JPH03266995A - Production of 4-hydroxy-l-proline - Google Patents
Production of 4-hydroxy-l-prolineInfo
- Publication number
- JPH03266995A JPH03266995A JP6678290A JP6678290A JPH03266995A JP H03266995 A JPH03266995 A JP H03266995A JP 6678290 A JP6678290 A JP 6678290A JP 6678290 A JP6678290 A JP 6678290A JP H03266995 A JPH03266995 A JP H03266995A
- Authority
- JP
- Japan
- Prior art keywords
- hydroxy
- proline
- microorganism
- acid
- oxoglutaric acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 title claims abstract description 38
- 229960002591 hydroxyproline Drugs 0.000 title claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- WXSKVKPSMAHCSG-UHFFFAOYSA-N 4-hydroxy-2-oxoglutaric acid Chemical compound OC(=O)C(O)CC(=O)C(O)=O WXSKVKPSMAHCSG-UHFFFAOYSA-N 0.000 claims abstract description 43
- 150000001875 compounds Chemical class 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 241000588722 Escherichia Species 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims description 36
- 229960002429 proline Drugs 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 19
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 12
- 235000003704 aspartic acid Nutrition 0.000 claims description 12
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 12
- 230000005764 inhibitory process Effects 0.000 claims description 10
- 229930182821 L-proline Natural products 0.000 claims description 9
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
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- 102000004190 Enzymes Human genes 0.000 claims description 4
- 230000001851 biosynthetic effect Effects 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 102000005133 Glutamate 5-kinase Human genes 0.000 claims description 3
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- 101710198928 Gamma-glutamyl phosphate reductase Proteins 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 15
- 229940024606 amino acid Drugs 0.000 abstract description 7
- 150000001413 amino acids Chemical class 0.000 abstract description 7
- 239000000203 mixture Substances 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 abstract 6
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 17
- 239000002609 medium Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 14
- PMMYEEVYMWASQN-IMJSIDKUSA-N cis-4-Hydroxy-L-proline Chemical compound O[C@@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-IMJSIDKUSA-N 0.000 description 11
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000004098 Tetracycline Substances 0.000 description 5
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
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- 239000008272 agar Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000019270 ammonium chloride Nutrition 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 239000012736 aqueous medium Substances 0.000 description 3
- 230000003570 biosynthesizing effect Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- -1 nitrogen-containing organic compounds Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 238000005119 centrifugation Methods 0.000 description 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 101150046501 proB gene Proteins 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- UWDMKTDPDJCJOP-UHFFFAOYSA-N 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-ium-4-carboxylate Chemical compound CC1(C)CC(O)(C(O)=O)CC(C)(C)N1 UWDMKTDPDJCJOP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- WXSKVKPSMAHCSG-UWTATZPHSA-N D-4-hydroxy-2-oxoglutaric acid Chemical compound OC(=O)[C@H](O)CC(=O)C(O)=O WXSKVKPSMAHCSG-UWTATZPHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 1
- 229930182847 D-glutamic acid Natural products 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
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- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
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- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、ＤまたはＬ −４−ヒドロキシ−２オキソグ
ルタル酸（以下単に４−ヒドロキシル２オキソグルタル
酸と称す）からトランス−４ヒドロキシ−し−プロリン
および／またはシス４−ヒドロキシ−し−プロリン（以
下単に４−ヒドロキシ−し−プロリンと称す）を生合成
する能力を有する微生物を用いた４−ヒドロキシ−Ｌプ
ロリンの製造法に関する。４−ヒドロキシ−Ｌプロリン
は、医薬品の合成中間体などとして有用なアミノ酸であ
る。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to the production of trans-4-hydroxy-di-proline and/or Alternatively, the present invention relates to a method for producing 4-hydroxy-L-proline using a microorganism capable of biosynthesizing cis-4-hydroxy-l-proline (hereinafter simply referred to as 4-hydroxy-l-proline). 4-Hydroxy-L-proline is an amino acid useful as a synthetic intermediate for pharmaceuticals.
従来の技術
従来の４−ヒドロキシ−Ｌ−プロリンの製造法としては
、コラーゲンを加水分解し、その構成アミノ酸であるト
ランス−４−ヒドロキシ−Ｌ−プロリンを回収、分離す
る方法が知られている。またＤ−グルタミン酸から合成
する方法〔ビュレッティン・オブ・ケミカル・ソザエテ
イ・オブ・ジャパン（Ｂｕｌｌ、 Ｃｈｅｍ、Ｓｏｃ
、Ｊａｐ、） ４７． １７０４（１９７４） 〕や、
グリオキサールとオキサロ酢酸から合成する方法〔ジャ
ーナル・オブ・オルガニック・ケミストリイ（Ｊ、Ｏｒ
ｇ、 ［ｈｅｍ、＞ ４２ 、３４４０（１９７７＞
〕なども知られている。2. Description of the Related Art As a conventional method for producing 4-hydroxy-L-proline, a method is known in which collagen is hydrolyzed and its constituent amino acid trans-4-hydroxy-L-proline is recovered and separated. In addition, a method for synthesizing from D-glutamic acid [Bull, Chem, Soc.
, Jap, ) 47. 1704 (1974)] and
Synthesis method from glyoxal and oxaloacetic acid [Journal of Organic Chemistry (J, Or
g, [hem, > 42 , 3440 (1977 >
] are also known.
発明が解決しようとする課題
従来の製造法は、（１）原料が高価である、（２）反応
工程が多い、（３）回収精製の工程に問題があるなどの
点で、工業的な製法として必ずしも満足できる方法では
なく、工業的に安価に４−ヒドロキシトープロリンを製
造する方法の開発が求められている。Problems to be Solved by the Invention Conventional manufacturing methods are not industrially viable due to the following points: (1) raw materials are expensive, (2) there are many reaction steps, and (3) there are problems with the recovery and purification process. However, there is a need to develop a method for industrially producing 4-hydroxytoproline at low cost.
課題を解決するための手段
本発明者は、微生物の生合成系を利用して、４ヒドロキ
シ−２−オキソグルタル酸から４−ヒドロキシ−し−プ
ロリンを製造できることを見出し、本発明を完成した。Means for Solving the Problems The present inventors have discovered that 4-hydroxy-proline can be produced from 4-hydroxy-2-oxoglutaric acid using a microbial biosynthetic system, and have completed the present invention.
本発明は、エシェリヒア属に属し、４−ヒドロキシ−２
−オキソグルタル酸から４−ヒドロキシＬ−プロリンを
生合成する能力を有する微生物を用いて、アミノ基供与
体の存在下４−ヒドロキシ−２−オキソグルタル酸もし
くは該微生物によって４−ヒドロキシ−２−オキソグル
タル酸に転換される化合物から４−ヒドロキシ−し−プ
ロリンを製造する方法を提供する。The present invention belongs to the genus Escherichia, and 4-hydroxy-2
- Using a microorganism capable of biosynthesizing 4-hydroxy L-proline from oxoglutaric acid, 4-hydroxy-2-oxoglutaric acid or 4-hydroxy-2-oxoglutaric acid is converted by the microorganism into 4-hydroxy-2-oxoglutaric acid in the presence of an amino group donor. A method for producing 4-hydroxy-proline from converted compounds is provided.
以下に本発明の詳細な説明する。The present invention will be explained in detail below.
本発明は、具体的には、エシェリヒア属に属し、４−ヒ
ドロキシ−２−オキソグルタル酸から４ヒドロキシ−し
−プロリンを生合成する能力を有する微生物を、４−ヒ
ドロキシ−２−オキソグルタル酸もしくは該微生物によ
って４−ヒドロキシ２−オキソグルタル酸に転換される
化合物を添加した培地で培養し、培養物中に４−ヒドロ
キシトープロリンを生成蓄積させ、該培養物から４ヒド
ロキシ−し−プロリンを採取するか、またはエシェリヒ
ア属に属し、４−ヒドロキシ−２オキソグルタル酸から
４−ヒドロキシ−し−プロリンを生合成する能力を有す
る微生物の培養物、菌体またはそれらの処理物を、水性
媒体中でアミノ基供与体の存在下４−ヒドロキシ−２−
オキソグルタル酸もしくは該微生物によって４−ヒドロ
キシ−２−オキソグルタル酸に転換される化合物に作用
させて、反応液中に生成した４−ヒドロキシ−Ｌ−プロ
リンを採取することにより実施できる。Specifically, the present invention relates to a microorganism that belongs to the genus Escherichia and has the ability to biosynthesize 4-hydroxy-2-oxoglutaric acid from 4-hydroxy-2-oxoglutaric acid or the microorganism. culturing in a medium supplemented with a compound that is converted to 4-hydroxy-2-oxoglutaric acid, producing and accumulating 4-hydroxy-to-proline in the culture, and collecting 4-hydroxy-to-proline from the culture; Alternatively, a culture, bacterial cells, or a treated product of a microorganism belonging to the genus Escherichia and having the ability to biosynthesize 4-hydroxy-thi-proline from 4-hydroxy-2oxoglutaric acid is used as an amino group donor in an aqueous medium. 4-hydroxy-2- in the presence of
This can be carried out by reacting oxoglutaric acid or a compound that is converted to 4-hydroxy-2-oxoglutaric acid by the microorganism and collecting 4-hydroxy-L-proline produced in the reaction solution.
本発明に用いられる微生物としては、エシェリヒア属に
属し、４−ヒドロキシ−２−オキソグルタル酸から４−
ヒドロキシ−Ｌ−プロリンを生合成する能力を有する微
生物なら、いかなる微生物でも使用できる。具体的には
、エシェリヒア・コリΔＴＣＣ３３６２５などがあげら
れる。なかでも■、−プロリンによるフィードバック阻
害が解除されたＴ−グルタミルキナーゼを持ち、Ｌ−プ
ロリンを過剰生産する能力を獲得した該菌種の変異株、
あるいは遺伝子組換え等の手法によりＬ−プロリン生合
成酵素活性が増強された該菌種株を用いれば、より高い
収率で４−ヒドロキシ〜Ｌ−プロリンを得ることができ
る。このような組換え株は、トイチエらの記述した方法
〔ヌクレイツク・アシッヅ・リサーチ（Ｎｕｃｌｅｉｃ
Ａｃ１ｄｓ Ｒｅｓ、） １２ 。The microorganisms used in the present invention belong to the genus Escherichia, and include 4-hydroxy-2-oxoglutaric acid to 4-hydroxy-2-oxoglutaric acid.
Any microorganism can be used as long as it has the ability to biosynthesize hydroxy-L-proline. Specifically, Escherichia coli ΔTCC33625 and the like can be mentioned. Among these, ■, a mutant strain of the strain that has T-glutamyl kinase in which the feedback inhibition by proline has been released and has acquired the ability to overproduce L-proline;
Alternatively, 4-hydroxy to L-proline can be obtained at a higher yield by using the strain whose L-proline biosynthetic enzyme activity has been enhanced by techniques such as genetic recombination. Such recombinant strains can be obtained by the method described by Toichie et al.
Ac1ds Res, ) 12.
６３３７ （１９８４） ］に従えば取得可能である。6337 (1984)].
あるいは本明細書の実施例に記述した方法に従ってもよ
い。Alternatively, the method described in the Examples of this specification may be followed.
Ｌ−プロリンによるフィードバック阻害が解除されたＴ
−グルタミルキナーゼを持ち、Ｌ−プロリンを過剰生産
する能力を獲得したエシェリヒア属菌種の具体例として
は、■、−プロリンによるフィードバック阻害が解除さ
れたエシェリヒア・コｌＪＭＭ２９４株由来のｐｒｏＢ
遺伝子を組み込んだ組換え体ＤＮＡを保有するエシェリ
ヒア・コリに８３があげられる。該菌株は、平成２年３
月１５日付で、ブダペスト条約に基づいて、工業技術院
微生物工業技術研究所（微工研）に、エシェリヒア・コ
リＫ ８３ （ＦＢＲＭ ＢＰ−２８０７）として寄託
されている。T where feedback inhibition by L-proline is released
- Specific examples of Escherichia species that possess glutamyl kinase and have acquired the ability to overproduce L-proline include: - proB derived from Escherichia coli JMM294 strain in which feedback inhibition by proline has been released;
There are 83 examples of Escherichia coli that carry recombinant DNA that incorporates genes. The strain is from March 1990.
It has been deposited as Escherichia coli K 83 (FBRM BP-2807) with the Institute of Microbiology and Technology (Feikoken), Agency of Industrial Science and Technology, on the basis of the Budapest Treaty, dated May 15, 2015.
微生物中で４−ヒドロキシ−２−オキソグルタル酸に変
換される化合物としては、例えばエシェリヒア・コリの
場合には、４−ヒドロキシ−２オキソグルタル酸の生成
酵素Ｄ−４−ヒドロキシ２−オキソグルタル酸アルドラ
ーゼの基質であるピルビン酸とグリオキシル酸があげら
れる〔ジャーナル・オブ・バイオロジカル・ケミストリ
ー（、Ｊ、Ｂｉｏｌ、Ｃｈｅｍ、） ２４７ 、５０７
９（１９７２））。Compounds that are converted to 4-hydroxy-2-oxoglutarate in microorganisms include, for example, in the case of Escherichia coli, substrates for the 4-hydroxy-2oxoglutarate producing enzyme D-4-hydroxy-2-oxoglutarate aldolase. Examples include pyruvic acid and glyoxylic acid [Journal of Biological Chemistry (J. Biol. Chem.) 247, 507
9 (1972)).
また、培地または反応液に、さらにアスパラギン酸ある
いはアスパラギン、または微生物中でアスパラギン酸に
変換される化合物を添加すれば、より高収率で４−ヒド
ロキシ−Ｌ−プロリンが生成される。微生物中でアスパ
ラギン酸に変換される化合物としては、例えばエシェリ
ヒア・コリの場合には、その生成酵素アスパルターゼの
基質であるフマル酸とアンモニアがあげられる。Furthermore, if aspartic acid, asparagine, or a compound that is converted to aspartic acid in microorganisms is further added to the medium or reaction solution, 4-hydroxy-L-proline can be produced in a higher yield. Examples of compounds that are converted to aspartic acid in microorganisms include fumaric acid and ammonia, which are substrates for the production enzyme aspartase, in the case of Escherichia coli.
本発明に用いられる微生物の培養は、通常用いられる合
成ないし天然培地を用いて行うことができる。培地中の
炭素源としては、グルコース、フラクトース、シューク
ロース、マルトース、ラクトース、澱粉、澱粉加水分解
物、糖蜜、セルロース加水分解物などの糖類や、酢酸、
乳酸などの有機酸、あるいはエタノールなどのアルコー
ルなどが用いられる。窒素源としては、アンモニア、硫
酸アンニウム、塩化アンモニウム、酢酸アンモニウムな
どのアンモニウム塩や尿素、硝酸塩ならびにペプトン、
肉エキス、酵母エキス、コーン・スチープ・リカーなど
の各種の窒素含有有機化合物が使用可能である。無機塩
としては、リン酸カリウム、硫酸アンモニウム、塩化ア
ンモニウム、塩化ナトリウム、硫酸マグネシウム、硫酸
第１鉄、硫酸マンガンなどが使用できる。他に微量元素
としてカルシウム、亜鉛、はう素、銅、コバルト、モリ
ブデンなどの塩類を加えてもよい。また微生物によって
はビタミン、アミノ酸、核酸関連物質などの添加により
生育がより良好になるが、前記したような他の培地成分
にしたがって培地に供給されれば、特に加えなくてもよ
い。The microorganisms used in the present invention can be cultured using commonly used synthetic or natural media. Carbon sources in the medium include sugars such as glucose, fructose, sucrose, maltose, lactose, starch, starch hydrolysates, molasses, and cellulose hydrolysates, acetic acid,
Organic acids such as lactic acid or alcohols such as ethanol are used. Nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium acetate, urea, nitrates and peptone,
Various nitrogen-containing organic compounds can be used, such as meat extract, yeast extract, corn steep liquor, etc. As the inorganic salt, potassium phosphate, ammonium sulfate, ammonium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, etc. can be used. In addition, salts such as calcium, zinc, borosilicate, copper, cobalt, and molybdenum may be added as trace elements. Also, depending on the microorganism, the growth may be improved by adding vitamins, amino acids, nucleic acid-related substances, etc., but it is not necessary to add them as long as they are supplied to the medium according to the other medium components as described above.
上記培地に４−ヒドロキシ−２−オキソグルタル酸もし
くは微生物中で該化合物に変換される化合物を添加して
、上記微生物の培養を行うことにより、４−ヒドロキシ
−し−プロリンを培養液中に生成蓄積させることができ
る。培地に添加する４−ヒドロキシ−２−オキソグルタ
ル酸もしくは微生物中で該化合物に変換される化合物の
濃度には特に制限はないが、一般には０．１〜５０ｇ／
Ｉｔの濃度を保持しながら培養する。アスパラギン酸、
アスパラギン、または微生物中でアスパラギン酸に変換
される化合物の添加量にも特に制限はないが、一般には
０．１〜５０ｇ／ｌｌの濃度になるように加える。By adding 4-hydroxy-2-oxoglutaric acid or a compound that is converted into this compound in a microorganism to the above medium and culturing the above microorganism, 4-hydroxy-thi-proline is produced and accumulated in the culture solution. can be done. There is no particular limit to the concentration of 4-hydroxy-2-oxoglutaric acid added to the medium or the compound converted to this compound in microorganisms, but it is generally 0.1 to 50 g/
Culture is carried out while maintaining the concentration of It. aspartic acid,
There is no particular limit to the amount of asparagine or a compound that is converted to aspartic acid in microorganisms, but it is generally added at a concentration of 0.1 to 50 g/1.
培養液からの４−ヒドロキシ−Ｌ−プロリンの採取方法
は、通常培養液からアミノ酸採取に用いられる方法にし
たがって実施することができる。A method for collecting 4-hydroxy-L-proline from a culture solution can be carried out according to a method normally used for collecting amino acids from a culture solution.
例えば、遠心分離により菌体を除いた培養液上清から、
イオン交換樹脂膜処理法などの操作を組み合わせて、４
−ヒドロキシ−し−プロリンを単離することができる。For example, from the culture supernatant after removing bacterial cells by centrifugation,
By combining operations such as ion exchange resin membrane treatment, 4
-Hydroxy-proline can be isolated.
さらに、上記したような通常の培地を使用して培養して
得た微生物の培養物、菌体もしくはそれらの処理物と、
４−ヒドロキシ−２−オキソグルタル酸もしくは微生物
中で該化合物に変換される化合物とを、アミノ基供与体
の存在下に反応させて４−ヒドロキシ−Ｌ−プロリンを
生成させることもできる。反応は、水性媒体中４−ヒド
ロキシ２−オキソグルタル酸もしくは微生物中で該化合
物に変換される化合物１〜２００ｇ／β、微生物の菌体
またはその処理物０１〜２００ ｇ／Ｒ（微生物菌体換
算）、これにアミノ基供与体としてアスパラギン酸やア
スパラギン、各種アミノ酸、アンモニウム塩、尿素など
を加え、温度２０〜６０℃、ｐＨ６〜１１の条件で行い
、反応時間は０．１〜２００時間程度である。水性媒体
としては、例えばリン酸バッファーや生理食塩水などが
使用できる。また反応液から４−ヒドロキシ−し−プロ
リンを取得する方法としては、前記した方法が同様に使
用できる。Furthermore, microbial cultures, microbial cells, or processed products thereof obtained by culturing using the above-mentioned ordinary medium,
4-Hydroxy-L-proline can also be produced by reacting 4-hydroxy-2-oxoglutaric acid or a compound that is converted to this compound in a microorganism in the presence of an amino group donor. The reaction is carried out using 1 to 200 g/β of 4-hydroxy 2-oxoglutaric acid in an aqueous medium or a compound converted into the compound in a microorganism, and 01 to 200 g/R of microorganism cells or a processed product thereof (in terms of microorganism cells). To this, aspartic acid, asparagine, various amino acids, ammonium salts, urea, etc. are added as amino group donors, and the reaction is carried out at a temperature of 20 to 60°C and a pH of 6 to 11, and the reaction time is about 0.1 to 200 hours. . As the aqueous medium, for example, phosphate buffer, physiological saline, etc. can be used. Further, as a method for obtaining 4-hydroxy-proline from the reaction solution, the above-mentioned method can be similarly used.
以下に本発明の実施例を示す。Examples of the present invention are shown below.
実施例
１）エシェリヒア・コリのｐｒｏＡＢ遺伝子のクローニ
ング
エシェリヒア・コリに１２株のｐｒｏΔＢ遺伝子を含む
染色体ＤＮＡは、エシェリヒア・コＩＪ Ｋ１２株亜株
ＭＭ２９４ （ＡＴＣＣ３３６２５）より常法〔バイオ
キミカ・工・バイオフィジカ・アクタ（Ｂｉｏｃｈｉｍ
、 Ｂｉｏｐｈｙｓ、八ｃｔａ）７２．６１９（１９６
３） ：］により単離した。ベクターとして使用したｐ
ＢＲ３２２（アンピシリン耐性、テトラサイクリン耐性
）は、全酒造社製の市販品を用いた。ｐ Ｂ Ｒ３２２
プラスミドＤＮＡ １ｇｇおよびＭＭ２９４株染色体Ｄ
ＮＡ３μｇを含む制限酵素ＰｓｔＩ用反応被反応液ｍＭ
）リス、５０ｍＭ Ｎａ（、ｆｌ、７ｍＭ Ｍｇ（
、Ｌ、ｐＨ７，５）１００ｎ（ｌに１６単位のＰｓｔＩ
（全酒造社製）を添加し、３７℃で６０分間反応後、６
５℃で４０分間加温して反応を停止させた。該反応消化
物に、１０倍濃度のＴ４１Ｊガーゼ緩衝液２
（６６０ｍＭ）リス、６６ｍＭ ＭｇＣｊ！、 、１
００ｍＭジチオスレイトール、ｐＨ７，６）１２ｕＱ、
１００ｍＭＡＴＰ３μｇおよびＴ４１Ｊガーゼ（全
酒造社製）３５０単位を加え、１５℃で１６時間反応さ
せた。Example 1) Cloning of the proAB gene of Escherichia coli Chromosomal DNA containing the proΔB gene of 12 strains of Escherichia coli was cloned from Escherichia coli IJ K12 substrain MM294 (ATCC 33625) using a conventional method [Biochimica, Engineering, Biophysics, Acta (Biochim
, Biophys, octa) 72.619 (196
3) :]. p used as a vector
As BR322 (ampicillin resistant, tetracycline resistant), a commercial product manufactured by Zenshuzo Co., Ltd. was used. pB R322
1 gg of plasmid DNA and MM294 strain chromosome D
Reaction solution for restriction enzyme PstI containing 3 μg of NA (mM)
) squirrel, 50mM Na(, fl, 7mM Mg(
, L, pH 7,5) 100 n (16 units of PstI in l)
(manufactured by Zenshuzo Co., Ltd.) and reacted at 37°C for 60 minutes.
The reaction was stopped by heating at 5° C. for 40 minutes. The reaction digest was supplemented with 10x T41J gauze buffer 2 (660mM), 66mM MgCj! , ,1
00mM dithiothreitol, pH 7,6) 12uQ,
3 μg of 100 mM ATP and 350 units of T41J gauze (manufactured by Zenshuzo Co., Ltd.) were added, and the mixture was allowed to react at 15° C. for 16 hours.
このリガーゼ反応混合物をｐｒｏＡの欠損変異を有する
エシェリヒア・コリに１２株亜株ＨＢＩＯＩ（ＡＴＣＣ
３３６９４）Ｃマニアナイスら；モレキュラー・クロー
ニング（Ｍｏｌｅｃｕｌａｒ ［：ｌｏｎｉｎｇ、 Ａ
Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ）（１９Ｂ２Ｌ
５０４頁］の形質転換に供した。エシェリヒア・コリの
形質転換は常法〔モレキュラー・クローニング（Ｍｏｌ
ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ。This ligase reaction mixture was applied to 12 Escherichia coli substrains HBIOI (ATCC
33694) C Maninais et al.; Molecular cloning (Molecular [:loning, A
Laboratory Manual) (19B2L
p. 504] was used for transformation. Transformation of Escherichia coli is carried out using a conventional method [Molecular Cloning (Mol)].
ecular Cloning.
Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ）（１９８２
）、 ２５０−２５１頁］に従った。選択培地には、２
０μｇ／−のテトラサイクリンを含むデービス最小寒天
培地〔グルコース２ｇ、（ＮＨ４）２ＳＯ４１ｇ、に２
ＨＰ０４７ｇ。A Laboratory Manual) (1982
), pp. 250-251]. The selective medium contains 2
Davis minimal agar medium containing 0 μg/- of tetracycline [2 g of glucose, 1 g of (NH4)2SO4,
HP047g.
ＫＨ２ＰＯ−２ｇ５Ｍｇ５○４ ・７Ｈ２００，１ｇ。KH2PO-2g5Mg5○4・7H200,1g.
クエン酸３ナトリウム塩０．５ｇ、サイアミン塩酸塩４
ｍｇおよび寒天１６ｇを水１１−に含み、ｐＨ７２に調
整した培地〕を用いた。出現した形質転換株の培養菌体
からマニアナイスらにより記述された方法〔モレキュラ
ー・クローニング（ＭｏｌｅｃｕｌａｒＣｌｏｎｉｎｇ
、Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ）（１９Ｂ
２）、 ８６−９６頁〕に従って、プラスミドＤＮＡ
を単離した。形質転換株の１株から得られｐＰＲＣ）−
１と命名されたプラスミドは、各種制限酵素での消化と
アガロスゲルミ気泳動による解析の結果、ｐＢＲ３２２
の唯一のＰｓｔｌ切断部位に、トイチエらの報告したｐ
ｒｏＡＢオペロン〔ヌクレイツク・アシッヅ・リサーチ
（Ｎｕｃｌｅｉｃ Ａｃ１ｄｓ Ｒｅｓ、） １２．６
３３７（１９８４）］と同一の構造を有する３、０キロ
ベースのＰｓｌｌＤＮＡ断片が挿入された構造であるこ
とが示された。ｐＰＲＯ−１を用い８８１０１株を再形
質転換した。選択培地には、２０μｇ／噌のテトラサイ
クリンを含むし培地〔バクトドリプトン１０ｇ、酵母エ
キス５ｇ、グルコース１ｇ、ＮａＣｊ！ ５ｇを水１
１に含み、ｐ Ｈ７，２に調整した培地〕を用いた。得
られたテトラサイクリン耐性形質転換株はすべてプロリ
ン非要求性を示し、エシェリヒア・コリに１２株のｐｒ
ｏＡＢ遺伝子がクローニングされたことが確認された。Citric acid trisodium salt 0.5g, thiamine hydrochloride 4
A medium containing 16 g of agar and 16 g of agar in 11 g of water and adjusted to pH 72] was used. The method described by Manianais et al. [Molecular Cloning]
, A Laboratory Manual) (19B
2), pp. 86-96].
was isolated. pPRC obtained from one of the transformed strains)
As a result of digestion with various restriction enzymes and analysis by agarose gel electrophoresis, the plasmid named 1 was found to be pBR322.
The pstl cleavage site reported by Toichie et al.
roAB Operon [Nucleic Acids Research (Nucleic Ac1ds Res,) 12.6
337 (1984)] into which a 3,0 kilobase Psll DNA fragment was inserted. The 88101 strain was retransformed using pPRO-1. The selective medium contains 20 μg/ml of tetracycline [Bactodryptone 10 g, yeast extract 5 g, glucose 1 g, NaCj! 5g to 1 water
1 and adjusted to pH 7.2] was used. All of the obtained tetracycline-resistant transformants showed proline non-auxotrophy, and 12 strains of pr
It was confirmed that the oAB gene had been cloned.
ｐＰＲ０１の作製過程を第１図に示す。The production process of pPR01 is shown in FIG.
２）プロリンによるフィードバック阻害が弱められた変
異型ｐＰＲＯ−１の取得
プロリンによるフィードバック阻害が弱められた変異型
ｐＰＲｃ）−１を、プロリンのアナログである３、４−
デヒドロ−ＤＬ−プロリン（ＤＨＰ）を用いて、以下の
ようにして取得した。ｐＰＲｏｌを保有するＨ８１０１
株にＮ−メチル−Ｎ′ニトロ−Ｎ−ニトロソグアニジン
（ＮＴＧ）４００μｇ／―を用いて通常の変異処理操作
を施した後、１００μｇ／＋ｍｆ＋のＤＨＰを含むデー
ビス最小寒天培地に塗布した。出現したＤ ＨＰ ｉＪ
性株について、スミスらにより記述された方法〔シャー
ナＪＬ、・オブーハクテリオロシー（Ｊ、Ｂａｃｔｅｒ
ｉｏｌ、 ）１５７ 、５４５（１９８４） ］に従っ
て、ｐｒｏＢ遺伝子によってコードされ、通常はプロリ
ンによるフィードバック阻害を受ける酵素γ−グルタミ
ルキナゼの活性を測定した。その結果、ＤＨＰ耐性株の
１株であるＤＨＰＩ株の該酵素については、プロリンに
よる阻害が約１００倍弱狛られていることが判明した。2) Obtaining mutant pPRO-1 with weakened feedback inhibition by proline. Mutant pPRc)-1 with weakened feedback inhibition by proline was obtained from proline analogues 3,4-
It was obtained as follows using dehydro-DL-proline (DHP). H8101 carrying pPRol
The strain was subjected to the usual mutation treatment using 400 μg/- of N-methyl-N'nitro-N-nitrosoguanidine (NTG) and then plated on Davis minimal agar medium containing 100 μg/+mf+ of DHP. Appeared D HP iJ
For bacterial strains, the method described by Smith et al.
Iol, ) 157, 545 (1984)], the activity of the enzyme γ-glutamylkinase, which is encoded by the proB gene and is normally subject to feedback inhibition by proline, was measured. As a result, it was found that the inhibition of the enzyme by proline in the DHPI strain, which is one of the DHP-resistant strains, was about 100 times weaker.
このＤＨＰＩ株より前述の方法に従って調製されｐＰＲ
Ｏ−１１と命名したプラスミドを用い、Ｈ８１０１株を
再形質転換したところ、得られたテトラサイクリン耐性
株はすべて１００μｇ／ｍｆｆのＤＨＰに耐性であった
。このようにして、プロリンによるフィードバッグ阻害
の弱杓られた変異型Ｔ−グルタミルキナーゼ遺伝子を含
むプラスミドを取得した。pPR was prepared from this DHPI strain according to the method described above.
When the H8101 strain was retransformed using the plasmid designated O-11, all of the resulting tetracycline-resistant strains were resistant to 100 μg/mff of DHP. In this way, a plasmid containing a mutant T-glutamyl kinase gene in which feedback inhibition by proline was weakly inhibited was obtained.
３）４−ヒドロキシ−２−オキソグルタル酸の調製
ルッフォらの方法〔バイオケミカル・ジャーナル（Ｂｉ
ｏｃｈｅｍ、 Ｊ、） ８５ 、５８８（１９６２）］
に従い、４ヒドロキシ−２−オキソグルタル酸を調製し
た。3) Preparation of 4-hydroxy-2-oxoglutaric acid The method of Ruffo et al. [Biochemical Journal (Bi
Ochem, J.) 85, 588 (1962)]
4-hydroxy-2-oxoglutaric acid was prepared according to.
即ち、１．３２ ｇのオキシロ酢酸および１．１４ ｇ
のグリオキシル酸ナトリウムを純水に溶解し、水酸化ナ
トリウムでｐＨを７．４に調整した後、容量を５０ｍ１
とした。４０℃で３時間加温した後、塩化水素でｐＨを
３に調整した。室温で３０分放置した後、水酸化す）
ＩＪウムでｐＨを再び中性にした。i.e. 1.32 g oxyloacetic acid and 1.14 g
of sodium glyoxylate was dissolved in pure water, the pH was adjusted to 7.4 with sodium hydroxide, and the volume was reduced to 50ml.
And so. After heating at 40° C. for 3 hours, the pH was adjusted to 3 with hydrogen chloride. After leaving it at room temperature for 30 minutes, it is hydroxylated)
The pH was made neutral again with IJum.
グルタミン酸デヒドロゲナーセによる定量〔バ５
６
グマイヤー編；メソッヅ・オブ・エンザイマティック・
アナリシス（Ｍｅｔｈｏｄｓ ｏｆ Ｂｎｚｙｍａｔｉ
ｃ＾ｎａｌｙｓｉｓ）第２版、第１巻、ｐ４６１ （１
９７４） ］の結果、該反応液中には０．１８ ｍｏｌ
ｅ／ Ｉｌ （７） Ｄ Ｌ−，４−ヒドロキシ−２−
オキソグルタル酸が生成していた。Determination by glutamate dehydrogenase [edited by Gmeyer; Methods of Enzymatics]
Analysis (Methods of Bnzymati)
c^nalysis) 2nd edition, Volume 1, p461 (1
974) ] As a result, 0.18 mol was present in the reaction solution.
e/Il (7) D L-,4-hydroxy-2-
Oxoglutaric acid was produced.
４）４−ヒドロキシ−２−オキソグルタル酸からの４−
ヒドロキシ−Ｌ−プロリンの生産（１）３）項で調製し
たＤＬ−４−ヒドロキシ−２オキソグルタル酸を用いて
、以下のようにトランス−４−ヒドロキシ−し−プロリ
ンおよびシス４−ヒドロキシ−し−プロリンの生産試験
を行った。4) 4- from 4-hydroxy-2-oxoglutaric acid
Production of hydroxy-L-proline Using DL-4-hydroxy-2oxoglutaric acid prepared in section (1) 3), trans-4-hydroxy-di-proline and cis-4-hydroxy-di-proline were produced as follows. A proline production test was conducted.
３ｍｌのＬ培地を試験管に分注し、滅菌後、エシェリヒ
ア・コリに１２株亜株ＭＭ ２９４’ （ＡＴＣＣ３３
６２５）、および常法によりｐＰＲｏ−１１を導入した
ＭＭ２９４株（Ｋ２Ｓ株）を接種し、３０℃で１６時間
振とう培養した。培養終了後、遠心分離操作にて該培養
物よりエシェリヒア・コリの菌体を集め、滅菌した純水
にて洗浄後、滅菌した１０ｍ１のＡ培地〔グルコース３
０ｇ１ＫＨ２Ｐ０４ １ｇ、 （ＮＨ４）２ＳＯ４１
０ｇ。Dispense 3 ml of L medium into test tubes, sterilize them, and inoculate Escherichia coli with 12 substrains MM 294' (ATCC33
625) and MM294 strain (K2S strain) into which pPRo-11 had been introduced by a conventional method, and cultured with shaking at 30°C for 16 hours. After culturing, Escherichia coli cells were collected from the culture by centrifugation, washed with sterilized pure water, and then added to 10 ml of sterilized A medium [Glucose 3
0g1KH2P04 1g, (NH4)2SO41
0g.
Ｍｇ５Ｏ＜’ ７Ｈ２０１ｇ、Ｆ ｅＳＯ４’ ７
Ｈ７Ｈ２Ｏ２，ＭｎＳＯ４・ ７Ｈ２０２ｍｇ、ＣａＣ
Ｃ）＋ ３０ｇを純水１βに含みｐ Ｈ７，４に調整
した培地〕に懸濁し、大形試験管中で３０℃で振とう培
養した。Mg5O<' 7H201g, FeSO4' 7
H7H2O2, MnSO4・7H202mg, CaC
C) +30g was suspended in a medium containing pure water 1β and adjusted to pH 7.4, and cultured with shaking at 30°C in a large test tube.
培養開始８時間後に３）項で調製したＤＬ−４ヒドロキ
シ−２−オキソグルタル酸（ＨＯＧ）を液中濃度が１０
ｍＭになるように添加し、さらに６０時間培養した。ベ
ロンの記述した方法〔アナリテイカル・バイオケミスト
リー（Ａｎａｌ、Ｂｌｏｃｈｅｍ、）１３７ 、１５１
（１９８４） ：ｌに従い、薄層クロマトグラフィーに
て培養物中の２級アミンを分析したところ、ＨＯＧを添
加したに８３株の培養物上清からトランス−４−ヒドロ
キシ−Ｌ−プロリンおよびシス４−ヒドロキシ−Ｌ−プ
ロリンが検出された。8 hours after the start of culture, DL-4 hydroxy-2-oxoglutaric acid (HOG) prepared in section 3) was added to the solution at a concentration of 10
The solution was added to give a concentration of mM, and the cells were further cultured for 60 hours. The method described by Bellon [Analytical Biochemistry (Anal, Blochem,) 137, 151
(1984): When secondary amines in the culture were analyzed by thin layer chromatography, trans-4-hydroxy-L-proline and cis-4 -Hydroxy-L-proline was detected.
ＨＰＬＣにて定量した培養液中のトランス−４ヒドロキ
シ−Ｌ−プロリンおよびシス−４−ヒドロキシ−Ｌ−プ
ロリンの量を第１表に示す。Table 1 shows the amounts of trans-4-hydroxy-L-proline and cis-4-hydroxy-L-proline in the culture solution determined by HPLC.
第
１
表
第
表
５）４−ヒドロキシ−２−オキソグルタル酸からの４−
ヒドロキシ−Ｌ−プロリンの生産（２）４）項と同様に
してＭＭ２９４株およびに８３株をＡ培地にて培養した
。培養開始８時間後、ＨＯＧ ］、ＯｍＭ、ＨＯＧ１０
ｍＭおよびアスパラギン酸（△ＳＰ）またはアスパラギ
ン（、へＳＮ）２０ｍＭまたはピルビン酸（ＰＹＲ）と
グツオシシル酸（ＧＯＸ）を同時に各５０ｍ八１をそれ
ぞれ添加して、さらに６０時間培養した。ＨＰＬＣにて
定量した培養液中のトランス−４−ヒドロキシ−Ｌ−プ
ロリンおよびシス−４−ヒドロキシ−Ｌ−プロリンの量
を第２表に示す。Table 1 Table 5) 4- from 4-hydroxy-2-oxoglutaric acid
Production of hydroxy-L-proline (2) MM294 strain and MM83 strain were cultured in A medium in the same manner as in section 4). 8 hours after the start of culture, HOG], OmM, HOG10
50mM and 20mM of aspartic acid (ΔSP) or asparagine (SN) or pyruvate (PYR) and gastrointestinal acid (GOX) were added at the same time, and the cells were further cultured for 60 hours. Table 2 shows the amounts of trans-4-hydroxy-L-proline and cis-4-hydroxy-L-proline in the culture solution determined by HPLC.
９
６）４−ヒドロキシ−２−オキソグルタル酸からの４−
ヒドロキシ−し−プロリンの生産（３）Ｂ培地〔クルコ
ース１０ｇ、クエン酸２ｇ、ＮａＮＨｌＨＰＯ＜ ・
４ Ｈ２Ｏ３ｇ、に２ＨＰ０゜５ ｇ、 Ｍｇ Ｓ Ｏ
４・７ Ｈ２００，５ｇ １酵母工キスＩｇを純水ｌｌ
中に含みｐ Ｈ７，０に調整した培地〕１００ｍＱを三
角フラスコにとり、１２０℃、２０分間殺菌した後、Ｋ
８３株を１白金耳植菌し、３０℃で２４時間培養した。9 6) 4- from 4-hydroxy-2-oxoglutaric acid
Production of hydroxy-proline (3) B medium [10 g of crucose, 2 g of citric acid, NaNHlHPO<・
4 H2O3g, 2HP0゜5g, Mg SO
4.7 H200, 5g 1 yeast extract Ig with pure water
100 mQ of the medium contained in the medium adjusted to pH 7.0 was placed in an Erlenmeyer flask, sterilized at 120°C for 20 minutes, and then
One platinum loop of 83 strains was inoculated and cultured at 30°C for 24 hours.
培養後遠心分離し、５０ｍＭＩＪン酸バッファー（ｐＨ
７，５）で洗浄して得た０
湿菌体を、グルコース１％、４−ヒドロキシ−２オキソ
グルタル酸４０ ｍＭ、塩化アンモニウム０．１％、ア
スパラギン酸０．１％を含有する予めｐ Ｈ７，５に調
整した反応液に懸濁し、３０℃で２日間反応を行った。After culturing, centrifuge and add 50mM IJ acid buffer (pH
The wet bacterial cells obtained by washing with 7,5) were pretreated with pH 7, containing 1% glucose, 40 mM 4-hydroxy-2oxoglutaric acid, 0.1% ammonium chloride, and 0.1% aspartic acid. The suspension was suspended in a reaction solution adjusted to a temperature of 5, and the reaction was carried out at 30°C for 2 days.
培養液中のトランス−４−ヒドロキシ−Ｌ−プロリンお
よびシス−４−ヒドロキシ−Ｌ−プロリンをＨＰＬＣに
て定量したところ、０．７ｍＭのトランス−４−ヒドロ
キシ−し−プロリンおよび０．７ ｍＭのシス−４−ヒ
ドロキシ−Ｌプロリンの生成が認められた。Trans-4-hydroxy-L-proline and cis-4-hydroxy-L-proline in the culture solution were quantified by HPLC. Production of cis-4-hydroxy-L proline was observed.
発明の効果
本発明によれば、４−ヒドロキシ−２−オキソグルタル
酸から４−ヒドロキシ−Ｌ−プロリンを生合成する能力
を有する微生物を、４−ヒドロキシ−２−オキソグルタ
ル酸もしくは微生物中で該化合物に変換される化合物と
作用させることにより、４−ヒドロキシ−し−プロリン
を効率よく製造することができる。Effects of the Invention According to the present invention, a microorganism capable of biosynthesizing 4-hydroxy-L-proline from 4-hydroxy-2-oxoglutaric acid is converted into 4-hydroxy-2-oxoglutaric acid or the compound in the microorganism. By reacting with the compound to be converted, 4-hydroxy-proline can be efficiently produced.
よび５ａｌｌによる切断地図とｐ Ｐ ＲＣＩ−１の作
製過程を示す。図中ＰはＰｓｔｌを、Ｓは５ａ１１を表
ず。プラスミドの大きさは、キロベース（ｋｂ）で表示
されている。ｐＰＲＯ−１の太い実線部分は、ＭＭ２９
４株染色体ＤＮＡよりクローニングしたｐ ｒ ｏＡＢ
遺伝子を含むＤＮＡ断片を表している。Figure 5 shows the cleavage map by and 5all and the production process of pPRCI-1. In the figure, P stands for Pstl and S stands for 5a11. Plasmid sizes are expressed in kilobases (kb). The thick solid line part of pPRO-1 is MM29
p r oAB cloned from 4-strain chromosomal DNA
Represents a DNA fragment containing a gene.
Claims (7)
キソグルタル酸から４−ヒドロキシ−Ｌ−プロリンを生
合成する能力を有する微生物を用いて、アミノ基供与体
の存在下４−ヒドロキシ−２−オキソグルタル酸もしく
は該微生物によって４−ヒドロキシ−２−オキソグルタ
ル酸に転換される化合物から４−ヒドロキシ−Ｌ−プロ
リンを製造する方法。(1) Using a microorganism that belongs to the genus Escherichia and has the ability to biosynthesize 4-hydroxy-L-proline from 4-hydroxy-2-oxoglutaric acid, 4-hydroxy-2-oxoglutaric acid was synthesized in the presence of an amino group donor. A method for producing 4-hydroxy-L-proline from an acid or a compound that is converted to 4-hydroxy-2-oxoglutaric acid by the microorganism.
シ−２−オキソグルタル酸から４−ヒドロキシ−Ｌ−プ
ロリンを生合成する能力を有する微生物を、４−ヒドロ
キシ−２−オキソグルタル酸もしくは該微生物によって
４−ヒドロキシ−２−オキソグルタル酸に転換される化
合物を添加した培地で培養し、培養物中に４−ヒドロキ
シ−Ｌ−プロリンを生成蓄積させ、該培養物から４−ヒ
ドロキシ−Ｌ−プロリンを採取することからなる方法で
ある請求項１記載の方法。(2) The method is a method for converting a microorganism belonging to the genus Escherichia and having the ability to biosynthesize 4-hydroxy-L-proline from 4-hydroxy-2-oxoglutaric acid using 4-hydroxy-2-oxoglutaric acid or the microorganism. Cultivate in a medium supplemented with a compound that is converted to 4-hydroxy-2-oxoglutaric acid, produce and accumulate 4-hydroxy-L-proline in the culture, and collect 4-hydroxy-L-proline from the culture. 2. The method of claim 1, which comprises:
は該微生物によってアスパラギン酸に転換される化合物
を添加して培養する請求項２記載の方法。(3) The method according to claim 2, wherein aspartic acid, asparagine, or a compound that is converted to aspartic acid by the microorganism is added to the medium for culturing.
シ−２−オキソグルタル酸から４−ヒドロキシ−Ｌ−プ
ロリンを生合成する能力を有する微生物の培養物、菌体
またはそれらの処理物を、水性媒体中でアミノ基供与体
の存在下４−ヒドロキシ−２−オキソグルタル酸もしく
は該微生物によって４−ヒドロキシ−２−オキソグルタ
ル酸に転換される化合物に作用させて、反応液中に生成
した４−ヒドロキシ−Ｌ−プロリンを採取することから
なる方法である請求項１記載の方法。(4) The method comprises aqueous culture, cells, or processed products of a microorganism belonging to the genus Escherichia and having the ability to biosynthesize 4-hydroxy-L-proline from 4-hydroxy-2-oxoglutaric acid. 4-Hydroxy-2-oxoglutaric acid or a compound that is converted to 4-hydroxy-2-oxoglutaric acid by the microorganism is reacted with 4-hydroxy-2-oxoglutaric acid in the presence of an amino group donor in a medium to produce 4-hydroxy-2-oxoglutaric acid in the reaction solution. The method according to claim 1, which comprises collecting L-proline.
たは該微生物によってアスパラギン酸に転換される化合
物を添加して反応する請求項４記載の方法。(5) The method according to claim 4, wherein aspartic acid, asparagine, or a compound that is converted to aspartic acid by the microorganism is added to the reaction solution.
阻害から解除されたＬ−プロリン生合成系を有する微生
物であるか、またはＬ−プロリン生合成系の酵素活性量
が増強された微生物である請求項１〜５記載の方法。(6) A claim in which the microorganism is a microorganism that has an L-proline biosynthetic system that has been released from feedback inhibition by L-proline, or a microorganism that has an enhanced enzyme activity of the L-proline biosynthetic system. 5. The method described in 1 to 5.
ードバック阻害が解除されたγ−グルタミルキナーゼを
コードする遺伝子を組み込んだ組換え体ＤＮＡを保有す
る微生物。(7) A microorganism belonging to the genus Escherichia and containing a recombinant DNA incorporating a gene encoding γ-glutamyl kinase in which feedback inhibition by L-proline has been released.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6678290A JP3078296B2 (en) | 1990-03-19 | 1990-03-19 | Method for producing 4-hydroxy-L-proline |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6678290A JP3078296B2 (en) | 1990-03-19 | 1990-03-19 | Method for producing 4-hydroxy-L-proline |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03266995A true JPH03266995A (en) | 1991-11-27 |
| JP3078296B2 JP3078296B2 (en) | 2000-08-21 |
Family
ID=13325782
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6678290A Expired - Lifetime JP3078296B2 (en) | 1990-03-19 | 1990-03-19 | Method for producing 4-hydroxy-L-proline |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3078296B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0547898A2 (en) * | 1991-12-17 | 1993-06-23 | Sankyo Company Limited | Microbial process for the production of trans-4-hydroxy-L-proline |
| US5607848A (en) * | 1994-03-01 | 1997-03-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing optically active 4-hydroxy-2-ketoglutaric acid using microorganisms |
| JP2011097903A (en) * | 2009-11-09 | 2011-05-19 | Kyowa Hakko Bio Co Ltd | Method for producing cis-hydroxy-l-proline |
-
1990
- 1990-03-19 JP JP6678290A patent/JP3078296B2/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0547898A2 (en) * | 1991-12-17 | 1993-06-23 | Sankyo Company Limited | Microbial process for the production of trans-4-hydroxy-L-proline |
| EP0547898A3 (en) * | 1991-12-17 | 1994-08-03 | Sankyo Co | |
| US5607848A (en) * | 1994-03-01 | 1997-03-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing optically active 4-hydroxy-2-ketoglutaric acid using microorganisms |
| JP2011097903A (en) * | 2009-11-09 | 2011-05-19 | Kyowa Hakko Bio Co Ltd | Method for producing cis-hydroxy-l-proline |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3078296B2 (en) | 2000-08-21 |
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