JP2023540292A - L-ヒスチジン排出蛋白質およびこれを利用したl-ヒスチジン生産方法 - Google Patents
L-ヒスチジン排出蛋白質およびこれを利用したl-ヒスチジン生産方法 Download PDFInfo
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- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- LVCQAASWWXWFTQ-UHFFFAOYSA-L magnesium;sulfate;pentahydrate Chemical compound O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O LVCQAASWWXWFTQ-UHFFFAOYSA-L 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 238000000520 microinjection Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229940111688 monobasic potassium phosphate Drugs 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 230000006798 recombination Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
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- 239000008117 stearic acid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000020238 sunflower seed Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 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
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- 101150062776 yccA gene Proteins 0.000 description 1
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Abstract
Description
配列番号41、配列番号42、またはその組み合わせ、および、
配列番号44、配列番号45、またはその組み合わせ。
L-ヒスチジン生産能を有する微生物が前記L-ヒスチジン排出蛋白質を発現するように改変、例えば、1)前記L-ヒスチジン排出蛋白質を追加的に発現したり、2)内在的L-ヒスチジン排出蛋白質を代替して発現するように改変されることによって、非改変微生物に比べて増加されたL-ヒスチジン生産能を有する場合、および/または
L-ヒスチジン生産能を有さない微生物が前記L-ヒスチジン排出蛋白質を発現するように改変されることによってL-アミノ酸生産能を有するようになる場合、を意味するために使用され得る。
L-ヒスチジンは、アミノ酸分類の中で主に塩基性(Basic)アミノ酸に分類されるが、芳香族(Aromatic)アミノ酸または分枝鎖(Branched chain)アミノ酸に分類されたりもする。L-ヒスチジン特異排出能を有する蛋白質候補を選別するために各分類別アミノ酸(塩基性アミノ酸:L-リジン、芳香族アミノ酸:Trp、分枝鎖アミノ酸:イソロイシン)に対する排出蛋白質(LysE(Arch Microbiol 180:155-160)、Wex(大韓民国登録特許第10-1968317号)、BrnFE(Arch Microbiol 180:155-160))のアミノ酸配列をquery配列とし、NCBIとKegg databaseを基盤としてPSI-BLAST探索結果、L-ヒスチジンを排出する可能性のある膜蛋白質として予測される候補遺伝子とこれを保有する微生物を選定した。
前記実施例1で選定した外来L-ヒスチジン排出遺伝子候補6種をコリネバクテリウム属菌株に導入するためのベクター6種を作製した。
前記実施例2で作製された7種の組換えコリネバクテリウム・グルタミカム菌株(ATCC13032ΔN2131、ATCC13032ΔN2131::Haq、ATCC13032ΔN2131::Cpi、ATCC13032ΔN2131::Kcr、ATCC13032ΔN2131::Cst、ATCC13032ΔN2131::Lsa、およびATCC13032ΔN2131::Dva)のL-ヒスチジン排出能活性の保有有無の確認のために、L-ヒスチジンを利用した最小阻止濃度(minimum inhibitory concentration、MIC)実験を行った。7種の菌株を最小液体培地に30℃で24時間培養した後、1×103と1×104個の細胞で希釈してL-ヒスチジンが添加された最小固体培地でスポッティング(spotting)培養した。前記使用された最小固体培地の組成は次のとおりである。
ブドウ糖10g、KH2PO4 1g、K2HPO4 2g、MgSO4 7H2O 0.4g、尿素2g、(NH4)2SO4 5g、NaCl 0.5g、ニコチンアミド5μg、カルシウム-パントテン酸0.1μg、ビオチン0.2μg、チアミンHCl 3μg、Trace elements solution* 1ml(蒸溜水1リットル基準)、20g Agar
Na2B4O7 10H2O 0.09g、(NH4)6Mo7O27 4H2O 0.04 g、ZnSO4 7H2O 0.01 g、CuSO4 5H2O 0.27g、MnCl2 4H2O 0.01g、FeCl3 6H2O 1g、CaCl2 0.01g(蒸溜水1リットル基準)
+:single colonyは形成されないが、heavy(single colonyとして成長されずにかたまって成長する形態)は形成される
++:heavyが形成され、single colonyが5個未満形成される
+++:heavyが形成され、single colonyが50個未満形成される
++++:heavyがsingle colonyと区分されないように形成される)
Dermabacter vaginalis由来蛋白質DvaのL-ヒスチジン排出能を確認するために、Dermabacter vaginalis由来遺伝子dvaをL-ヒスチジン生産菌株KCCM 80179(大韓民国出願特許第10-2019-004693414-682号)菌株に導入した。
肉汁1%(w/v)、ポリペプトン1%(w/v)、塩化ナトリウム0.5%(w/v)、酵母エキス1%(w/v)、寒天2%(w/v)、pH7.2
ブドウ糖5%(w/v)、バクトペプトン1%(w/v)、塩化ナトリウム0.25%(w/v)、酵母エキス1%(w/v)、尿素0.4%(w/v)、pH7.2
ブドウ糖10%(w/v)、硫酸アンモニウム2%(w/v)、第1リン酸カリウム0.1%(w/v)、硫酸マグネシウム7水塩0.05%(w/v)、CSL(とうもろこし浸漬液)2.0%(w/v)、ビオチン200μg/L、炭酸カルシウム、pH7.2
前記実施例3と4でDermabacter vaginalis由来蛋白質のL-ヒスチジン排出能を確認したため、前記蛋白質とアミノ酸の配列相同性が高い類似蛋白質を追加的に確保するために、DvaFE中のDvaFの配列(配列番号12)をqueryで利用してBLAST探索を行った(表4参照)。
前記実施例5で追加選定されたL-ヒスチジン排出遺伝子候補2種をコリネバクテリウム属菌株に導入するためのベクター2種を作製した。実施例2と同一にNCgl2131遺伝子を欠損siteとして、PgapAをプロモーターとして使用した。
Helcobacillus massiliensis由来蛋白質HmaおよびMycobacterium abscessus subsp. abscessus由来蛋白質MabがL-ヒスチジン排出能を有するか否かを確認するために、hmaとmabをそれぞれL-ヒスチジン生産菌株KCCM 80179菌株に導入した。
前記Dermabacter vaginalis由来蛋白質Dva、Helcobacillus massiliensis由来蛋白質Hma、およびMycobacterium abscessus subsp. abscessus由来蛋白質MabのL-ヒスチジン排出能を再度確認するために、野生型のコリネバクテリウム・グルタミカムATCC13032由来でL-ヒスチジンによるフィードバック制限解消HisGポリペプチド変異導入、L-ヒスチジン生合成遺伝子が強化されたL-ヒスチジン生産菌株CA14-737(大韓民国出願特許第10-2019-004693414-682号)菌株に導入した。
前記選別されたL-ヒスチジン排出体が多様な菌株でL-ヒスチジン生産能を示すか否かを確認した。このために、大腸菌でDermabacter vaginalis由来、Helcobacillus massiliensis由来、Mycobacterium abscessus subsp. abscessus由来蛋白質をそれぞれ発現させることができるベクターを作製した。それぞれの遺伝子は大腸菌発現ベクターであるpCC1BAC(以下、pBAC、Epicenter corp.)にクローニングされ、大腸菌菌株MG1655のyccAプロモーター(以下、PyccA、配列番号53)下で発現させた。
大腸菌由来L-ヒスチジン生産菌株を基盤として新たなヒスチジン排出体3種(Dva、Hma、Mab)のL-ヒスチジン排出能を確認するために、前記作製されたベクター3種を既報告された遺伝子型(purR欠損、hisL欠損、hisGr; The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants; Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 2, pp. 130-135)を有するCA14-9003e菌株(MG1655+ hisGr hisL’_Δ ΔpurR)に導入した。このために前記実施例9で作製されたベクター3種(pBAC-PyccA-Dva、pBAC-PyccA-Hma、pBAC-PyccA-Mab)とpBACベクターをそれぞれ導入して、CA14-9003e/pBAC、CA14-9003e/pBAC-PyccA-Dva、CA14-9003e/pBAC-PyccA-Hma、およびCA14-9003e/pBAC-PyccA-Mab菌株を作製した。
ブドウ糖4%(w/v)、酵母抽出液0.2%(w/v)、硫酸アンモニウム1.6%(w/v)、第2リン酸カリウム3水塩0.06%(w/v)、硫酸鉄7水塩0.0005%(w/v)、硫酸マグネシウム5水塩0.0005%(w/v)、炭酸カルシウム、pH7.2
寄託機関名:韓国微生物保存センター
受託番号:KCCM12793P
受託日付:20200921
Claims (15)
- 配列番号12、配列番号13、またはその組み合わせと60%以上の配列相同性を有する蛋白質を発現するように改変された、L-ヒスチジン生産微生物。
- 前記蛋白質は、外来の蛋白質である、請求項1に記載のL-ヒスチジン生産微生物。
- 前記改変は、配列番号12、配列番号13、またはその組み合わせと60%以上の配列相同性を有するアミノ酸配列をコードする遺伝子の導入によるものである、請求項1に記載のL-ヒスチジン生産微生物。
- 前記蛋白質は、配列番号12および13、配列番号41および42、または配列番号44および45のアミノ酸配列で表わされるものである、請求項1に記載のL-ヒスチジンを生産する微生物。
- 前記改変は、配列番号12および13、配列番号41および42、または配列番号44および45のアミノ酸配列をコードする遺伝子の導入によるものである、請求項4に記載のL-ヒスチジン生産微生物。
- 前記微生物は、コリネバクテリウム属またはエスケリキア属である、請求項1~5のいずれか一項に記載のL-ヒスチジン生産微生物。
- 前記微生物は、コリネバクテリウム・グルタミカムまたはエスケリキアコリである、請求項6に記載のL-ヒスチジン生産微生物。
- 配列番号12、配列番号13、またはその組み合わせと60%以上の配列相同性を有する蛋白質、
前記蛋白質をコードする遺伝子、
前記遺伝子を含む組換えベクター、または、
前記遺伝子または前記組換えベクターを含む組換え微生物、
を含む、L-ヒスチジン生産用組成物。 - 前記蛋白質は、配列番号12および13、配列番号41および42、または配列番号44および45のアミノ酸配列で表わされるものである、請求項8に記載のL-ヒスチジン生産用組成物。
- 前記微生物は、コリネバクテリウム属またはエスケリキア属である、請求項8または9に記載のL-ヒスチジン生産用組成物。
- 前記微生物は、コリネバクテリウム・グルタミカムまたはエスケリキアコリである、請求項10に記載のL-ヒスチジン生産用組成物。
- 請求項1~5のいずれか一項に記載のL-ヒスチジン生産微生物を培地で培養する工程を含む、L-ヒスチジン生産方法。
- 前記培養する工程の後、培養した微生物または培地からL-ヒスチジンを回収する工程を追加的に含む、請求項12に記載のL-ヒスチジン生産方法。
- 前記微生物は、コリネバクテリウム属またはエスケリキア属である、請求項12に記載のL-ヒスチジン生産方法。
- 前記微生物は、コリネバクテリウム・グルタミカムまたはエスケリキアコリである、請求項14に記載のL-ヒスチジン生産方法。
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KR1020200183702A KR20220092182A (ko) | 2020-12-24 | 2020-12-24 | L-히스티딘 배출 단백질 및 이를 이용한 l-히스티딘 생산 방법 |
PCT/KR2021/019769 WO2022139523A1 (ko) | 2020-12-24 | 2021-12-23 | L-히스티딘 배출 단백질 및 이를 이용한 l-히스티딘 생산 방법 |
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DE19951708A1 (de) * | 1999-10-27 | 2001-05-03 | Degussa | Für den Export verzweigtkettiger Aminosäuren kodierende Nikleotidsequenzen, Verfahren zu deren Isolierung und ihre Verwendung |
AU2007295159B2 (en) | 2006-09-15 | 2012-09-13 | Cj Cheiljedang Corporation | A Corynebacteria having enhanced L-lysine productivity and a method of producing L-lysine using the same |
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KR101904666B1 (ko) * | 2017-08-02 | 2018-11-29 | 씨제이제일제당 (주) | Atp 포스포리보실기 전이효소 변이체 및 이를 이용한 l-히스티딘 생산방법 |
KR101887821B1 (ko) * | 2017-08-11 | 2018-08-13 | 대상 주식회사 | 비산화 펜토오스 인산 경로 관련 효소의 불활성화에 의한 히스티딘 생산능 변이 균주 |
KR101968317B1 (ko) | 2018-02-23 | 2019-04-11 | 씨제이제일제당 주식회사 | 신규 l-트립토판 배출 단백질 및 이를 이용한 l-트립토판을 생산하는 방법 |
KR102204917B1 (ko) * | 2019-04-22 | 2021-01-20 | 씨제이제일제당 주식회사 | L-히스티딘 생산능이 강화된 미생물 및 이를 이용한 히스티딘 생산방법 |
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CN116615548A (zh) | 2023-08-18 |
KR20230149787A (ko) | 2023-10-27 |
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CA3197710A1 (en) | 2022-06-30 |
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