JP2017525381A - L−リジン生産能が向上した微生物及びそれを用いたl−リジン生産方法 - Google Patents
L−リジン生産能が向上した微生物及びそれを用いたl−リジン生産方法 Download PDFInfo
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- JP2017525381A JP2017525381A JP2017512724A JP2017512724A JP2017525381A JP 2017525381 A JP2017525381 A JP 2017525381A JP 2017512724 A JP2017512724 A JP 2017512724A JP 2017512724 A JP2017512724 A JP 2017512724A JP 2017525381 A JP2017525381 A JP 2017525381A
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- Prior art keywords
- amino acid
- siga
- replaced
- lysine
- arginine
- 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
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Abstract
Description
本実施例では、変異型SigAを獲得するために、下記方法で染色体内の1次交差挿入用ベクターのライブラリを作製した。コリネバクテリウムSigA(配列番号2)をコードするrpoD遺伝子(配列番号1)を対象に、Error−prone PCR法を行い、塩基置換変異がランダムに導入されたrpoD遺伝子変異体の断片(1497bp)を獲得した。前記Error−prone PCRは、GenemorphII Random Mutagenesis Kit(Stratagene)を使用して行い、コリネバクテリウム・グルタミクムATCC13032のゲノムDNAを鋳型としてプライマー1(配列番号3)及びプライマー2(配列番号4)を使用した。増幅された遺伝子断片内に1kb当り0〜4.5個の変異が導入されるようにし、PCR条件は、変性96℃、30秒;アニーリング53℃、30秒;及び重合反応72℃、2分を30回繰り返した。
KCCM11016P(特許文献3)菌株を親菌株にして、前記製作されたpCR2.1−rpoD(mt)ライブラリを相同染色体組換えにより形質転換し、カナマイシン(25mg/l)及び下記のような成分が含まれた複合平板培地に塗抹して、約25,000個のコロニーを確保した。これをKCCM11016P/pCR2.1−rpoD(mt)−1〜KCCM11016P/pCR2.1−rpoD(mt)−25000と命名した。また、前記製作されたpCR2.1−rpoD(WT)ベクターをKCCM11016P菌株に形質転換して対照群菌株を製作し、KCCM11016P/pCR2.1−rpoD(WT)と命名した。
グルコース10g、ペプトン10g、牛肉抽出物5g、酵母抽出物5g、ブレインハートインフュージョン18.5g、NaCl 2.5g、尿素2g、ソルビトール91g、寒天20g(蒸留水1リットル基準)
前記実施例2で確保された約25,000個のコロニーをそれぞれ下記のような成分が含まれた300μlの選別培地に接種し、96−ディープウェルプレートで32℃、1000rpmで約24時間培養した。培養中に生産されたL−リジンの生産量を分析するためにニンヒドリン方法を用いた(非特許文献6)。培養が完了した後、培養上層液10μlとニンヒドリン反応溶液190μl(63%のグリセロール、27%のニンヒドリン溶液(7.1g/L in 0.5M citrate buffer pH5.5))とを65℃で30分間反応させた。その後、570nmの波長で分光光度計を用いて吸光度を測定して、対照群であるKCCM11016P/pCR2.1−rpoD(WT)菌株の吸光度と比較し、10%以上増加した吸光度を示す約935個の変異菌株のコロニーを選別した。その他のコロニーは対照群に比べて類似または減少した吸光度を示した。
グルコース10g、硫酸アンモニウム 5.5g、MgSO4・7H2O 1.2g、KH2PO4 0.8g、K2HPO4 16.4g、ビオチン100μg、チアミンHCl1000μg、カルシウム−パントテン酸2000μg、ニコチン酸アミド2000μg(蒸留水1リットル基準)
前記実施例3で選別した231種の菌株のL−リジン生産能を以下のような方法で培養して分析した。
グルコース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100μg、チアミンHCl 1000μg、カルシウム−パントテン酸2000μg、ニコチン酸アミド2000μg(蒸留水1リットル基準)
グルコース100g、(NH4)2SO4 40g、大豆タンパク質2.5g、トウモロコシ浸漬固形分(Corn Steep Solids)5g、尿素3g、KH2PO4 1g、MgSO4・7H2O 0.5g、ビオチン100μg、チアミン塩酸塩1000μg、カルシウム−パントテン酸2000μg、ニコチン酸アミド3000μg、CaCO3 30g(蒸留水1リットル基準)。
前記実施例4で選別した17種のうち、対照群に比べてL−リジン生産能が増加した15種の菌株のSigAに導入された変異を確認するために、rpoD変異体の塩基配列を分析した。塩基配列を決定するためにプライマー1(配列番号3)及びプライマー3(配列番号5)を使用してPCRを行った。
前記実施例4で確認されたSigA変異を適用した効果を確認するために、これを染色体上に導入することができるベクターを作製した。
前記実施例6で製作した新規変異の導入ベクター15種及び既に報告された変異の導入ベクター1種を2段階の相同染色体組換えによりL−リジン生産菌株であるコリネバクテリウム・グルタミクムKCCM11016Pに形質転換した。その後、染色体上のSigA変異が導入された菌株を塩基配列の分析により選別し、前記SigA変異が導入された菌株をKCCM11016P::SigA(D136G、T281S)、KCCM11016P::SigA(L381R)、KCCM11016P::SigA(M230T)、KCCM11016P::SigA(M455V)、KCCM11016P::SigA(S488T)、KCCM11016P::SigA(R279L)、KCCM11016P::SigA(I254N)、KCCM11016P::SigA(A268S)、KCCM11016P::SigA(L451I、Q491R)、KCCM11016P::SigA(Q429R)、KCCM11016P::SigA(K90E、K105Y、D250G、I254L)、KCCM11016P::SigA(L447H)、KCCM11016P::SigA(K483R)、KCCM11016P::SigA(K479R)、KCCM11016P::SigA(D238V、N263S、E358D)、KCCM11016P::SigA(A414V)と命名した。
コリネバクテリウム・グルタミクムに属する他の菌株において、前記実施例7で選別したSigA変異11種の導入効果を確認するために、前記実施例7と同様の方法でL−リジン生産菌株であるコリネバクテリウム・グルタミクムKFCC10750(KCCM11347P、特許文献5)に11種のSigA変異がそれぞれ導入された菌株を製作して、KFCC10750::SigA(D136G、T281S)、KFCC10750::SigA(L381R)、KFCC10750::SigA(M455V)、KFCC10750::SigA(S488T)、KFCC10750::SigA(I254N)、KFCC10750::SigA(A268S)、KFCC10750::SigA(L451I、Q491R)、KFCC10750::SigA(Q429R)、KFCC10750::SigA(L447H)、KFCC10750::SigA(K483R)、KFCC10750::SigA(K479R)と命名した。また、既に報告した変異SigA(A414V)を導入した菌株も製作し、これをKFCC10750::SigA(A414V)と命名した。
コリネバクテリウム・グルタミクムに属する他の菌株において、前記実施例7で選別したSigA変異11種の効果を確認するために、前記実施例7と同様の方法でL−リジン生産菌株であるコリネバクテリウム・グルタミクムKCCM10770P(特許文献4)にSigA変異が導入された菌株を製作し、KCCM10770P::SigA(D136G、T281S)、KCCM10770P::SigA(L381R)、KCCM10770P::SigA(M455V)、KCCM10770P::SigA(S488T)、KCCM10770P::SigA(I254N)、KCCM10770P::SigA(A268S)、KCCM10770P::SigA(L451I、Q491R)、KCCM10770P::SigA(Q429R)、KCCM10770P::SigA(L447H)、KCCM10770P::SigA(K483R)、KCCM10770P::SigA(K479R)と命名した。また、既に報告した変異SigA(A414V)を導入した菌株も製作し、KCCM10770P::SigA(A414V)と命名した。
コリネバクテリウム・グルタミクムに属する他の菌株における効果も確認するために、前記実施例7と同様の方法でL−リジン生産菌株であるコリネバクテリウム・グルタミクムCJ3P(非特許文献7)にSigA変異が導入された菌株を製作し、CJ3P::SigA(D136G、T281S)、CJ3P::SigA(L381R)、CJ3P::SigA(M455V)、CJ3P::SigA(S488T)、CJ3P::SigA(I254N)、CJ3P::SigA(A268S)、CJ3P::SigA(L451I、Q491R)、CJ3P::SigA(Q429R)、CJ3P::SigA(L447H)、CJ3P::SigA(K483R)、CJ3P::SigA(K479R)と命名した。また、既に報告した変異SigA(A414V)を導入した菌株も製作し、CJ3P::SigA(A414V)と命名した。CJ3P菌株は、公知となった技術に基づいて野生株に3種の変異を導入して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・グルタミクム菌株である。
Claims (11)
- 配列番号2のアミノ酸配列からなるポリペプチドにおいて下記位置のアミノ酸のうち、一つ以上のアミノ酸が他のアミノ酸で置換された、RNAポリメラーゼシグマ因子A活性を有する変異型ポリペプチド:
開始メチオニンを1番目のアミノ酸にして、そこから136番目のアミノ酸;254番目のアミノ酸;268番目のアミノ酸;281番目のアミノ酸;381番目のアミノ酸;429番目のアミノ酸;及び445番目〜495番目のアミノ酸。 - 前記アミノ酸の置換が、136番目のアミノ酸;254番目のアミノ酸;268番目のアミノ酸;281番目のアミノ酸;381番目のアミノ酸;429番目のアミノ酸;447番目のアミノ酸;451番目のアミノ酸;455番目のアミノ酸;479番目のアミノ酸;483番目のアミノ酸;488番目のアミノ酸;及び491番目のアミノ酸からなるアミノ酸のうち、1種以上のアミノ酸が他のアミノ酸で置換された、請求項1に記載の変異型ポリペプチド。
- 下記アミノ酸の置換のうち、1種以上が組み合わされた、請求項1に記載の変異型ポリペプチド:
136番目のアミノ酸がグリシンで置換;254番目のアミノ酸がアスパラギンで置換;268番目のアミノ酸がセリンで置換;281番目のアミノ酸がセリンで置換;381番目のアミノ酸がアルギニンで置換;429番目のアミノ酸がアルギニンで置換;447番目のアミノ酸がヒスチジンで置換;451番目のアミノ酸がイソロイシンで置換;455番目のアミノ酸がバリンで置換;479番目のアミノ酸がアルギニンで置換;483番目のアミノ酸がアルギニンで置換;488番目のアミノ酸がトレオニンで置換;及び491番目のアミノ酸がアルギニンで置換。 - 前記変異型ポリペプチドが、配列番号12〜22のアミノ酸配列のいずれか一つのアミノ酸配列を有するものである、請求項1に記載の変異型ポリペプチド。
- 請求項1に記載の変異型ポリペプチドをコードするポリヌクレオチド。
- 請求項5に記載のポリヌクレオチドを含む宿主細胞。
- 配列番号2のアミノ酸配列からなるポリペプチドにおいて下記位置のアミノ酸のうち、一つ以上のアミノ酸が他のアミノ酸で置換された、RNAポリメラーゼシグマ因子A活性を有する変異型ポリペプチドを含むように形質転換されたL−リジン生産能が向上したコリネバクテリウム属微生物:
開始メチオニンを1番目のアミノ酸にして、そこから136番目のアミノ酸;254番目のアミノ酸;268番目のアミノ酸;281番目のアミノ酸;381番目のアミノ酸;429番目のアミノ酸;及び445番目〜495番目のアミノ酸。 - 前記アミノ酸の置換が、136番目のアミノ酸;254番目のアミノ酸;268番目のアミノ酸;281番目のアミノ酸;381番目のアミノ酸;429番目のアミノ酸;447番目のアミノ酸;451番目のアミノ酸;455番目のアミノ酸;479番目のアミノ酸;483番目のアミノ酸;488番目のアミノ酸;及び491番目のアミノ酸からなるアミノ酸のうち、1種以上のアミノ酸が他のアミノ酸で置換された、請求項7に記載のL−リジン生産能が向上したコリネバクテリウム属微生物。
- 下記アミノ酸の置換のうち、1種以上が組み合わされた、請求項7に記載のL−リジン生産能が向上したコリネバクテリウム属微生物:
136番目のアミノ酸がグリシンで置換;254番目のアミノ酸がアスパラギンで置換;268番目のアミノ酸がセリンで置換;281番目のアミノ酸がセリンで置換;381番目のアミノ酸がアルギニンで置換;429番目のアミノ酸がアルギニンで置換;447番目のアミノ酸がヒスチジンで置換;451番目のアミノ酸がイソロイシンで置換;455番目のアミノ酸がバリンで置換;479番目のアミノ酸がアルギニンで置換;483番目のアミノ酸がアルギニンで置換;488番目のアミノ酸がトレオニンで置換;及び491番目のアミノ酸がアルギニンで置換。 - 前記微生物が、コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)である、請求項7に記載のL−リジン生産能が向上したコリネバクテリウム属微生物。
- 請求項7〜10のいずれか一項による微生物を培養する段階;及び前記培養された微生物または培養培地からL−リジンを回収する段階を含む、L−リジンを生産する方法。
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