JP6892395B2 - 乳酸を生産する微生物及びそれを用いた乳酸の製造方法 - Google Patents
乳酸を生産する微生物及びそれを用いた乳酸の製造方法 Download PDFInfo
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Description
クエン酸 + ATP + CoA + H2O→OAA + アセチルCoA + A + Pi
本発明で使用する代表的な乳酸生産菌株を作製するために、Euroscarfから分譲された野生型酵母の代表的なサッカロマイセス・セレビシエ(Saccharomyces cerevisiae)CEN.PK2-1Dに一連の遺伝子操作を加えた。
ベース菌株である前記実施例1で製造されたCC02-0064菌株にPDCアイソザイムであるPDC5を欠損した菌株及びPDC5とPDC6の両方を欠損した菌株を作製することにより、PDCの力価を減少あるいは不活性化した菌株を作製した。
菌株の評価のために用いられた培地は、酵母の制限培地であるSC培地(Synthetic Complex media)である。この培地を作製するために0.67%アミノ酸を含まない酵母窒素塩基(yeast nitrogen base without amino acid)に基づいて、ここにアミノ酸dropout mix(Sigma)をメーカーのプロトコールに基づいて混合して、必要に応じて、除外されたアミノ酸を添加した。ロイシン(leucine)は380mg/L、ウラシル(Uracil)、トリプトファン(Tryptophan)及びヒスチジン(Histidine)は76mg/Lになるように添加し、炭素源としてグルコース8%と中和剤として1%CaCO3を添加した。このように製造した培地を、酵母菌株乳酸発酵の評価に用いた。
(1)外来ACLを乳酸生産菌株に導入するためのベクター作製
外来のACL酵素を導入してピルビン酸生合成経路の一つであるPCK1とPYK2を同時に過発現するための組換えベクターを作製した。
ピルビン酸生合成経路を強化するためのPCK1及びPYK2の同時過発現のための組換えベクターを作製した。
前記実施例2で製造したCC02-0553菌株をベースに、実施例4-(1)で作製された外来ACLの導入、及び実施例4-(2)で作製されたPCK1/PYK2同時過発現ベクターを形質転換を通じて挿入した。
前記実施例4-(3)で製造したPDC力価不活性化菌株でのACL-PCK1-PYK2強化菌株の評価を、前記実施例3で使用した方法と同様に適用して、乳酸発酵能を評価した。前記実験の結果をまとめると下記表7の通りである。
前記実施例5の結果を通じ、外来ACL導入による新たな経路のアセチルCoA生産及びピルビン酸生合成強化戦略が効果的な乳酸発酵収率の増加、生産微生物の生長を強化及び乳酸発酵生産性の増加方法であることを確認して、他の遺伝子を用いたピルビン酸生合成強化の際にも、このような結果があるかどうかを確認しようとした。
外来ACL導入によって生成されたOAAは、別の経路を介してピルビン酸で生合成されることがあるが、本来の細胞基質(cytosol)に位置するMDH2を過発現して、ミトコンドリア内に位置する酵素であるMAE1を細胞基質に位置を変更して過発現しようとした。このため、組換えベクターを作製した。
前記実施例2で製造したCC02-0553菌株をベースに、実施例4-(1)で作製された外来ACL導入ベクター及び実施例6-(1)で作製されたMDH2過発現ベクターと細胞基質MAE1過発現ベクターを形質転換により挿入した。形質転換は、前記実施例4-(3)で説明した方法を使用した。前記製造された菌株をCC02-0821と命名し、その遺伝形質をまとめると、下記の表9の通りである。
前記実施例6で製造されたPDC不活性化菌株でのACL-MDH2-細胞基質MAE1強化を通じた乳酸生産性の強化株の評価を前記実施例2で使用した方法で乳酸発酵能を評価した。前記実験の結果をまとめると、表10のとおりである。
前記実施例の結果を総合して、PDCの力価が除去された菌株で外来ACL導入及びピルビン酸生合成経路の強化を通じて、酵母微生物で乳酸発酵の生産性を向上させることができることを確認した。そこで、PDC力価が削除された菌株だけでなく、PDCの力価が減少された菌株でも同じ結果を得ることができるかの検証を行った。
前記実施例8で製造したCC02-0819、CC02-0820菌株を対照群であるCC02-0256と共に前記実施例3で使用した方法で乳酸発酵能を評価した。前記実験の結果をまとめると、表12のとおりである。
前記実施例8で製造したCC02-0831菌株は対照群であるCC02-0256、CC02-0819と共に前記実施例3で使用した方法で乳酸発酵能を評価した。前記実験の結果をまとめると、表13のとおりである。
(構成1)
ピルビン酸デカルボキシラーゼ(Pyruvate decarboxylase、PDC)の活性が内在的活性に比べて不活性化されて、ATP-クエン酸分解酵素(ATP-citrate lyase、ACL)活性が導入され、ピルビン酸生合成経路が内在的生合成経路に比べて強化されるように変異された、乳酸生産能を有するサッカロマイセス属(Saccharomyces sp.)微生物。
(構成2)
前記ピルビン酸生合成経路の強化が、ホスホエノールピルビン酸カルボキシキナーゼ1(Phosphoenolpyruvate carboxykinase 1、PCK1)またはピルビン酸キナーゼ(Pyruvate kinase 2、PYK2)、または両方の活性が内在的活性に比べて強化されるように変異させるものである、構成1に記載の微生物。
(構成3)
前記ピルビン酸生合成経路の強化が、リンゴ酸デヒドロゲナーゼ2(Malate dehydrogenase 2、MDH2)または細胞基質リンゴ酸酵素1(cytosolic Malic enzyme 1、cytosolic MAE1)、または両方の活性が内在的活性に比べて強化されるように変異させるものである、構成1に記載の微生物。
(構成4)
前記ピルビン酸デカルボキシラーゼが、配列番号39、41及び43のアミノ酸配列からなる群から選択された一つ以上のアミノ酸配列で表される酵素である、構成1に記載の微生物。
(構成5)
前記ATP-クエン酸分解酵素が、配列番号29のアミノ酸配列で表される酵素である、構成1に記載の微生物。
(構成6)
前記ホスホエノールピルビン酸カルボキシキナーゼ1が、配列番号31のアミノ酸配列で表される酵素であり、ピルビン酸キナーゼ2が、配列番号33のアミノ酸配列で表される酵素である、構成2に記載の微生物。
(構成7)
前記リンゴ酸デヒドロゲナーゼ2が、配列番号35のアミノ酸配列で表される酵素であり、細胞基質リンゴ酸酵素1が、配列番号37のアミノ酸配列または配列番号52のアミノ酸配列で表される酵素である、構成3に記載の微生物。
(構成8)
前記微生物が、乳酸デヒドロゲナーゼ(Lactate dehydrogenase、LDH)活性がさらに導入されるものである、構成1に記載の微生物。
(構成9)
前記乳酸デヒドロゲナーゼが、配列番号49のアミノ酸配列で表される酵素である、構成8に記載の微生物。
(構成10)
前記微生物がさらに、
(i)アルコールデヒドロゲナーゼ1(Alcohol dehydrogenase 1、ADH1)の活性が内在的活性に比べて不活性化されるように変異されたもの;
(ii)ピルビン酸デカルボキシラーゼ1(Pyruvate decarboxylase 1、PDC1)の活性が内在的活性に比べて不活性化されるように変異されたもの;及び
(iii)D−乳酸デヒドロゲナーゼ1(D-lactate dehydrogenase 1、DLD1)の活性が内在的活性に比べて不活性化されるように変異されたものである、構成1に記載の微生物。
(構成11)
前記サッカロマイセス属微生物が、サッカロマイセス・セレビシエ(Saccharomyces cerevisiase)である、構成1に記載の微生物。
(構成12)
(i)構成1〜11のいずれか一項に記載のサッカロマイセス属(Saccharomyces sp.)微生物を培地で培養する段階;及び
(ii)前記培養による培地または前記微生物から乳酸を回収する段階を含む、乳酸製造方法。
Claims (10)
- 変異されていない微生物に比べて、向上した乳酸生産能を有するサッカロマイセス属(Saccharomyces sp.)微生物であって、ピルビン酸デカルボキシラーゼ(Pyruvate decarboxylase、PDC)の活性がその内在的活性に比べて不活性化され、ATP-クエン酸分解酵素(ATP-citrate lyase、ACL)の活性が導入され、ピルビン酸生合成経路が内在的生合成経路に比べて強化され、アルコールデヒドロゲナーゼ1(Alcohol dehydrogenase 1、ADH1)の活性が内在的活性に比べて不活性化され、D‐乳酸デヒドロゲナーゼ1(D-lactate dehydrogenase 1、DLD1)の活性が内在的活性に比べて不活性化され、かつ、ラクトバシルス・プランタラム由来の乳酸デヒドロゲナーゼ(Lactate dehydrogenase、LDH)活性が導入されるように変異され、
前記ピルビン酸生合成経路の強化が、i)ホスホエノールピルビン酸カルボキシキナーゼ1(Phosphoenolpyruvate carboxykinase 1、PCK1)、及びピルビン酸キナーゼ2(Pyruvate kinase 2、PYK2)の活性強化により、または、ii)リンゴ酸デヒドロゲナーゼ2(Malate dehydrogenase 2、MDH2)、及び細胞基質リンゴ酸酵素1(cytosolic Malic enzyme 1、cytosolic MAE1)の活性強化により達成されたものである、前記サッカロマイセス属微生物。 - 前記ピルビン酸デカルボキシラーゼが、配列番号39、41及び43のアミノ酸配列からなる群から選択された少なくとも1つのアミノ酸配列で表される酵素である、請求項1に記載の微生物。
- 前記ATP-クエン酸分解酵素が、配列番号29のアミノ酸配列で表される酵素である、請求項1に記載の微生物。
- 前記ホスホエノールピルビン酸カルボキシキナーゼ1が、配列番号31のアミノ酸配列で表される酵素であり、ピルビン酸キナーゼ2が、配列番号33のアミノ酸配列で表される酵素である、請求項1に記載の微生物。
- 前記リンゴ酸デヒドロゲナーゼ2が、配列番号35のアミノ酸配列で表される酵素であり、かつ細胞基質リンゴ酸酵素1が、配列番号37のアミノ酸配列または配列番号52のアミノ酸配列で表される酵素である、請求項1に記載の微生物。
- 前記乳酸デヒドロゲナーゼが、配列番号49のアミノ酸配列で表される酵素である、請求項1に記載の微生物。
- 前記ピルビン酸デカルボキシラーゼが、ピルビン酸デカルボキシラーゼ1(Pyruvate decarboxylase 1、PDC1)である、請求項1に記載の微生物。
- 前記サッカロマイセス属微生物が、サッカロマイセス・セレビシエ(Saccharomyces cerevisiase)である、請求項1に記載の微生物。
- a)請求項1〜8のいずれか一項に記載のサッカロマイセス属(Saccharomyces sp.)微生物を培地で培養する段階;及び
b)該培地から乳酸を回収する段階を含む、乳酸製造方法。 - 乳酸の製造における、変異されたサッカロマイセス属(Saccharomyces sp.)微生物の使用であって、該微生物がピルビン酸デカルボキシラーゼ(Pyruvate decarboxylase、PDC)の活性がその内在的活性に比べて不活性化されて、ATP-クエン酸分解酵素(ATP-citrate lyase、ACL)の活性が導入され、ピルビン酸生合成経路が内在的生合成経路に比べて強化され、アルコールデヒドロゲナーゼ1(Alcohol dehydrogenase 1、ADH1)の活性が内在的活性に比べて不活性化され、D‐乳酸デヒドロゲナーゼ1(D-lactate dehydrogenase 1、DLD1)の活性が内在的活性に比べて不活性化され、かつ、ラクトバシルス・プランタラム由来の乳酸デヒドロゲナーゼ(Lactate dehydrogenase、LDH)活性が導入されるように変異され、
該ピルビン酸生合成経路の強化が、i)ホスホエノールピルビン酸カルボキシキナーゼ1(Phosphoenolpyruvate carboxykinase 1、PCK1)、及びピルビン酸キナーゼ2(Pyruvate kinase 2、PYK2)、または、ii)リンゴ酸デヒドロゲナーゼ2(Malate dehydrogenase 2、MDH2)、及び細胞基質リンゴ酸酵素1(cytosolic Malic enzyme 1、cytosolic MAE1)の活性強化により達成されたものである、前記使用。
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