JP7447294B2 - シュワネラ・アトランティカ由来蛋白質を発現する微生物、およびこれを利用したl-アミノ酸生産方法 - Google Patents
シュワネラ・アトランティカ由来蛋白質を発現する微生物、およびこれを利用したl-アミノ酸生産方法 Download PDFInfo
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- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Description
コリネバクテリウム属微生物が内在的に保有するL-リジン排出体(lysE、Ncgl1214)と比較してL-リジン排出活性が高い排出体を探索するために、NCBI CDD(Common Domain Database)を利用したRPS-BLAST探索とKEGG Protein Databaseを利用したBLAST探索を行った。L-リジンを排出できる膜蛋白質として考慮される候補蛋白質を選定した。
前記実施例1で選定されたシュワネラ・アトランティカ(Shewanella atlantica、Sat)由来の膜蛋白質は、配列番号1のアミノ酸配列を有する。米国国立衛生研究所ジェンバンク(NIH GenBank)から前記膜蛋白質をコーディングする遺伝子および周辺核酸配列に対する情報(登録番号NZ_RXNV01000002.1)を獲得した。当該遺伝子配列に基づいてDNA合成を行った(Cosmo genetech、Korea)。遺伝子を増幅させるために、合成されたDNAを鋳型として配列番号3と4のプライマー(表2)を利用してPCR(SolgTM Pfu-X DNA polymerase)を行った(表3)。その結果、609bpの遺伝子(配列番号2)を含む639bpの遺伝子断片を得た。
作製されたpDZTn-PgapA-Satベクターを利用してL-リジンを生産するコリネバクテリウム・グルタミクムKCCM11016P菌株(大韓民国登録特許第10-0159812号)を電気穿孔法(Appl. Microbiol. Biotechnol.(1999)52:541-545)で形質転換した後、2次交差過程を経てトランスポゾン遺伝子の間にPgapA-Satが挿入された菌株を獲得した。当該遺伝子が挿入された位置を含む隣接部位を増幅できる配列番号9と配列番号10のプライマー(表4)を利用してPCRおよび塩基配列分析を行い、当該遺伝的操作を確認した。このように得た菌株をコリネバクテリウム・グルタミクムKCCM11016P::PgapA-Satと命名した。
KCCM11016P::PgapA-Sat菌株と対照群KCCM11016Pを以下のような方法で培養して、菌体量、糖消耗能、アミノ酸生産能を比較した。
まず、種培地25mlを含有する250mlコーナーバッフルフラスコに各菌株を接種し、30℃で20時間、200rpmで振とう培養した。生産培地24mlを含有する250mlコーナーバッフルフラスコに1mlの種培養液を接種して37℃で42時間、200rpmで振とう培養した。培養終了後、HPLCによりL-アミノ酸の生産量を測定した。前記実験は3回繰り返し、培養結果(平均値)は表5に示した。
ブドウ糖20g、ペプトン10g、酵母抽出物5g、尿素1.5g、KH2PO4 4g、K2HPO4 8g、MgSO4・7H2O 0.5g、ビオチン0.1mg、チアミンHCl 1mg、カルシウム-パントテン酸22mg、ニコチンアミド2mg(蒸溜水1リットル基準)
ブドウ糖45g、(NH4)2SO4 15g、大豆蛋白質10g、糖蜜10g、KH2PO4 0.55g、MgSO4・7H2O 0.6g、ビオチン0.9mg、チアミン塩酸塩4.5mg、カルシウム-パントテン酸4.5mg、ニコチンアミド30mg、MnSO4 9mg、FeSO4 9mg、ZnSO4 0.45mg、CuSO4 0.45mg、CaCO3 30g(蒸溜水1リットル基準)。
前記実施例3に記述した方法を利用してコリネバクテリウム・グルタミクムKCCM10770P(大韓民国登録特許第10-0924065号)菌株のゲノム上のトランスポゾン位置にPgapA-Sat遺伝子が挿入された菌株を獲得した。このように得た菌株をコリネバクテリウム・グルタミクムKCCM10770P::PgapA-Satと命名した。
野生株に3種の変異[pyc(P458S)、hom(V59A)、lysC(T311I)]を導入してL-リジン生産能を有するようになったコリネバクテリウム・グルタミクムCJ3P(US 9556463 B2)菌株のゲノム上のトランスポゾン位置に前記実施例3に記述した方法を利用してPgapA-Sat遺伝子が挿入された菌株を獲得した。このように得た菌株をコリネバクテリウム・グルタミクムCJ3P::PgapA-Satと命名した。
Claims (10)
- 配列番号1のシュワネラ・アトランティカ(Shewanella atlantica)由来のL-アミノ酸排出能を有する膜蛋白質、またはこれと90%以上の配列同一性を有し、シュワネラ属微生物由来のL-アミノ酸排出能を有する膜蛋白質を発現する、コリネバクテリウム属微生物。
- 前記コリネバクテリウム属微生物は、前記膜蛋白質をコードするポリヌクレオチドが導入されたものである、請求項1に記載のコリネバクテリウム属微生物。
- 前記ポリヌクレオチドは、配列番号2またはこれと75%以上の配列同一性を有する核酸配列で表現されるものである、請求項2に記載のコリネバクテリウム属微生物。
- 前記膜蛋白質を発現しないコリネバクテリウム属微生物と比較して、L-アミノ酸生産能が増加された、請求項1~3のいずれか一項に記載のコリネバクテリウム属微生物。
- 前記L-アミノ酸は、L-リジンまたはL-アルギニンである、請求項4に記載のコリネバクテリウム属微生物。
- 前記コリネバクテリウム属微生物は、コリネバクテリウム・グルタミクムである、請求項1~3のいずれか一項に記載のコリネバクテリウム属微生物。
- 請求項1~3のいずれか一項に記載のコリネバクテリウム属微生物を含む、L-アミノ酸生産用組成物。
- 前記L-アミノ酸は、L-リジンまたはL-アルギニンである、請求項7に記載の組成物。
- 請求項1~3のいずれか一項に記載のコリネバクテリウム属微生物を培地で培養する工程、および
前記培養された微生物、培地、またはこれらの全てからL-アミノ酸を回収する工程を含む、L-アミノ酸の生産方法。 - 前記L-アミノ酸は、L-リジンまたはL-アルギニンである、請求項9に記載の方法。
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KR1020200171739A KR102616694B1 (ko) | 2020-12-09 | 2020-12-09 | 쉬와넬라 아틀란티카 유래 단백질을 발현하는 미생물, 및 이를 이용한 l-아미노산 생산 방법 |
PCT/KR2021/017793 WO2022124670A1 (ko) | 2020-12-09 | 2021-11-29 | 쉬와넬라 아틀란티카 유래 단백질을 발현하는 미생물, 및 이를 이용한 l-아미노산 생산 방법 |
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