JP4463871B2 - 組換え微生物 - Google Patents
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- JP4463871B2 JP4463871B2 JP2009095379A JP2009095379A JP4463871B2 JP 4463871 B2 JP4463871 B2 JP 4463871B2 JP 2009095379 A JP2009095379 A JP 2009095379A JP 2009095379 A JP2009095379 A JP 2009095379A JP 4463871 B2 JP4463871 B2 JP 4463871B2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
一態様において、前記異種のタンパク質又はポリペプチドは、バチルス属細菌由来のアルカリセルラーゼ又はアルカリアミラーゼである。
枯草菌168株から抽出したゲノムDNAを鋳型とし、表2に示したccpA-AFとccpA-A/CmR、及びccpA-B/CmFとccpA-BRの各プライマーセットを用いて、ゲノム上のccpA遺伝子の上流に隣接する0.6kb断片(A)、及び下流に隣接する0.6kb断片(B)をそれぞれ調製した。一方、プラスミドpC194(J. Bacteriol. 150 (2), 815 (1982))のクロラムフェニコール耐性遺伝子をプラスミドpUC18のXbaI−BamHI切断点に挿入した組換えプラスミドpCBB31を鋳型とし、表2に示したCmFとCmRプライマーセットを用いて、クロラムフェニコール耐性遺伝子を含む1kb断片(C)を調製した。次に、得られた(A)(B)(C)3断片を混合して鋳型とし、表2のプライマーccpA-AFとccpA-BRを用いたSOE−PCRを行うことによって、3断片を(A)(C)(B)の順になる様に結合させ、2.2kbのDNA断片を得た(図1参照)。このDNA断片を用いてコンピテント法により枯草菌168株の形質転換を行い、クロラムフェニコールを含むLB寒天培地上に生育したコロニーを形質転換体として分離した。得られた形質転換体のゲノムを抽出し、PCRによってccpA遺伝子が削除され、クロラムフェニコール耐性遺伝子に置換していることを確認した。
一方、実施例1と同様に、表2に示した各遺伝子-AF、各遺伝子-A/CmR、各遺伝子-B/CmF、各遺伝子-BR、CmF、CmRのプライマーセットにより調製した削除用DNA断片を用いて、ゲノム上のcomA、yopO、treR、yvbA、yvaN、yttP、yurK、yozA、licR、sigL、mntR、glcT、ykvE、slr、rocR、yyaA、及びrsiX、遺伝子が削除され、クロラムフェニコール耐性
遺伝子に置換した胞子形成遺伝子削除株をそれぞれ分離した。
また、表2に示した各遺伝子-AF、各遺伝子-A/Cm2R、各遺伝子-B/Cm2F、各遺伝子-BR、Cm2F、Cm2Rのプライマーセットにより、実施例2と同様に調製した削除用DNA断片を用いて、ゲノム上のcspB、yvdE、yaaT、yycH、及びylbO、各遺伝子が削除され、クロラムフェニコール耐性遺伝子に置換した胞子形成遺伝子削除株をそれぞれ分離した。
更に、表2に示した各遺伝子-AF、各遺伝子-A/Cm4R、各遺伝子-B/Cm4F、各遺伝子-BR、各遺伝子-A/Cm4F、各遺伝子-B/Cm4Rのプライマーセットにより、実施例2と同様に調製した削除用DNA断片を用いて、ゲノム上のyacP、hprK、及びyhdK、各遺伝子が削除され、クロラムフェニコール耐性遺伝子に置換した胞子形成遺伝子削除株をそれぞれ分離した。
実施例1〜4にて得られた各遺伝子削除株、及び対照として枯草菌168株に、バチルス エスピー(Bacillus sp.)KSM−S237株由来のアルカリセルラーゼ遺伝子(特開2000-210081号公報)をコードするDNA断片(3.1kb)がシャトルベ
クターpHY300PLKのBamHI制限酵素切断点に挿入された組換えプラスミドpHY−S237を、プロトプラスト形質転換法によって導入した。これによって得られた菌株を5mLのLB培地で一夜30℃で振盪培養を行い、更にこの培養液0.03mLを30mLの2×L−マルトース培地(2%トリプトン、1%酵母エキス、1%NaCl、7.5%マルトース、7.5ppm硫酸マンガン4−5水和物、15ppmテトラサイクリン)に接種し、30℃で3日間、振盪培養を行った。培養後、遠心分離によって菌体を除いた培養液上清のアルカリセルラーゼ活性を測定し、培養によって菌体外に分泌生産されたアルカリセルラーゼの量を求めた。この結果、表3に示した様に、宿主として各遺伝子削除株を用いた場合、対照の168株(野生型)の場合と比較して高いアルカリセルラーゼの分泌生産が認められた。
実施例1〜4にて得られた各遺伝子削除株、及び対照として枯草菌168株に、バチルス エスピー(Bacillus sp.)KSM−K38株由来のアルカリアミラーゼ遺伝子(特開2000−184882号公報、Eur. J. Biochem., 268, 2974 (2001))の成熟酵素領域(Asp1−Gln480)をコードするDNA断片(1.5kb)の上流に配列番号3に示されるアルカリセルラーゼ遺伝子のプロモーター領域とシグナル配列領域の一部を含む上流側0.6kb断片を結合して成る2.1kb断片(配列番号5)をシャトルベクターpHY300PLKのBglII−XbaI制限酵素切断部位に挿入された組換えプラスミドpHSP−K38を、プロトプラスト形質転換法によって導入した。これによって得られた菌株を5mLのLB培地で一夜30℃で振盪培養を行い、更にこの培養液0.6mLを30mLの2×L−マルトース培地(2%トリプトン、1%酵母エキス、1%NaCl、7.5%マルトース、7.5ppm硫酸マンガン4−5水和物、15ppmテトラサイクリン)に接種し、30℃で3〜6日間、振盪培養を行った。培養後、遠心分離によって菌体を除いた培養液上清のアルカリアミラーゼ活性を測定し、培養によって菌体外に分泌生産されたアルカリアミラーゼの量を求めた。この結果、表4に示した様に、各遺伝子削除株を宿主として用いた場合、対照の168株(野生型)の場合と比較して高いアルカリアミラーゼの分泌生産が認められた。
Claims (2)
- 枯草菌の遺伝子yopO、treR、yvbA、cspB、yvaN、licR、sigL、glcT、yvdE、slr、rocR、yycH及びyacPのいずれか1以上の遺伝子が削除又は不活性化された枯草菌又はその他のバチルス属細菌株に、転写開始制御領域、翻訳開始制御領域及び分泌用シグナル領域が上流に結合したバチルス属細菌由来のアルカリセルラーゼ又はアルカリアミラーゼをコードする遺伝子を導入した組換え微生物であって、当該転写開始制御領域、翻訳開始制御領域及び分泌シグナル領域が、配列番号1で示される塩基配列からなるセルラーゼ遺伝子の塩基番号1〜659の塩基配列、配列番号3で示される塩基配列からなるセルラーゼ遺伝子の塩基番号1〜696の塩基配列又は当該塩基配列のいずれかと90%以上の同一性を有する塩基配列からなり、且つ転写開始制御機能、翻訳開始制御機能及び分泌用シグナル機能を有するDNA断片である、組換え微生物。
- 請求項1記載の組換え微生物を用いるバチルス属細菌由来のアルカリセルラーゼ又はアルカリアミラーゼの製造方法。
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