norleucine, 100 mM ⁇ -ketoglutarate, 1 mM sodium ascorbate, 10 mM FeSO 4 7H 2 O.
  • the E. coli AG6772 cells were collected form 10 ml culture broth as described above, and resuspended in 2 ml of reaction mixture containing 100 mM KPB (pH7.0), 100 mM L-Met, 100 mM ⁇ -ketoglutarate, 1 mM sodium ascorbate, 10 mM FeSO 4 7H 2 O.
  • the reaction mixture was centrifused at 8,000 rpm for 10 min.
  • the eution fractions were assayed using TLC, and fractions containing oxidized or oxidated L-Met were collected, evaporated, and analyzed by NMR and ESI-MS. The result indicated the purified compound was L-methionine sulfoxide.
  • dioxygenase from Bacillus thuringiensis 2e2 strain was determined.
  • Oxidizing reaction of L- Met by IDO was performed at 25°C for 2 h with shaking.
  • the reaction mixture containined 20 mM L-Met, 10 mM ascorbata, 100 mM ⁇ -ketoglutarate, 1 mM FeSO 4 , 100 mM KPB (pH 6.0), 20 mg/ml His-tagged IDO .
  • the enzymatic reaction product was further derivatized by
  • Marfey's reagent (l-fluoro-2,4-dinitrophenyl-5-L-alanine amide) according to the procedures described by Joseph T. Dever et al (Drug Metabolism and Disposition 34, 2036-2043 (2006)), followed by HPLC analysis.
  • a typical HPLC chromatogram of racemic L-methionine sulfoxide derivatized by Marfery's reagent is shown in Fig. 23. R-enantimer and S-enantiomer can be separated.
  • the chromatgrams of IDO reaction products after Marfey's reagent treatment are shown in Fig. 24. Only S-enantiomer of L-methionine sulfoxide (approximately 19 mM) was detected in the reaction mixture.
  • the hydroxylated L-Thr was collected, eluated by water, and freezed dried.
  • the oxidized L-Met was collected, eluated by water, and freezed dried.
  • the NMR and ESI-MS analysis indicated that the purified compound was L- methionine sulfoxide.

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Abstract

La présente invention concerne un procédé de fabrication d'acides aminés hydroxylés, en particulier l'isoleucine hydroxylée, la méthionine hydroxylée, la thréonine hydroxylée, la leucine hydroxylée ou la valine hydroxylée, par conversion enzymatique à l'aide d'une dioxygénase.
PCT/JP2010/064323 2009-08-21 2010-08-18 Procédé de production d'acides aminés hydroxylés WO2011021717A2 (fr)

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CN105543128A (zh) * 2015-12-24 2016-05-04 山东大学 一种极地适冷耐盐褐藻酸裂解酶及其编码基因c3与应用
CN113355371A (zh) * 2021-05-27 2021-09-07 无锡晶海氨基酸股份有限公司 一种全细胞催化制备4-羟基异亮氨酸的方法

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Publication number Priority date Publication date Assignee Title
CN105543128A (zh) * 2015-12-24 2016-05-04 山东大学 一种极地适冷耐盐褐藻酸裂解酶及其编码基因c3与应用
CN113355371A (zh) * 2021-05-27 2021-09-07 无锡晶海氨基酸股份有限公司 一种全细胞催化制备4-羟基异亮氨酸的方法

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