JP3967812B2 - Production method of organic acid by pyruvate carboxylase genetically modified cells - Google Patents

Description

【００４６】
実際のＰＣＲは、パーキンエルマーシータス社製の「ＤＮＡサーマルサイクラー」を用い、反応試薬として、レコンビナント・タックＤＮＡ・ポリメラーゼ・タカラ・タック（Recombinant TaqDNA Polymerase TaKaRa Taq）（宝酒造製）を用いて下記の条件で行った。
【００４７】
反応液：
（１０×）ＰＣＲ緩衝液 １０μｌ
１．２５ｍＭ ｄＮＴＰ混合液 １６μｌ
鋳型ＤＮＡ １０μｌ（DNA 含有量 １μＭ以下）
上記記載のａ−１，ｂ−１プライマー 各々１μｌ（最終濃度０．２５μＭ）
レコンビナント・タックＤＮＡ・ポリメラーゼ ０．５μｌ
滅菌蒸留水 ６１．５μｌ
以上を混合し、この１００μｌの反応液をＰＣＲにかけた。
【００４８】
ＰＣＲサイクル：
デナチュレーション過程：９４℃ ６０秒
アニーリング過程 ：５２℃ ６０秒
エクステンション過程 ：７２℃ １２０秒
【００４９】
以上を１サイクルとし、２５サイクル行った。
上記で生成した反応液１０μｌを０．８％アガロースゲルにより電気泳動を行い、約３．５６ｋｐのＤＮＡ断片が検出できた。
【００５０】
〔実施例２〕ＰＣ遺伝子によるコリネ型細菌組み換え体の作製
（Ａ）シャトルベクターの構築
特開平３−２１０１８４号公報に記載のプラスミドｐＣＲＹ３０内に存在する、コリネ型細菌内でのプラスミドの安定化に必要な領域の配列をもとに、下記の１対のプライマーを、アプライド・バイオシステムズ（Applied Biosystems）社製「３９４ ＤＮＡ／ＲＮＡシンセサイザー（synthesizer）」を用いて合成した。
【００５１】

【００５２】
上記プラスミドｐＣＲＹ３０は、次のようにして構築されたプラスミドである。ブレビバクテリウム・スタチオニス（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｓｔａｔｉｏｎｉｓ） ＩＦＯ１２１４４（工業技術院生命工学工業技術研究所にＦＥＲＭ ＢＰ−２５１５の受託番号で寄託されている）からプラスミドｐＢＹ５０３（このプラスミドの詳細については特開平１−９５７８５号公報参照）ＤＮＡを抽出し、制限酵素ＸｈｏＩで大きさが約４．０ｋｂのプラスミドの複製増殖機能を司る遺伝子を含むＤＮＡ断片（複製領域）を切り出し、制限酵素ＥｃｏＲＩおよびＫｐｎＩで大きさが約２．１ｋｂのプラスミドの安定化機能を司る遺伝子を含むＤＮＡ断片（安定化領域）を切り出す。これらの両ＤＮＡ断片をプラスミドｐＨＳＧ２９８（宝酒造製）のＥｃｏＲＩ−ＫｐｎＩ部位及びＳａｌＩ部位にそれぞれ組み込むことにより、プラスミドベクターｐＣＲＹ３０を調製することができる。
【００５３】
実際のＰＣＲは、パーキンエルマーシータス社製の「ＤＮＡサーマルサイクラー」を用い、反応試薬として、レコンビナント・タックＤＮＡ・ポリメラーゼ・タカラ・タック（Recombinant TaqDNA Polymerase TaKaRa Taq）（宝酒造製）を用いて下記の条件で行った。
【００５４】
反応液：
（１０×）ＰＣＲ緩衝液 １０μｌ
１．２５ｍＭ ｄＮＴＰ混合液 １６μｌ
鋳型ＤＮＡ １０μｌ（DNA 含有量 １μＭ以下）
上記記載のａ−２，ｂ−２プライマー 各々１μｌ（最終濃度０．２５μＭ）
レコンビナント・タックＤＮＡ・ポリメラーゼ ０．５μｌ
滅菌蒸留水 ６１．５μｌ
以上を混合し、この１００μｌの反応液をＰＣＲにかけた。
【００５５】
ＰＣＲサイクル：
デナチュレーション過程：９４℃ ６０秒
アニーリング過程 ：５２℃ ６０秒
エクステンション過程 ：７２℃ １２０秒
【００５６】
以上を１サイクルとし、２５サイクル行った。
上記で生成した反応液１０μｌを０．８％アガロースゲルにより電気泳動を行い、約１．１ｋｂのＤＮＡ断片が検出できた。
【００５７】
上記で増幅産物を確認できた反応液 １０μｌ、プラスミドｐＢｌｕｅｓｃｒｉｐｔＩＩＳＫ＋ １μｌを各々制限酵素ＥｃｏＲＩおよびＸｈｏＩで完全に切断し、７０℃で１０分間処理させることにより制限酵素を失活させた後、両者を混合し、これに、Ｔ4 ＤＮＡリガーゼ１０×緩衝液 １μｌ、Ｔ4 ＤＮＡリガーゼ１ｕｎｉｔの各成分を添加し、滅菌蒸留水で１０μｌにして、１５℃で３時間反応させ、結合させた。
【００５８】
得られたプラスミド混液を用い、塩化カルシウム法〔Journal of Molecular Biology, 53, 159(1970)〕によりエシェリヒア・コリＪＭ１０９（宝酒造製）を形質転換し、アンピシリン ５０ｍｇを含む培地〔トリプトン １０ｇ、イーストエキストラクト ５ｇ、ＮａＣｌ ５ｇ及び寒天 １６ｇを蒸留水１Ｌに溶解〕に塗抹した。
【００５９】
この培地上の生育株を常法により液体培養し、培養液よりプラスミドＤＮＡを抽出し、該プラスミドを制限酵素（ＥｃｏＲＩ，ＸｈｏＩ）により切断し、挿入断片を確認した。この結果、プラスミドｐＢｌｕｅｓｃｒｉｐｔＩＩＳＫ＋の長さ３．０ｋｂのＤＮＡ断片に加え、長さ１．１ｋｂの挿入ＤＮＡ断片が認められた。本プラスミドをｐＢＳｐａｒと命名した。
【００６０】
米国特許５，１８５，２６２号明細書記載のプラスミドｐＣＲＹ３１内に存在する、コリネ型細菌内でのプラスミドの複製に必要な領域の配列をもとに、下記の１対のプライマーを、アプライド・バイオシステムズ（Applied Biosystems）社製「３９４ ＤＮＡ／ＲＮＡシンセサイザー（synthesizer ）」を用いて合成した。
【００６１】

【００６２】
上記プラスミドｐＣＲＹ３１は、次のようにして構築されたプラスミドである。前記ｐＢＹ５０３由来の複製領域とプラスミドｐＨＳＧ３９８（宝酒造製）とを連結したプラスミドｐＣＲＹ３（このプラスミドを保持するブレビバクテリウム・フラバム MJ233 GE102は、ＦＥＲＭ ＢＰ−２５１３として工業技術院生命工学工業技術研究所に寄託されている）をＫｐｎＩで部分分解してＤＮＡ断片を得る。一方、ｐＢＹ５０３をブレビバクテリウム・ラクトファーメンタムＩＦＯ１２１４４（ＦＥＲＭ ＢＰ−２５１５）から調製し、ＫｐｎＩで完全分解し、約７ｋｂのＤＮＡ断片を精製する。これらのＤＮＡ断片を連結し、各種制限酵素で切断したときに下記表に示す切断パターンを示すプラスミドを選択することによって、ｐＣＲＹ３１が得られる。
【００６３】
【表１】

【００６４】
実際のＰＣＲは、パーキンエルマーシータス社製の「ＤＮＡサーマルサイクラー」を用い、反応試薬として、レコンビナント・タックＤＮＡ・ポリメラーゼ・タカラ・タック（Recombinant TaqDNA Polymerase TaKaRa Taq）（宝酒造製）を用いて下記の条件で行った。
【００６５】
反応液：
（１０×）ＰＣＲ緩衝液 １０μｌ
１．２５ｍＭ ｄＮＴＰ混合液 １６μｌ
鋳型ＤＮＡ １０μｌ（DNA 含有量 １μＭ以下）
上記記載のａ−３，ｂ−３プライマー 各々１μｌ（最終濃度０．２５μＭ）
レコンビナント・タックＤＮＡ・ポリメラーゼ ０．５μｌ
滅菌蒸留水 ６１．５μｌ
以上を混合し、この１００μｌの反応液をＰＣＲにかけた。
【００６６】
ＰＣＲサイクル：
デナチュレーション過程：９４℃ ６０秒
アニーリング過程 ：５２℃ ６０秒
エクステンション過程 ：７２℃ １２０秒
【００６７】
以上を１サイクルとし、２５サイクル行った。
上記で生成した反応液１０μｌを０．８％アガロースゲルにより電気泳動を行い、約１．８ｋｂのＤＮＡ断片が検出できた。
【００６８】
上記で増幅産物を確認できた反応液１０μｌ、プラスミドｐＢＳｐａｒ １μｌを各々制限酵素ＸｈｏＩおよびＫｐｎＩで完全に切断し、７０℃１０分処理させることにより制限酵素を失活させた後、両者を混合し、Ｔ4 ＤＮＡリガーゼ １０×）緩衝液 １μｌ、Ｔ4 ＤＮＡリガーゼ１ｕｎｉｔの各成分を添加し、滅菌蒸留水で１０μｌにして、１５℃で３時間反応させ、結合させた。
【００６９】
得られたプラスミド混液を用い、塩化カルシウム法〔Journal of Molecular Biology, 53, 159(1970)〕によりエシェリヒア・コリＪＭ１０９（宝酒造製）を形質転換し、アンピシリン ５０ｍｇを含む培地〔トリプトン １０ｇ、イーストエキストラクト ５ｇ、ＮａＣｌ ５ｇ及び寒天 １６ｇを蒸留水１Ｌに溶解〕に塗抹した。
【００７０】
この培地上の生育株を常法により液体培養し、培養液よりプラスミドＤＮＡを抽出し、該プラスミドを制限酵素（ＸｈｏＩ，ＫｐｎＩ）により切断し、挿入断片を確認した。この結果、プラスミドｐＢＳｐａｒの長さ４．１ｋｂのＤＮＡ断片に加え、長さ１．８ｋｂの挿入ＤＮＡ断片が認められた。本プラスミドをｐＢＳｐａｒ−ｒｅｐと命名した。
【００７１】
上記で作製したプラスミドｐＢＳｐａｒ−ｒｅｐ １μｌ、ｐＨＳＧ２９８（宝酒造社製） １μｌを各々制限酵素ＫｐｎＩおよびＥｃｏＲＩで完全に切断し、７０℃で１０分処理させることにより制限酵素を失活させた後、両者を混合し、これに、Ｔ4 ＤＮＡリガーゼ１０×緩衝液 １μｌ、Ｔ4 ＤＮＡリガーゼ１ｕｎｉｔの各成分を添加し、滅菌蒸留水で１０μｌにして、１５℃で３時間反応させ、結合させた。
【００７２】
得られたプラスミド混液を用い、塩化カルシウム法〔Journal of Molecular Biology, 53, 159(1970)〕によりエシェリヒア・コリＪＭ１０９（宝酒造製）を形質転換し、カナマイシン ５０ｍｇを含む培地〔トリプトン １０ｇ、イーストエキストラクト ５ｇ、ＮａＣｌ ５ｇ及び寒天 １６ｇを蒸留水１Ｌに溶解〕に塗抹した。
【００７３】
この培地上の生育株を常法により液体培養し、培養液よりプラスミドＤＮＡを抽出し、該プラスミドを制限酵素により切断し、挿入断片を確認した。この結果、プラスミドｐＨＳＧ２９８の長さ２．６ｋｂのＤＮＡ断片に加え、長さ２．９ｋｂの挿入ＤＮＡ断片が認められた。本プラスミドをｐＨＳＧ２９８ｐａｒ−ｒｅｐと命名した。
【００７４】
（Ｂ）ｔａｃプロモーターの挿入
ｔａｃプロモーターを含有するプラスミドｐＴｒｃ９９Ａ（ファルマシア社製）を鋳型としたＰＣＲ法により、ｔａｃプロモーター断片を増幅させるべく、下記の１対のプライマーを、アプライド・バイオシステムズ（Applied Biosystems）社製「３９４ ＤＮＡ／ＲＮＡシンセサイザー（synthesizer ）」を用いて合成した。
【００７５】

【００７６】
実際のＰＣＲは、パーキンエルマーシータス社製の「ＤＮＡサーマルサイクラー」を用い、反応試薬として、レコンビナント・タックＤＮＡ・ポリメラーゼ・タカラ・タック（Recombinant TaqDNA Polymerase TaKaRa Taq）（宝酒造製）を用いて下記の条件で行った。
【００７７】
反応液：
（１０×）ＰＣＲ緩衝液 １０μｌ
１．２５ｍＭ ｄＮＴＰ混合液 １６μｌ
鋳型ＤＮＡ １０μｌ（DNA 含有量 １μＭ以下）
上記記載のａ−４，ｂ−４プライマー 各々１μｌ（最終濃度０．２５μＭ）
レコンビナント・タックＤＮＡ・ポリメラーゼ ０．５μｌ
滅菌蒸留水 ６１．５μｌ
以上を混合し、この１００μｌの反応液をＰＣＲにかけた。
【００７８】
ＰＣＲサイクル：
デナチュレーション過程：９４℃ ６０秒
アニーリング過程 ：５２℃ ６０秒
エクステンション過程 ：７２℃ １２０秒
【００７９】
以上を１サイクルとし、２５サイクル行った。
上記で生成した反応液１０μｌを３％アガロースゲルにより電気泳動を行い、約１００ｂｐのＤＮＡ断片が検出できた。
【００８０】
上記で増幅産物を確認できた反応液１０μｌ、上記（Ａ）で作製したプラスミドｐＨＳＧ２９８ｐａｒ−ｒｅｐ ５μｌを各々制限酵素ＢａｍＨＩおよびＫｐｎＩで完全に切断し、７０℃で１０分処理させることにより制限酵素を失活させた後、両者を混合し、これに、Ｔ4 ＤＮＡリガーゼ１０×緩衝液 １μｌ、Ｔ4 ＤＮＡリガーゼ１ｕｎｉｔの各成分を添加し、滅菌蒸留水で１０μｌにして、１５℃で３時間反応させ、結合させた。
【００８１】
得られたプラスミド混液を用い、塩化カルシウム法〔Journal of Molecular Biology, 53, 159(1970)〕によりエシェリヒア・コリＪＭ１０９（宝酒造製）を形質転換し、カナマイシン ５０ｍｇを含む培地〔トリプトン １０ｇ、イーストエキストラクト ５ｇ、ＮａＣｌ ５ｇ及び寒天 １６ｇを蒸留水１Ｌに溶解〕に塗抹した。
【００８２】
この培地上の生育株を常法により液体培養し、培養液よりプラスミドＤＮＡを抽出し、該プラスミドを制限酵素により切断し、挿入断片を確認した。この結果、上記（Ａ）作製のプラスミドの長さ５．５ｋｂのＤＮＡ断片に加え、長さ０．１ｋｂの挿入ＤＮＡ断片が認められた。このプラスミドをｐＨＳＧ２９８ｔａｃと命名した。
【００８３】
（Ｃ）ＰＣ遺伝子のシャトルベクターへの挿入
実施例１（Ｂ）で増幅産物を確認できた反応液１０μｌ、上記（Ｂ）で作製したプラスミドｐＨＳＧ２９８ｔａｃ ５μｌを各々制限酵素ＢｇｌII、ＳｓｅＩまたはＢａｍＨＩ、ＳｓｅＩで各々切断し、７０℃で１０分処理させることにより制限酵素を失活させた後、両者を混合し、これに、Ｔ4 ＤＮＡリガーゼ１０×緩衝液 １μｌ、Ｔ4 ＤＮＡリガーゼ１ｕｎｉｔの各成分を添加し、滅菌蒸留水で１０μｌにして、１５℃で３時間反応させ、結合させた。
【００８４】
得られたプラスミド混液を用い、塩化カルシウム法〔Journal of Molecular Biology, 53, 159(1970)〕によりエシェリヒア・コリＪＭ１０９（宝酒造製）を形質転換し、カナマイシン ５０ｍｇを含む培地〔トリプトン １０ｇ、イーストエキストラクト ５ｇ、ＮａＣｌ ５ｇ及び寒天 １６ｇを蒸留水１Ｌに溶解〕に塗抹した。
【００８５】
この培地上の生育株を常法により液体培養し、培養液よりプラスミドＤＮＡを抽出し、該プラスミドを制限酵素（ＳｓｅＩ，ＮｄｅＩ）により切断し、挿入断片を確認した。この結果、上記（Ｂ）作製のプラスミドの長さ５．６ｋｂのＤＮＡ断片に加え、長さ３．５６ｋｂの挿入ＤＮＡ断片が認められた。
このプラスミドをｐＰＣ−ＰＹＣ２と命名した。
【００８６】
（Ｄ）ブレビバクテリウム・フラバム MJ-233-AB-41株の形質転換
本プラスミドを米国特許第５，１８５，２６２号明細書記載の方法に従って、ブレビバクテリウム・フラバム MJ-233-AB-41（ＦＥＲＭ ＢＰ−１４９８）に導入した。
【００８７】
ブレビバクテリウム・フラバムＭＪ−２３３−ＡＢ−４１は、１９７６年１１月１７日に、通商産業省工業技術院生命工学工業技術研究所(日本国茨城県つくば市東１丁目１番３号、郵便番号305)に受託番号ＦＥＲＭ Ｐ−３８１２として寄託され、１９８１年５月１日にブダペスト条約に基づく国際寄託に移管され、受託番号ＦＥＲＭ ＢＰ−１４９８が付与されている。
【００８８】
〔実施例３〕有機酸の製造（Ｉ）
尿素：４ｇ、（ＮＨ₄）₂ＳＯ₄：１４ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、酵母エキス１ｇ、カザミノ酸１ｇ及び蒸留水：１０００ｍｌ（ｐＨ６．６）の培地を１００ｍｌずつ５００ｍｌ容の三角フラスコに分注し、１２０℃、１５分間滅菌処理したものに滅菌済み５０％グルコース水溶液４ｍｌを加え、上記ｐＰＣプラスミドを導入し形質転換させたブレビバクテリウム フラバムＭＪ−２３３−ＡＢ−４１菌株を植菌し、３３℃にて２４時間振とう培養した（好気的培養）。培養終了後、遠心分離(8000g、２０分)により菌体を回収した。得られた菌体全量を以下の反応に供試した。
【００８９】
（ＮＨ₄）₂ＳＯ₄：２３ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、炭酸ナトリウム２０ｇ／Ｌ、蒸留水：１０００ｍｌの培地を２Ｌ容のジャーファーメンターに入れ、上記菌体とグルコース５０％液１２０ｍｌを添加し、密閉した状態で（溶存酸素濃度０．１ｐｐｍ）、これを３０℃にて２４時間ゆるく(200rpm)攪拌し、反応させた。得られた培養液を遠心分離（８０００ｒｐｍ、１５分、４℃）して得られた上清液を分析したところ、乳酸が２８．５ｇ／Ｌ、酢酸が３．５ｇ／Ｌ、コハク酸が１６ｇ／Ｌ、リンゴ酸が１．２ｇ／Ｌ生成していた。
【００９０】
〔実施例４〕有機酸の製造（II）
実施例３と同様に培養した菌体を、以下の反応に供試した。
【００９１】
（ＮＨ₄）₂ＳＯ₄：２３ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、蒸留水：１０００ｍｌの培地を２Ｌ容のジャーファーメンターに入れ、上記菌体とグルコース５０％液１２０ｍｌを添加し、ここに１０％二酸化炭素ガス(９０％窒素ガス）を0.1vvmの速度で供給しながら３０℃にて２４時間ゆるく(200rpm)攪拌し、反応させた。得られた培養液を遠心分離（８０００ｒｐｍ、１５分、４℃）して得られた上清液を分析したところ、乳酸が２７ｇ／Ｌ、酢酸が２．５ｇ／Ｌ、コハク酸が１７ｇ／Ｌ、リンゴ酸が１．５ｇ／Ｌ生成していた。
【００９２】
〔比較例１〕
形質転換してないブレビバクテリウム・フラバム ＭＪ−２３３−ＡＢ−４１株を用いた以外は、実施例４と同様に行った。
【００９３】
尿素：４ｇ、（ＮＨ₄）₂ＳＯ₄：１４ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、酵母エキス１ｇ、カザミノ酸１ｇ及び蒸留水：１０００ｍｌ（ｐＨ６．６）の培地を１００ｍｌずつ５００ｍｌ容の三角フラスコに分注し、１２０℃、１５分間滅菌処理したものに滅菌済み５０％グルコース水溶液４ｍｌを加え、ブレビバクテリウム・フラバム ＭＪ−２３３−ＡＢ−４１菌株を植菌し、３３℃にて２４時間振とう培養した（好気的培養）。培養終了後、遠心分離(8000g、２０分)により菌体を回収した。得られた菌体全量を以下の反応に供試した。
【００９４】
（ＮＨ₄）₂ＳＯ₄：２３ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、炭酸ナトリウム２０ｇ／ｌ、蒸留水：１０００ｍｌの培地を２Ｌ容のジャーファーメンターに入れ、上記菌体とグルコース５０％液１２０ｍｌを添加し、密閉した状態で（溶存酸素濃度０．１ｐｐｍ）、これを３０℃にて２４時間ゆるく(200rpm)攪拌し、反応させた。得られた培養液を遠心分離（８０００ｒｐｍ、１５分、４℃）して得られた上清液を分析したところ、乳酸が３３．４ｇ／Ｌ、酢酸が５ｇ／Ｌ、コハク酸が１０ｇ／Ｌ、リンゴ酸が０．５ｇ／Ｌ生成していた。
【００９５】
〔比較例２〕
炭酸イオンを添加しない以外は、実施例４と同様に行った。
【００９６】
（ＮＨ₄）₂ＳＯ₄：２３ｇ，ＫＨ₂ＰＯ₄：０．５ｇ、Ｋ₂ＨＰＯ₄：０．５ｇ， ＭｇＳＯ₄・７Ｈ₂Ｏ：０．５ｇ，ＦｅＳＯ₄・７Ｈ₂Ｏ：２０ｍｇ，ＭｎＳＯ₄・ｎＨ₂Ｏ：２０ｍｇ、Ｄ−ビオチン：２００μｇ、塩酸チアミン：１００μｇ、蒸留水：１０００ｍｌの培地を２Ｌ容のジャーファーメンターに入れ、実施例４と同様に培養した菌体とグルコース５０％液１２０ｍｌを添加し、密閉した状態で（溶存酸素濃度０．１ｐｐｍ）、３０℃で２４時間ゆるく(200rpm)攪拌し、反応させた。得られた培養液を遠心分離（８０００ｒｐｍ、１５分、４℃）して得られた上清液を分析したところ、乳酸が１４ｇ／Ｌ、酢酸が３．６ｇ／Ｌ、コハク酸が２．４ｇ／Ｌ生成していた。
【００９７】
〔実施例５〕酵母（Saccharomyces cerevisiae）由来ＰＣ遺伝子（ＰＹＣ１）を用いた有機酸製造
酵母（Saccharomyces cerevisiae）由来のＰＣ遺伝子の一つであるＰＹＣ１はその配列が既知（Mol.Gen.Genet., 229, 307-315, (1991)）であるため、実施例１（Ａ）に示したように菌体を培養し、染色体ＤＮＡを調製し、これをＰＣＲの鋳型として用いて、実施例１（Ｂ）で使用したプライマーの代わりに、次の２つのプライマー（ａ−４，ｂ−４）により、ＰＣをコードする遺伝子部分を増幅させることができる。以下実施例２（Ｄ）と同様の手法で、酵母（Saccharomyces cerevisiae）由来のＰＣ遺伝子（ＰＹＣ１）で組み換えた好気性コリネ型細菌を得ることができる。
【００９８】

【００９９】
得られたコリネ型細菌を実施例３の方法に従って培養したところ、コハク酸が１５．５ｇ／Ｌ生成していた。
【０１００】
〔実施例６〕リゾビウム・エトリ由来ＰＣ遺伝子を用いた有機酸製造
リゾビウム・エトリ由来のＰＣ遺伝子はその配列が既知（J.Bacteriol., 178, 5960-5970, (1996)）であるため、実施例１（Ａ）に示したように菌体を培養し、染色体ＤＮＡを調製し、これをＰＣＲの鋳型として用いて、実施例１（Ｂ）で使用したプライマーの代わりに、次の２つのプライマー（ａ−５，ｂ−５）を用いて、ＰＣをコードする遺伝子部分を増幅させることができる。以下実施例２（Ｄ）と同様の手法で、Rhizobium etli由来のＰＣ遺伝子で組み換えた好気性コリネ型細菌を得ることができる。
【０１０１】

【０１０２】
得られたコリネ型細菌を実施例３の方法に従って培養したところ、コハク酸が１４．５ｇ／Ｌ生成していた。
【０１０３】
〔実施例７〕バチルス・ステアロサーモフィルス由来ＰＣ遺伝子を用いた有機酸製造
バチルス・ステアロサーモフィルス由来のＰＣ遺伝子はその配列が既知（GENE, 191, 47-50, (1997)）であるため、実施例１（Ａ）に示したように菌体を培養し、染色体ＤＮＡを調製し、これをＰＣＲの鋳型として用いて、実施例２（Ｄ）で使用したプライマーの代わりに、次の２つのプライマー（ａ−６，ｂ−６）を用いて、ＰＣをコードする遺伝子部分を増幅させることができる。以下実施例２（Ｄ）と同様の手法で、Bacillus atearothermophilus由来のＰＣ遺伝子で組み換えた好気性コリネ型細菌を得ることができる。
【０１０４】

【０１０５】
得られたコリネ型細菌を実施例３の方法に従って培養したところ、コハク酸が１４．０ｇ／Ｌ生成していた。
【０１０６】
【発明の効果】
本発明の方法によれば、ＰＣ遺伝子で組み換えた好気性コリネ型細菌を用いた培養法あるいは酵素法により、効率よく、かつ 高収率でコハク酸等の有機酸を製造することができる。
【０１０７】
【配列表】

【０１０８】

【０１０９】

【０１１０】

【０１１１】

【０１１２】

【０１１３】

【０１１４】

【０１１５】

【０１１６】

【０１１７】

【０１１８】

【０１１９】

【０１２０】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an organic acid production method using an aerobic coryneform bacterium recombined with a pyruvate carboxylase gene or a preparation thereof.
[0002]
[Prior art]
Pyruvate carboxylase (hereinafter sometimes abbreviated as “PC”) generates oxaloacetate by fixing carbon dioxide (bicarbonate ion) to pyruvate, which is a metabolic intermediate compound of glycolysis. 4 carbon (C) in acid (TCA) cycle _Four ) It is said to play a physiological role in supplementing compounds.
[0003]
The presence of pyruvate carboxylase has been widely confirmed in microorganisms, animals and plants.
Regarding the gene encoding the enzyme, many studies have been conducted on mammals, but regarding microorganisms, genes derived from Bacillus stearothermophilus [see GENE, 191, 47-50, (1997)], Gene derived from Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)], gene derived from yeast Saccharomyces serevisiae [Mol. Gen. Genet., 229, 307-315, (1991) Is isolated and its base sequence has only been determined.
[0004]
The TCA cycle is an important metabolic pathway in the biosynthesis system of various useful substances such as amino acids. By utilizing this gene, the supply of substances to the TCA cycle is strengthened, and it is expected that the ability to produce amino acids (aspartic acid, threonine, etc.) biosynthesized from oxaloacetic acid will be enhanced.
[0005]
As far as the present inventors know, from an industrial point of view, a method for producing amino acids (aspartic acid, threonine, etc.) biosynthesized from oxaloacetic acid using bacterial cells recombined with the phosphoenolpyruvate carboxylase gene 7-83714, Japanese Patent Laid-Open No. 9-121872], but the use of PC recombinant strains is hardly studied.
[0006]
That is, an efficient production method for producing malic acid, fumaric acid, succinic acid and the like by reversing the TCA cycle from oxaloacetic acid using an aerobic coryneform bacterium with enhanced PC gene has never been known.
[0007]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for producing organic acids such as malic acid, fumaric acid and succinic acid using aerobic coryneform bacteria recombined with a PC gene.
[0008]
[Means for Solving the Problems]
Accordingly, the present inventors examined a method for producing these organic acids using an aerobic coryneform bacterium. As a result, the aerobic coryneform bacterium or a preparation thereof was converted into carbonate ion, bicarbonate ion, or carbon dioxide gas. It is found that these organic acids can be produced efficiently by anaerobically acting on organic raw materials in a reaction solution containing, and further, PC activity is obtained by recombination of aerobic coryneform bacteria with the PC gene. It was found that the productivity of these organic acids was remarkably improved by using cells having enhanced ability to incorporate CO2 into pyruvic acid, and completed the present invention.
[0009]
That is, the gist of the present invention is that an aerobic coryneform bacterium recombined with a pyruvate carboxylase gene or a preparation thereof acts on an organic raw material anaerobically in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas. And producing an organic acid.
[0010]
In the above-described method, as a method for adding carbonate ion or bicarbonate ion or carbon dioxide gas to the reaction solution, carbon dioxide or bicarbonate or a salt thereof is added to the reaction solution, or carbon dioxide gas is supplied to the reaction solution. A method is mentioned.
[0011]
Examples of the pyruvate carboxylase gene include genes derived from microorganisms or animals and plants.
Examples of the pyruvate carboxylase gene include genes derived from humans, mice, rats, yeasts, or microorganisms belonging to the genus Corynebacterium, Bacillus, Rhizobium or Escherichia.
[0012]
Examples of the aerobic coryneform bacterium include Brevibacterium flavum. Moreover, Brevibacterium flavum MJ-233 is mentioned as Brevibacterium flavum.
Hereinafter, the present invention will be described in detail.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The PC gene used in the present invention is a gene whose base sequence has already been determined, or a DNA fragment encoding a protein having PC activity is isolated from a chromosome of a microorganism, animal or plant, etc., by a conventional method. The sequence determined can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used.
[0014]
The DNA fragment containing the PC gene of the present invention exists on chromosomes derived from microorganisms, animals and plants. The basic operation for preparing a PC gene from these source microorganisms and animals and plants is described as follows, taking as an example one derived from the yeast Saccharomyces cerevisiae, whose sequence is known.
[0015]
There are at least two types of PC genes on the Saccharomyces cerevisiae chromosome, and their sequences are known [Mol. Gen. Genet., 229, 307-315, (1991)]. Genes can be isolated and acquired.
[0016]
For example, when PCR is carried out using a oligonucleotide derived from Saccharomyces cerevisiae as a template using oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 as primers for PCR, a PC gene (PYC2) consisting of 3.56 kb is amplified. Can be made.
[0017]
Moreover, when PCR was performed using the oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 9 and 10 as a primer for PCR and a chromosome derived from Saccharomyces cerevisiae as a template, a PC gene (PYC1) consisting of 3.54 kb was amplified. Can be made.
[0018]
At this time, by adding an appropriate restriction enzyme site to the 5 ′ end of the primer used for PCR, it can be ligated to an appropriate site of the vector described later, and the resulting recombinant vector is used for coryneform type. Can be introduced into bacteria.
[0019]
Even if the gene sequence is unknown, the protein can be purified using PC activity as an index, and the gene fragment can be isolated from the N-terminal amino acid sequence or partially degraded sequence by a commonly used hybridization technique. Moreover, it is possible to obtain fragments by hybridization or PCR based on the amino acid sequence of the region conserved between PC proteins. The DNA fragment sequence of the obtained fragment can be determined by a usual method.
[0020]
In the present specification, the size of the “cleavage fragment” and the size of the plasmid can be obtained by cleaving Escherichia coli lambda phage DNA with restriction enzyme HindIII when using agarose gel electrophoresis. Based on a standard line drawn by the migration distance of DNA fragments of known molecular weight on the same agarose gel, and when polyacrylamide gel electrophoresis is used, DNA of Escherichia coli Phi-X 174 phage (φX174 phage) The size of each DNA fragment of the cleaved DNA fragment or plasmid can be calculated based on the standard line drawn by the migration distance on the same polyacrylamide gel of a DNA fragment of known molecular weight obtained by cleaving with restriction enzyme HaeIII it can. . In determining the size of each DNA fragment, the result obtained by 1% agarose gel electrophoresis is adopted for the size of a fragment of 1 kb or more, and the size of a fragment of about 0.1 kb to less than 1 kb is used. Results obtained by 4% polyacrylamide gel electrophoresis were employed.
[0021]
The DNA fragment used in the present invention including the PC gene is not only one isolated from Saccharomyces cerevisiae chromosomal DNA, but also a commonly used DNA synthesizer, such as 394 DNA / RNA synthesizer manufactured by Applied Biosystems. It may be synthesized using.
[0022]
In addition, as described above, a PC gene obtained from yeast (Saccharomyces serevisiae) chromosomal DNA is a part of the base sequence as long as it does not substantially impair the function of the encoded PC, that is, the property involved in carbon dioxide fixation. The base may be replaced with another base, may be deleted, a new base may be inserted, and a part of the base sequence may be rearranged. Well, any of these derivatives can be used in the present invention.
[0023]
In addition, a yeast gene other than Saccharomyces cerevisiae, or a PC gene derived from other microorganisms or animals and plants can also be used. In particular, the following microorganism or animal / plant-derived PC genes have known sequences (references are shown in parentheses), and amplify the ORF portion by hybridization or PCR as described above. Can be obtained. The obtained gene can be inserted downstream of the tac promoter of the vector prepared in Example 2 (B) described later. The inserted plasmid can be transformed into an aerobic coryneform bacterium according to the method of Example 2 (D) and used for the production of an organic acid.
[0024]
Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
yeast;
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]
[0025]
The DNA fragment containing the PC gene can be expressed by inserting it into an appropriate expression plasmid such as pUC118 (Takara Shuzo) and introducing it into an appropriate host microorganism such as Escherichia coli JM109 (Takara Shuzo). Confirmation of the expressed PC gene product, pyruvate carboxylase, was carried out by extracting a crude enzyme solution from the transformant and directly using the method of Fisher & Magasanik [J. Bacteriol., 158, 55-62, (1984)]. It can be confirmed by measuring the activity and comparing it with the PC activity of the crude enzyme solution extracted from the non-transformed strain.
[0026]
A DNA fragment containing a PC gene is introduced into a suitable plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in a coryneform bacterium, whereby recombination capable of highly expressing PC in the coryneform bacterium. A plasmid can be obtained.
[0027]
Here, in the above recombinant plasmid, the promoter for expressing the PC gene can be a promoter possessed by coryneform bacteria, but is not limited thereto, and has a base sequence for initiating transcription of the PC gene. Any promoter can be used.
[0028]
The plasmid vector into which the PC gene can be introduced is not particularly limited as long as it contains at least a gene that controls the replication growth function in coryneform bacteria. Specific examples thereof include, for example, a plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262. pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; PAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; described in JP-A-57-183799 PCG And pCG11, etc. can be mentioned.
[0029]
Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.
[0030]
A recombinant vector obtained by inserting a PC gene into a suitable plasmid vector replicable in an aerobic coryneform bacterium, such as a coryneform bacterium such as Brevibacterium flavum MJ-233 (FERM BP-1497) Aerobic coryneform bacterium recombined with the PEPC gene used in the present invention.
[0031]
The aerobic coryneform bacterium recombined with the PC gene used in the present invention or a preparation thereof is not particularly limited as long as it is a coryneform bacterium or a preparation thereof that can grow under normal aerobic conditions. However, examples thereof include coryneform bacteria such as Brevibacterium, Corynebacterium, and Arthrobacter, or preparations thereof. Of these, Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233-AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne Corynebacterium such as Corynebacterium glutamicum ATCC 31831, Brevibacterium lactofermentum ATCC 13869 or a preparation thereof is preferably used.
[0032]
In order to use an aerobic coryneform bacterium in the method of the present invention, it can be used after culturing the cells under normal aerobic conditions. As a medium used for culture, a medium usually used for culture of microorganisms can be used. For example, a general medium in which a natural nutrient source such as meat extract, yeast extract or peptone is added to a composition composed of inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used.
[0033]
The cultured cells are collected by centrifugation, membrane separation or the like, and used for the following reactions as they are or as preparations. The preparation here refers to, for example, an immobilized microbial cell immobilized with acrylamide, carrageenan, etc., a crushed product of the crushed cell, a supernatant of centrifugation thereof, or a part of the supernatant by ammonium sulfate treatment or the like. It refers to a fraction having purified PEPC activity.
[0034]
As the reaction solution, water, a buffer solution, a medium, or the like is used, and a medium containing an appropriate inorganic salt is most preferable. The reaction solution is characterized by containing an organic raw material such as glucose or ethanol and carbonate ion, bicarbonate ion or carbon dioxide gas, and reacting under anaerobic conditions. In this case, the organic raw material is not particularly limited and can be selected according to the target organic acid, and can be selected from general organic raw materials. Specifically, glucose and ethanol, which are inexpensive and have a high production rate of the target organic acid, are preferably used. In this case, the addition concentration of glucose is preferably 0.5 g / Ll to 500 g / L, and the addition concentration of ethanol is preferably 0.5 g / L to 30 g / L.
[0035]
Carbonate ions and bicarbonate ions are added at a concentration of 1 mM to 500 mM, preferably 2 to 300 mM, more preferably 3 to 200 mM. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas is contained per liter of the solution.
[0036]
Moreover, anaerobic conditions refer to making it react, keeping the dissolved oxygen concentration in a solution low. In this case, the dissolved oxygen concentration is desirably 0 to 2 ppm, preferably 0 to 1 ppm, more preferably 0 to 0.5 ppm. As a method therefor, for example, a method in which the container is sealed and reacted without aeration, an inert gas such as nitrogen gas is supplied and reacted, a carbon dioxide gas-containing inert gas is aerated, or the like is used. it can.
[0037]
The reaction temperature is usually 15 ° C to 45 ° C, preferably 25 ° C to 37 ° C. The pH is 5 to 9, preferably 6 to 8. The reaction is usually carried out for 5 to 120 hours. The amount of cells used in the reaction is not particularly limited, but 1 g / l to 700 g / l, preferably 10 g / l to 500 g / l, more preferably 20 g / l to 400 g / l is used. When using a preparation of bacterial cells, it is preferable to use an amount corresponding to the amount of bacterial cells of the above amount.
[0038]
The above-mentioned preparation means, for example, an immobilized microbial cell immobilized with acrylamide, carrageenan or the like, a crushed product obtained by crushing the microbial cell, a centrifugal supernatant thereof, or a part of the supernatant by ammonium sulfate treatment or the like It refers to a purified fraction.
[0039]
The organic acid produced by the method as described above can be separated and purified from the reaction solution by a normal separation and purification method as necessary. Specifically, it is separated from the cells and their preparation by ultrafiltration membrane separation, centrifugation, etc., and then purified by a known method such as a column method or a crystallization method, and then collected as crystals by drying. Methods and the like.
[0040]
In the present invention, the organic acid to be produced is not particularly limited, but from the effect of the present invention, a compound that cannot be efficiently produced by an aerobic corynebacterium or its preparation in an oxygen-containing atmosphere. Is particularly preferred.
[0041]
Specific examples include organic carboxylic acids, and more specific examples include succinic acid, malic acid, and fumaric acid.
[0042]
Although the present invention has been described above, the present invention will be described more specifically with reference to the following examples. However, it should be understood that the examples are to be regarded as an aid to obtaining specific recognition of the invention and are not intended to limit the scope of the invention.
[0043]
【Example】
[Example 1] Cloning of DNA fragment containing PC gene (PYC2) derived from yeast Saccharomyces cerevisiae
(A) Extraction of total DNA of Saccharomyces cerevisiae:
Saccharomyces cerevisiae W303-1A strain (Yeast, Vol. 2, 163-167 (1986)) in 1 L of yeast growth medium (YPAD) [composition: 10 g Yeast Extract, 20 g peptone, 20 g glucose, 100 mg adenine and distilled water: 1000 ml] ) Was inoculated using platinum ears and cultured at 30 ° C. until the late logarithmic growth phase to collect the cells.
[0044]
15 ml of a solution containing each component of 10 mg / ml lysozyme, 10 mM NaCl, 20 mM Tris buffer (pH 8.0) and 1 mM EDTA · 2Na so as to obtain a concentration of 10 mg / ml. Is the final concentration). Next, proteinase K was added to a final concentration of 100 μg / ml and incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate (SDS) was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal volume of phenol / chloroform solution, gently shake at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain the supernatant. The fraction was collected. To this supernatant, sodium acetate was added to a concentration of 0.3 M, and then twice the amount of ethanol was slowly added. DNA existing between the aqueous layer and the ethanol layer was scraped off with a glass rod, washed with 70% ethanol, and then air-dried. To the obtained DNA, 5 ml of 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and left overnight at 4 ° C., and used for the subsequent experiments.
[0045]
(B) Cloning of DNA fragment containing PC gene (PYC2) derived from Saccharomyces cerevisiae and creation of recombinant:
PCR was performed using the chromosomal DNA prepared in the above section (A) as a template. In the PCR, the following pair of primers were synthesized and used using “394 DNA / RNA synthesizer” (Applied Biosystems).

[0046]
The actual PCR uses a “DNA thermal cycler” manufactured by PerkinElmer Citas, Inc., and uses the following conditions as a reaction reagent: Recombinant TaqDNA Polymerase TaKaRa Taq (Takara Shuzo) I went there.
[0047]
Reaction solution:
(10 ×) PCR buffer 10 μl
1.25 mM dNTP mixture 16 μl
Template DNA 10μl (DNA content 1μM or less)
1 μl each of a-1 and b-1 primers described above (final concentration 0.25 μM)
Recombinant Tack DNA Polymerase 0.5 μl
Sterile distilled water 61.5 μl
The above was mixed and 100 μl of the reaction solution was subjected to PCR.
[0048]
PCR cycle:
Denaturation process: 94 ° C 60 seconds
Annealing process: 52 ° C 60 seconds
Extension process: 72 ° C 120 seconds
[0049]
The above is one cycle, and 25 cycles are performed.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 3.56 kp could be detected.
[0050]
[Example 2] Production of recombinant coryneform bacteria using PC gene
(A) Construction of shuttle vector
Based on the sequence of the region necessary for the stabilization of the plasmid in the coryneform bacterium present in the plasmid pCRY30 described in JP-A-3-210184, the following pair of primers was applied to Applied Biosystems. (Applied Biosystems) "394 DNA / RNA synthesizer" was used for synthesis.
[0051]

[0052]
The plasmid pCRY30 is a plasmid constructed as follows. Brevibacterium stathionis IFO 12144 (deposited with the accession number of FERM BP-2515 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology) from plasmid pBY503 (details of this plasmid are disclosed in JP-A-1-95785) (See the gazette) DNA is extracted, a DNA fragment (replication region) containing a gene responsible for the replication growth function of a plasmid having a size of about 4.0 kb is extracted with the restriction enzyme XhoI, and the size is about 2 with the restriction enzymes EcoRI and KpnI. A DNA fragment (stabilized region) containing a gene that controls the stabilizing function of a 1 kb plasmid is excised. A plasmid vector pCRY30 can be prepared by incorporating both of these DNA fragments into the EcoRI-KpnI site and SalI site of plasmid pHSG298 (Takara Shuzo).
[0053]
The actual PCR uses a “DNA thermal cycler” manufactured by PerkinElmer Citas, Inc., and uses the following conditions as a reaction reagent: Recombinant TaqDNA Polymerase TaKaRa Taq (Takara Shuzo) I went there.
[0054]
Reaction solution:
(10 ×) PCR buffer 10 μl
1.25 mM dNTP mixture 16 μl
Template DNA 10μl (DNA content 1μM or less)
1 μl each of the a-2 and b-2 primers described above (final concentration 0.25 μM)
Recombinant Tack DNA Polymerase 0.5 μl
Sterile distilled water 61.5 μl
The above was mixed and 100 μl of the reaction solution was subjected to PCR.
[0055]
PCR cycle:
Denaturation process: 94 ° C 60 seconds
Annealing process: 52 ° C 60 seconds
Extension process: 72 ° C 120 seconds
[0056]
The above is one cycle, and 25 cycles are performed.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 1.1 kb was detected.
[0057]
10 μl of the reaction solution in which the amplification product was confirmed as above and 1 μl of the plasmid pBluescriptIISK + were completely digested with the restriction enzymes EcoRI and XhoI, respectively, and the restriction enzyme was inactivated by treating at 70 ° C. for 10 minutes, and then both were mixed. To this, each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added, made 10 μl with sterile distilled water, reacted at 15 ° C. for 3 hours, and bound.
[0058]
Using the obtained plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg of ampicillin [tryptone 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].
[0059]
The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with a restriction enzyme (EcoRI, XhoI) to confirm the inserted fragment. As a result, in addition to the 3.0 kb DNA fragment of plasmid pBluescriptIISK +, an inserted DNA fragment of 1.1 kb was observed. This plasmid was named pBSpar.
[0060]
Based on the sequence of the region necessary for plasmid replication in the coryneform bacterium present in the plasmid pCRY31 described in US Pat. No. 5,185,262, the following pair of primers was applied to The synthesis was performed using “394 DNA / RNA synthesizer” (Applied Biosystems).
[0061]

[0062]
The plasmid pCRY31 is a plasmid constructed as follows. Plasmid pCRY3 (brevibacterium flavum MJ233 GE102 carrying this plasmid, deposited with the replication region derived from pBY503 and plasmid pHSG398 (manufactured by Takara Shuzo) was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as FERM BP-2513. Are partially digested with KpnI to obtain a DNA fragment. On the other hand, pBY503 is prepared from Brevibacterium lactofermentum IFO12144 (FERM BP-2515), completely digested with KpnI, and a DNA fragment of about 7 kb is purified. PCRY31 is obtained by ligating these DNA fragments and selecting a plasmid that shows the cleavage pattern shown in the following table when cleaved with various restriction enzymes.
[0063]
[Table 1]

[0064]
The actual PCR uses a “DNA thermal cycler” manufactured by PerkinElmer Citas, Inc., and uses the following conditions as a reaction reagent: Recombinant TaqDNA Polymerase TaKaRa Taq (Takara Shuzo) I went there.
[0065]
Reaction solution:
(10 ×) PCR buffer 10 μl
1.25 mM dNTP mixture 16 μl
Template DNA 10μl (DNA content 1μM or less)
1 μl each of the a-3 and b-3 primers described above (final concentration 0.25 μM)
Recombinant Tack DNA Polymerase 0.5 μl
Sterile distilled water 61.5 μl
The above was mixed and 100 μl of the reaction solution was subjected to PCR.
[0066]
PCR cycle:
Denaturation process: 94 ° C 60 seconds
Annealing process: 52 ° C 60 seconds
Extension process: 72 ° C 120 seconds
[0067]
The above is one cycle, and 25 cycles are performed.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 1.8 kb could be detected.
[0068]
After 10 μl of the reaction solution in which the amplification product was confirmed and 1 μl of the plasmid pBSpar were each completely digested with the restriction enzymes XhoI and KpnI and treated with 70 ° C. for 10 minutes to inactivate the restriction enzyme, both were mixed, Each component of T4 DNA ligase 10 ×) buffer solution 1 μl and T4 DNA ligase 1 unit was added to 10 μl with sterilized distilled water, reacted at 15 ° C. for 3 hours, and bound.
[0069]
Using the obtained plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg of ampicillin [tryptone 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].
[0070]
The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with restriction enzymes (XhoI, KpnI) to confirm the inserted fragment. As a result, in addition to the 4.1 kb DNA fragment of plasmid pBSpar, an inserted DNA fragment of 1.8 kb in length was observed. This plasmid was named pBSpar-rep.
[0071]
1 μl of the plasmid pBSpar-rep prepared above and 1 μl of pHSG298 (Takara Shuzo) were completely digested with the restriction enzymes KpnI and EcoRI, respectively, and the restriction enzymes were inactivated by treating them at 70 ° C. for 10 minutes. After mixing, each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added to 10 μl with sterilized distilled water, reacted at 15 ° C. for 3 hours, and bound.
[0072]
Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg kanamycin [tryptone 10 g, yeast extract]. 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].
[0073]
The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, in addition to the 2.6 kb DNA fragment of plasmid pHSG298, an inserted DNA fragment of 2.9 kb was observed. This plasmid was designated as pHSG298par-rep.
[0074]
(B) Insertion of tac promoter
In order to amplify the tac promoter fragment by the PCR method using the plasmid pTrc99A (Pharmacia) containing the tac promoter as a template, the following pair of primers was applied to “394 DNA / Applied Biosystems”. It was synthesized using an “synthesizer”.
[0075]

[0076]
The actual PCR uses a “DNA thermal cycler” manufactured by PerkinElmer Citas, Inc., and uses the following conditions as a reaction reagent: Recombinant TaqDNA Polymerase TaKaRa Taq (Takara Shuzo) I went there.
[0077]
Reaction solution:
(10 ×) PCR buffer 10 μl
1.25 mM dNTP mixture 16 μl
Template DNA 10μl (DNA content 1μM or less)
1 μl each of a-4 and b-4 primers described above (final concentration 0.25 μM)
Recombinant Tack DNA Polymerase 0.5 μl
Sterile distilled water 61.5 μl
The above was mixed and 100 μl of the reaction solution was subjected to PCR.
[0078]
PCR cycle:
Denaturation process: 94 ° C 60 seconds
Annealing process: 52 ° C 60 seconds
Extension process: 72 ° C 120 seconds
[0079]
The above is one cycle, and 25 cycles are performed.
10 μl of the reaction solution generated above was electrophoresed on a 3% agarose gel, and a DNA fragment of about 100 bp could be detected.
[0080]
10 μl of the reaction solution in which the amplification product was confirmed above and 5 μl of the plasmid pHSG298par-rep prepared in (A) above were completely digested with the restriction enzymes BamHI and KpnI, respectively, and treated with 70 ° C. for 10 minutes to thereby remove the restriction enzyme. After activation, both were mixed, and each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added to 10 μl with sterilized distilled water, reacted at 15 ° C. for 3 hours, and bound. I let you.
[0081]
Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg kanamycin [tryptone 10 g, yeast extract]. 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].
[0082]
The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, an inserted DNA fragment having a length of 0.1 kb was recognized in addition to the 5.5 kb DNA fragment of the plasmid prepared in (A). This plasmid was named pHSG298tac.
[0083]
(C) Insertion of PC gene into shuttle vector
10 μl of the reaction solution in which the amplification product was confirmed in Example 1 (B) and 5 μl of the plasmid pHSG298tac prepared in (B) above were cleaved with restriction enzymes BglII, SseI or BamHI and SseI, respectively, and treated at 70 ° C. for 10 minutes. After inactivating the restriction enzyme, both components were mixed, and each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added to 10 μl with sterilized distilled water at 15 ° C. Reacted for 3 hours and allowed to bind.
[0084]
Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg kanamycin [tryptone 10 g, yeast extract]. 5 g, NaCl 5 g and agar 16 g were dissolved in 1 L of distilled water].
[0085]
The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with restriction enzymes (SseI, NdeI) to confirm the inserted fragment. As a result, in addition to the 5.6 kb DNA fragment of the plasmid prepared in (B) above, an inserted DNA fragment of 3.56 kb in length was observed.
This plasmid was named pPC-PYC2.
[0086]
(D) Transformation of Brevibacterium flavum MJ-233-AB-41 strain
This plasmid was introduced into Brevibacterium flavum MJ-233-AB-41 (FERM BP-1498) according to the method described in US Pat. No. 5,185,262.
[0087]
Brevibacterium flavum MJ-233-AB-41 was inaugurated on November 17, 1976 at the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry (1-3 East, 1-3 Tsukuba, Ibaraki, Japan) 305) as deposit number FERM P-3812, transferred to an international deposit under the Budapest Treaty on May 1, 1981, and assigned deposit number FERM BP-1498.
[0088]
[Example 3] Production of organic acid (I)
Urea: 4 g, (NH _Four ) ₂ SO _Four : 14g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, yeast extract 1 g, casamino acid 1 g and distilled water: 1000 ml (pH 6.6) medium were dispensed in 100 ml portions into a 500 ml Erlenmeyer flask at 120 ° C. Sterilized for 15 minutes, 4 ml of a sterilized 50% aqueous glucose solution was added, and the Brevibacterium flavum MJ-233-AB-41 strain into which the pPC plasmid was introduced and transformed was inoculated, and the culture was continued at 33 ° C. for 24 hours. Shake culture (aerobic culture). After completion of the culture, the cells were collected by centrifugation (8000 g, 20 minutes). The total amount of the obtained microbial cells was subjected to the following reaction.
[0089]
(NH _Four ) ₂ SO _Four : 23g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, Thiamine hydrochloride: 100 μg, Sodium carbonate 20 g / L, Distilled water: 1000 ml of medium was placed in a 2 L jar fermenter, and the above cells and 120 ml of glucose 50% solution were added, In a sealed state (dissolved oxygen concentration of 0.1 ppm), this was allowed to react by stirring gently (200 rpm) at 30 ° C. for 24 hours. When the supernatant obtained by centrifuging the obtained culture broth (8000 rpm, 15 minutes, 4 ° C.) was analyzed, lactic acid was 28.5 g / L, acetic acid was 3.5 g / L, and succinic acid was 16 g. / L and malic acid were produced at 1.2 g / L.
[0090]
[Example 4] Production of organic acid (II)
The cells cultured in the same manner as in Example 3 were subjected to the following reaction.
[0091]
(NH _Four ) ₂ SO _Four : 23g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, distilled water: 1000 ml of the medium was put into a 2 L jar fermenter, and the above cells and 120 ml of glucose 50% solution were added, and 10% carbon dioxide was added thereto. While supplying gas (90% nitrogen gas) at a rate of 0.1 vvm, the reaction was performed by stirring gently (200 rpm) at 30 ° C. for 24 hours. When the supernatant obtained by centrifuging the obtained culture broth (8000 rpm, 15 minutes, 4 ° C.) was analyzed, lactic acid was 27 g / L, acetic acid was 2.5 g / L, and succinic acid was 17 g / L. , 1.5 g / L of malic acid was produced.
[0092]
[Comparative Example 1]
This was carried out in the same manner as in Example 4 except that the untransformed Brevibacterium flavum MJ-233-AB-41 strain was used.
[0093]
Urea: 4 g, (NH _Four ) ₂ SO _Four : 14g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, yeast extract 1 g, casamino acid 1 g and distilled water: 1000 ml (pH 6.6) medium were dispensed in 100 ml portions into a 500 ml Erlenmeyer flask at 120 ° C. Sterilized for 15 minutes, 4 ml of sterilized 50% aqueous glucose solution was added, Brevibacterium flavum MJ-233-AB-41 strain was inoculated, and cultured with shaking at 33 ° C. for 24 hours (aerobic culture). ). After completion of the culture, the cells were collected by centrifugation (8000 g, 20 minutes). The total amount of the obtained microbial cells was subjected to the following reaction.
[0094]
(NH _Four ) ₂ SO _Four : 23g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, Thiamine hydrochloride: 100 μg, Sodium carbonate 20 g / l, Distilled water: 1000 ml of medium was placed in a 2 L jar fermenter, and the above cells and 120 ml of glucose 50% solution were added, In a sealed state (dissolved oxygen concentration of 0.1 ppm), this was allowed to react by stirring gently (200 rpm) at 30 ° C. for 24 hours. When the supernatant obtained by centrifuging the obtained culture broth (8000 rpm, 15 minutes, 4 ° C.) was analyzed, lactic acid was 33.4 g / L, acetic acid was 5 g / L, and succinic acid was 10 g / L. , 0.5 g / L of malic acid was produced.
[0095]
[Comparative Example 2]
It carried out like Example 4 except not adding a carbonate ion.
[0096]
(NH _Four ) ₂ SO _Four : 23g, KH ₂ PO _Four : 0.5 g, K ₂ HPO _Four : 0.5 g, MgSO _Four ・ 7H ₂ O: 0.5 g, FeSO _Four ・ 7H ₂ O: 20 mg, MnSO _Four ・ NH ₂ O: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 100 μg, distilled water: 1000 ml of medium was placed in a 2 L jar fermenter, and cells cultured in the same manner as in Example 4 and 120 ml of 50% glucose solution were added. In a sealed state (dissolved oxygen concentration 0.1 ppm), the mixture was allowed to react by stirring gently (200 rpm) at 30 ° C. for 24 hours. When the supernatant obtained by centrifuging the obtained culture broth (8000 rpm, 15 minutes, 4 ° C.) was analyzed, lactic acid was 14 g / L, acetic acid was 3.6 g / L, and succinic acid was 2.4 g. / L was generated.
[0097]
[Example 5] Organic acid production using PC gene (PYC1) derived from yeast (Saccharomyces cerevisiae)
Since the sequence of PYC1, one of the PC genes derived from yeast (Saccharomyces cerevisiae), is known (Mol. Gen. Genet., 229, 307-315, (1991)), it is shown in Example 1 (A). The microbial cells were cultured as described above, and chromosomal DNA was prepared and used as a template for PCR. Instead of the primers used in Example 1 (B), the following two primers (a-4, b- According to 4), the gene part encoding PC can be amplified. Thereafter, an aerobic coryneform bacterium recombined with a PC gene (PYC1) derived from yeast (Saccharomyces cerevisiae) can be obtained in the same manner as in Example 2 (D).
[0098]

[0099]
When the obtained coryneform bacterium was cultured according to the method of Example 3, 15.5 g / L of succinic acid was produced.
[0100]
[Example 6] Organic acid production using Rhizobium etori-derived PC gene
Since the sequence of the Rhizobium etori-derived PC gene is known (J. Bacteriol., 178, 5960-5970, (1996)), the cells were cultured as shown in Example 1 (A), and chromosomes were obtained. DNA is prepared and used as a template for PCR, and the following two primers (a-5, b-5) are used in place of the primers used in Example 1 (B) to encode PC. The gene part can be amplified. Thereafter, an aerobic coryneform bacterium recombined with a PC gene derived from Rhizobium etli can be obtained in the same manner as in Example 2 (D).
[0101]

[0102]
When the obtained coryneform bacterium was cultured according to the method of Example 3, 14.5 g / L of succinic acid was produced.
[0103]
[Example 7] Organic acid production using PC gene derived from Bacillus stearothermophilus
Since the sequence of the PC gene derived from Bacillus stearothermophilus is known (GENE, 191, 47-50, (1997)), the cells were cultured as shown in Example 1 (A), and the chromosome DNA is prepared and used as a template for PCR, and the following two primers (a-6 and b-6) are used in place of the primers used in Example 2 (D) to encode PC. The gene part can be amplified. Thereafter, an aerobic coryneform bacterium recombined with a PC gene derived from Bacillus atearothermophilus can be obtained in the same manner as in Example 2 (D).
[0104]

[0105]
When the obtained coryneform bacterium was cultured according to the method of Example 3, 14.0 g / L of succinic acid was produced.
[0106]
【The invention's effect】
According to the method of the present invention, an organic acid such as succinic acid can be produced efficiently and in a high yield by a culture method or an enzymatic method using an aerobic coryneform bacterium recombined with a PC gene.
[0107]
[Sequence Listing]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

Claims (6)

   Aerobic coryneform bacterium recombined with pyruvate carboxylase gene or its preparation anaerobically acting on organic raw materials in a reaction solution containing carbonate ion, bicarbonate ion or carbon dioxide gas to produce succinic acid A process for producing succinic acid, characterized in that The method according to claim 1, wherein the aerobic coryneform bacterium recombined with a pyruvate carboxylase gene has enhanced pyruvate carboxylase activity.    3. Carbon dioxide, bicarbonate ion, or carbon dioxide gas is added to the reaction solution by adding carbonic acid or bicarbonate or a salt thereof to the reaction solution, or carbon dioxide gas is supplied to the reaction solution. The method described in 1.  The method according to any one of claims 1 to 3, wherein the pyruvate carboxylase gene is derived from a microorganism or an animal or plant.   The method according to claim 4, wherein the pyruvate carboxylase gene is derived from human, mouse, rat, yeast, or a microorganism belonging to the genus Corynebacterium, Bacillus, Rhizobium or Escherichia.  The method according to any one of claims 1 to 5, wherein the aerobic coryneform bacterium is Brevibacterium flavum.