JP7001596B2 - 3hh単位含有共重合phaを生産する形質転換体、及び当該phaの製造方法 - Google Patents
3hh単位含有共重合phaを生産する形質転換体、及び当該phaの製造方法 Download PDFInfo
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Classifications
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Description
本発明では、3HH単位を含有する共重合PHAを合成可能なPHA合成酵素遺伝子を有する原核微生物に対し、トランス-2-エノイル-CoAヒドラターゼ活性と(R)-3-ヒドロキシアシル-CoAデヒドロゲナーゼ活性を有する酵素をコードする遺伝子を導入することによって、3HH単位をより高い組成比率で含有する共重合PHAを生産する形質転換体を提供する。
また、非特許文献5において、3HHモノマーを取り込み可能なPHA合成酵素遺伝子を導入したカプリアビダス・ネカトールに、クロトニル-CoA還元酵素(CCR)遺伝子及びエチルマロニル-CoA脱炭酸酵素(EMD)遺伝子を導入することで、糖を炭素源としてP(3HB-co-3HH)を生産できることが報告されている。本発明の形質転換体においても、前記導入遺伝子に加えて、CCR遺伝子及び/又はEMD遺伝子が導入されていても良い。CCR遺伝子及び/又はEMD遺伝子の導入により、糖を炭素源とする場合において、上述した炭素数6の(R)-3-ヒドロキシアシル-CoAの合成経路が強化又は効率化され、生産される共重合PHAにおける3HH単位の組成比率が向上することになる。
本発明の形質転換体を培養することで、PHAを生産させ、得られたPHAを回収することでPHAを製造することができる。
この製造例では、MFE2yl発現用プラスミドの作製を行った。作製は以下のように行った。
この実施例では、製造例1で得たプラスミドベクターpCUP2-trc-MFE2ylをKNK005株に導入して、形質転換体pCUP2-trc-MFE2yl/KNK005株を得た。
まず、染色体置換用プラスミドの作製を行った。作製は以下のように行った。
製造例1で作製したプラスミドベクターpCUP2-trc-MFE2ylを、実施例1に記載の電気導入によって、製造例2に記載のKNK005ΔphaZ1,2,6/nagEG793C,dR/Z2,Z6::Poe1-ccr-emd株に導入し、pCUP2-trc-MFE2yl/KNK005ΔphaZ1,2,6/nagEG793C,dR/Z2,Z6::Poe1-ccr-emd株を取得した。
まず、特開2013-9627号公報に記載の染色体導入用プラスミドベクターbAO/pBlu/SacB-Kmを用いて、KNK005ΔphaZ1,2,6/nagEG793C,dR株を親株として、製造例2に記載の方法でプロモーターおよびSD配列挿入株ACP-bktB/ΔphaZ1,2,6/nagEG793C,dR株の作製を行った。
製造例1で作製したプラスミドベクターpCUP2-trc-MFE2ylを、実施例1に記載の電気導入によって、製造例3に記載のKNK143S/Z6::Poe1-ccr-emd株に導入し、pCUP2-trc-MFE2yl/KNK143S/Z6::Poe1-ccr-emd株を取得した。
種母培地の組成は1w/v% Meat-extract、1w/v% Bacto-Trypton、0.2w/v% Yeast-extract、0.9w/v% Na2HPO4・12H2O、0.15w/v% KH2PO4とした。
種母培地の組成は比較例1に記載のものと同様とした。種母培地でプラスミドベクター導入株を培養する場合には、カナマイシンを最終濃度100μg/mlとなるように種母培地に添加した。
種母培地の組成は比較例1に記載のものと同様とした。PHA生産に使用した生産培地の組成は比較例1に記載のものと同様とし、炭素源としてはパーム核油ではなく、40w/v%グルコース水溶液を単一炭素源として用い、グルコースが2.0w/v%となるように生産培地に添加した。
種母培地の組成は比較例1に記載のものと同様とした。種母培地でプラスミドベクター導入株を培養する場合には、カナマイシンを最終濃度100μg/mlとなるように種母培地に添加した。
種母培地の組成は比較例1に記載のものと同様とした。PHA生産に使用した生産培地の組成は比較例1に記載のものと同様とし、炭素源としてはパーム核油ではなく、40w/v%グルコース水溶液を単一炭素源として用い、グルコースが2.0w/v%となるように生産培地に添加した。
種母培地の組成は比較例1に記載のものと同様とした。PHA生産に使用した生産培地の組成は比較例1に記載のものと同様とし、炭素源としてはパーム核油ではなく、40w/v%グルコース水溶液を単一炭素源として用い、グルコースが2.0w/v%となるように生産培地に添加した。
Claims (7)
- 3HH単位を含有する共重合PHAを合成可能なPHA合成酵素遺伝子を有する原核微生物に対し、トランス-2-エノイル-CoAヒドラターゼ活性と(R)-3-ヒドロキシアシル-CoAデヒドロゲナーゼ活性を有する酵素をコードする遺伝子が導入されてなり、
前記トランス-2-エノイル-CoAヒドラターゼ活性と(R)-3-ヒドロキシアシル-CoAデヒドロゲナーゼ活性を有する酵素をコードする遺伝子が、ヤロウィア・リポリティカに由来する遺伝子、又は、ドロソフィラ・メラノガスターに由来する遺伝子であり、
前記原核微生物がカプリアビダス・ネカトールである、3HH単位を含有する共重合PHAを生産する形質転換体。 - さらに、クロトニル-CoA還元酵素(CCR)をコードする遺伝子が導入されてなる請求項1に記載の形質転換体。
- さらに、エチルマロニル-CoA脱炭酸酵素をコードする遺伝子が導入されてなる請求項2に記載の形質転換体。
- 請求項1~3いずれか1項に記載の形質転換体を培養する工程を含む、3HH単位を含有する共重合PHAの製造方法。
- 前記共重合PHAがP(3HB-co-3HH)である請求項4に記載の製造方法。
- 炭素源として油脂または脂肪酸を含む培地において前記形質転換体を培養する請求項4又は5に記載の製造方法。
- 炭素源として糖を含む培地において前記形質転換体を培養する請求項4又は5に記載の製造方法。
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