JP6853787B2 - スクロース資化性を有するpha生産微生物、及び該微生物を用いたphaの製造方法 - Google Patents
スクロース資化性を有するpha生産微生物、及び該微生物を用いたphaの製造方法 Download PDFInfo
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- JP6853787B2 JP6853787B2 JP2017556109A JP2017556109A JP6853787B2 JP 6853787 B2 JP6853787 B2 JP 6853787B2 JP 2017556109 A JP2017556109 A JP 2017556109A JP 2017556109 A JP2017556109 A JP 2017556109A JP 6853787 B2 JP6853787 B2 JP 6853787B2
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Description
(1)配列番号1に記載のアミノ酸配列をコードするスクロース加水分解酵素遺伝子、又は該アミノ酸配列に対して90%以上の配列同一性を有し、スクロース加水分解酵素活性を有するポリペプチドをコードする遺伝子
(2)配列番号2に記載のアミノ酸配列をコードするスクロース透過酵素遺伝子、又は該アミノ酸配列に対して90%以上の配列同一性を有し、スクロース透過酵素活性を有するポリペプチドをコードする遺伝子
好ましくは、前記微生物が、カプリアビダス属に属する微生物を宿主とする形質転換体であり、より好ましくは、前記カプリアビダス属に属する微生物が、カプリアビダス・ネカトールである。好ましくは、前記微生物はグルコース資化性が付与又は強化されている。好ましくは、前記PHA合成酵素遺伝子が、P(3HB−co−3HH)を合成可能なPHA合成酵素遺伝子である。好ましくは、前記微生物は、さらにクロトニル−CoA還元酵素遺伝子およびエチルマロニル−CoA脱炭酸酵素遺伝子を有する。好ましくは、前記微生物は、アセトアセチルCoA還元酵素遺伝子が欠失した、またはその発現量が抑制されている。
本発明では、PHA合成酵素遺伝子を有する微生物に対し、異種生物由来のスクロース加水分解酵素遺伝子と異種生物由来のスクロース透過酵素遺伝子の両方を導入することによって、スクロース資化性が付与または強化され、スクロースを炭素源としてPHAを生産する微生物を提供する。
本発明の微生物を、炭素源としてスクロースを含む培地で培養することで、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。
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。
まず、cscAおよびcscB遺伝子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。
製造例2で作製したプラスミドベクターpCUP2−lacUV5−cscABを、製造例2と同様の方法で、製造例1で作製したKNK005ΔphaZ1,2,6/nagEG793C,dR株へ導入し、形質転換体pCUP2−lacUV5−cscAB in KNK005ΔphaZ1,2,6/nagEG793C,dR株を得た。
まず、cscA遺伝子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。
まず、cscB遺伝子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。
まず、特開2013−9627号公報に記載のプラスミドbAO/pBlu/SacB−Kmを用いて、以下のようにしてプロモーターおよびリボソーム結合配列挿入株ACP−bktB/ΔphaZ1,2,6/nagEG793C,dR株の作製を行った。
まず、プロモーター、リボソーム結合配列及び遺伝子挿入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。
まず、遺伝子破壊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8に記載の遺伝子破壊用プラスミドベクターpNS2X−sacB+phaAB1UDを用いて、製造例7に記載のKNK−143S株を親株として、製造例1に記載の染色体置換株の作製と同様に、接合伝達、シモンズ寒天培地での選択、及び、15%のシュークロースを含むNutrient Agar培地での選択を行い、KNK142S株を作製した。KNK142S株はC.necator H16株の染色体上のphaZ6遺伝子及びphaZ1遺伝子の開始コドンから終止コドンまでを欠失し、さらにphaZ2遺伝子の16番目のコドンから終止コドンまでを欠失し、染色体上に配列番号4に記載のアミノ酸配列を有するPHA合成酵素をコードする遺伝子が導入され、nagE構造遺伝子の793番目の塩基であるGがCに置換され、nagR遺伝子の開始コドンから終止コドンまでを欠失し、bktB遺伝子の開始コドン直前にA. caviaeのphaC遺伝子のプロモーターおよびリボソーム結合配列を含む塩基配列からなるDNAが挿入され、phaJ4b遺伝子の開始コドン直前にtrcプロモーターおよびリボソーム結合配列を含む塩基配列からなるDNAが挿入され、元々はphaZ2遺伝子があった位置にtrcプロモーター、リボソーム結合配列、ccr遺伝子及びemd遺伝子が挿入され、さらにphaA遺伝子の開始コドンからphaB1遺伝子の終止コドンまでを欠失した株である。
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1〜4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1〜4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1〜4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。
Claims (7)
- PHA合成酵素遺伝子と、下記(1)及び(2)の異種生物由来遺伝子とを有し、
カプリアビダス・ネカトールを宿主とする形質転換体である、PHA生産微生物。
(1)配列番号1に記載のアミノ酸配列をコードするスクロース加水分解酵素遺伝子、又は該アミノ酸配列に対して90%以上の配列同一性を有し、スクロース加水分解酵素活性を有するポリペプチドをコードする遺伝子
(2)配列番号2に記載のアミノ酸配列をコードするスクロース透過酵素遺伝子、又は該アミノ酸配列に対して90%以上の配列同一性を有し、スクロース透過酵素活性を有するポリペプチドをコードする遺伝子 - グルコース資化性が付与又は強化されている、請求項1に記載の微生物。
- 前記PHA合成酵素遺伝子が、P(3HB−co−3HH)を合成可能なPHA合成酵素遺伝子である、請求項1又は2に記載の微生物。
- さらにクロトニル−CoA還元酵素遺伝子およびエチルマロニル−CoA脱炭酸酵素遺伝子を有する、請求項1〜3いずれか1項に記載の微生物。
- アセトアセチルCoA還元酵素遺伝子が欠失した、またはその発現量が抑制されている、請求項4に記載の微生物。
- 請求項1〜5いずれか1項に記載の微生物を、スクロースを炭素源として含む培地で培養する工程を含む、PHAの製造方法。
- PHAがP(3HB−co−3HH)である請求項6に記載の製造方法。
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