JP2007289193A - クラバム抗生物質の産生を強化する方法 - Google Patents
クラバム抗生物質の産生を強化する方法 Download PDFInfo
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Abstract
【解決手段】遺伝子挿入により分裂したストレプトマイセス・クラブリゲラスから由来のlat遺伝子を含有する組換えプラスミド。ならびに、分裂したlat遺伝子を有するpCF002と称される組換えプラスミド;および分裂したlat遺伝子を有するp486latapと称される組換えプラスミド;より選択される、プラスミド。
【選択図】なし
Description
該方法を開始するのに、pA2/119(A.K.Petrichの論文(1993);ストレプトマイセス・クラブリゲラスのイソペニシリン N シンセターゼ遺伝子の転写分析;アルバータ大学)をKpnIで消化し、その0.688kb挿入体を遊離させた。消化物をアガロースゲル電気泳動により分画し、0.688kbのフラグメントを溶出させてクレノー処理に付した。ついで、その平滑末端フラグメントをHincII消化pUC119にライゲーションし、それを用いてイー・コリ(E.coli)MV1193をアンピシリン耐性に形質転換させた。以下、pELK044と称される組換えプラスミドを含有する形質転換体を単離し、その同一性を制限分析により確認した。pELK004をBamHIおよびKpnIで消化し、BamHI−KpnI aprr−フラグメントにライゲーションした。そのライゲーション混合物を用いてイー・コリMV1193をアプラマイシン耐性に形質転換させた。得られた形質転換体をスクリーニングに付し、0.688kbの3’−latフラグメントの上流で、逆方向に挿入された、aprr−フラグメントを含有する組換えプラスミドpCF001Aを単離し、制限分析により確認した。
pCF002をEcoR1およびHindIIIで消化することで分裂したlat遺伝子をサブクローンし(分裂したlat遺伝子を有するDNAフラグメントを遊離させる)、その消化物を同様に消化したpIJ486にライゲーションし、p486latap[図1(b)]を形成させた。そのライゲーション混合物を用い、エス・リビダンス(S.lividans)プロトプラストをアプラマイシン耐性に形質転換させた(ストレプトマイセスの遺伝的操作;A Laboratory manual(1985)D.A.Hopwoodら)。組換えプラスミドp486latapを含有するクローンを選択し;ついで制限分析を用い、p486latap中に分裂したlat遺伝子があることを確認した。
抗生物質の産生における推定されるlat分裂変異の効果を測定するために、4つのすべての株(分裂体)を澱粉−アスパラギン(SA)液体培地(Paradkar,A.S.およびJesen,S.E.(1995)、J.Bacteriol.,177,1307−1314)に28℃で66時間培養した。上澄を調整して、種々の増殖速度を説明し、イー・コリESS(ParadkarおよびJensen、同書)に対してバイオアッセイに付し、野生型ストレプトマイセス・クラブリゲラスの培養物由来の同様に調整した上澄と比較した。lat−分裂体はすべて、約0.045μg/ml/セファロスポリンに相当する非常に小さな領域の阻害を生じ;同様の条件下で、野生型株は3.18μg/ml/セファロスポリンをもたらした。
lat分裂変異体を製造した。実験により該変異体がわずかな量のβ−ラクタム抗生物質(クラブラン酸を除く)を産生することがわかった。クラブラン酸については、該変異体は、澱粉−アスパラギン培地中、振とうフラスコ条件下で増殖させた野生型ストレプトマイセス・クラブリゲラスにより産生されるクラブラン酸の少なくとも2ないし3倍の量を産生した。
Claims (3)
- 遺伝子挿入により分裂したストレプトマイセス・クラブリゲラスから由来のlat遺伝子を含有する組換えプラスミド。
- 分裂したlat遺伝子を有し、図1(a)に示される制限部位の配置のpCF002と称される組換えプラスミド;および
分裂したlat遺伝子を有し、図1(b)に示される制限部位の配置のp486latapと称される組換えプラスミド;
より選択される、請求項1記載のプラスミド。 - セファロスポリン経路においてL−リジンのL−α−アミノアジピン酸への変換を妨げることにより、クラバム経路またはその一部およびセファロスポリン経路またはその一部の両方の経路を有する微生物において増量のクラバムを産生して単離するクラバムの生成方法であって、請求項1または2記載の組換えプラスミドを用いてlat遺伝子を分裂することでlat遺伝子の機能を除去することにより上記変換工程を妨げる、方法。
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Application Number | Priority Date | Filing Date | Title |
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GB9511679.4 | 1995-06-09 | ||
GBGB9511679.4A GB9511679D0 (en) | 1995-06-09 | 1995-06-09 | Novel process |
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JP50260797A Division JP3996640B2 (ja) | 1995-06-09 | 1996-06-06 | クラバム抗生物質の産生を強化する方法 |
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JP2007289193A true JP2007289193A (ja) | 2007-11-08 |
JP2007289193A6 JP2007289193A6 (ja) | 2008-02-07 |
JP2007289193A5 JP2007289193A5 (ja) | 2008-02-21 |
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JP50260797A Expired - Lifetime JP3996640B2 (ja) | 1995-06-09 | 1996-06-06 | クラバム抗生物質の産生を強化する方法 |
JP2007140125A Pending JP2007289193A (ja) | 1995-06-09 | 2007-05-28 | クラバム抗生物質の産生を強化する方法 |
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JP50260797A Expired - Lifetime JP3996640B2 (ja) | 1995-06-09 | 1996-06-06 | クラバム抗生物質の産生を強化する方法 |
Country Status (12)
Country | Link |
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US (1) | US6171814B1 (ja) |
EP (1) | EP0832247B1 (ja) |
JP (2) | JP3996640B2 (ja) |
KR (2) | KR100439081B1 (ja) |
AT (1) | ATE283362T1 (ja) |
AU (1) | AU6301796A (ja) |
CA (1) | CA2221518C (ja) |
DE (1) | DE69633921T2 (ja) |
ES (1) | ES2233969T3 (ja) |
GB (1) | GB9511679D0 (ja) |
HK (1) | HK1010087A1 (ja) |
WO (1) | WO1996041886A1 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1228218A2 (en) * | 1999-10-22 | 2002-08-07 | Smithkline Beecham Plc | 5r clavam producing microorganisms with deleted lat and cvm genes |
GB0402799D0 (en) * | 2004-02-09 | 2004-03-10 | Sandoz Ag | Pharmaceutical composition |
CN100355895C (zh) * | 2004-12-17 | 2007-12-19 | 天津科技大学 | 棒状链霉菌lat基因缺失的质粒、衍生物及其构建方法 |
EP2589663A1 (en) | 2011-11-04 | 2013-05-08 | LEK Pharmaceuticals d.d. | Process for production of clavulanic acid |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
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GB8813055D0 (en) * | 1988-06-02 | 1988-07-06 | Beecham Group Plc | Novel substance |
EP0467049A1 (en) * | 1990-07-16 | 1992-01-22 | American Cyanamid Company | DNA sequences and amino acid sequences of class b beta-lactamase enzymes from bacteroides fragilis |
GB9302041D0 (en) | 1993-02-02 | 1993-03-17 | Smithkline Peecham P L C | Novel compounds |
US5474912A (en) * | 1994-03-03 | 1995-12-12 | Sherman; David H. | Method for increasing production of microbial metabolites by genetic engineering |
CA2182951A1 (en) * | 1994-03-03 | 1995-09-08 | Juan F. Martin | A method of transformation of nocardia |
-
1995
- 1995-06-09 GB GBGB9511679.4A patent/GB9511679D0/en active Pending
-
1996
- 1996-06-06 CA CA002221518A patent/CA2221518C/en not_active Expired - Lifetime
- 1996-06-06 DE DE69633921T patent/DE69633921T2/de not_active Expired - Lifetime
- 1996-06-06 AU AU63017/96A patent/AU6301796A/en not_active Abandoned
- 1996-06-06 EP EP96921954A patent/EP0832247B1/en not_active Expired - Lifetime
- 1996-06-06 AT AT96921954T patent/ATE283362T1/de not_active IP Right Cessation
- 1996-06-06 KR KR1019970709202A patent/KR100439081B1/ko not_active IP Right Cessation
- 1996-06-06 WO PCT/EP1996/002497 patent/WO1996041886A1/en active IP Right Grant
- 1996-06-06 US US08/981,072 patent/US6171814B1/en not_active Expired - Lifetime
- 1996-06-06 JP JP50260797A patent/JP3996640B2/ja not_active Expired - Lifetime
- 1996-06-06 KR KR10-2004-7000216A patent/KR100468932B1/ko not_active IP Right Cessation
- 1996-06-06 ES ES96921954T patent/ES2233969T3/es not_active Expired - Lifetime
-
1998
- 1998-09-24 HK HK98110939A patent/HK1010087A1/xx not_active IP Right Cessation
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2007
- 2007-05-28 JP JP2007140125A patent/JP2007289193A/ja active Pending
Also Published As
Publication number | Publication date |
---|---|
GB9511679D0 (en) | 1995-08-02 |
WO1996041886A1 (en) | 1996-12-27 |
AU6301796A (en) | 1997-01-09 |
KR20040010848A (ko) | 2004-01-31 |
CA2221518C (en) | 2009-01-06 |
ATE283362T1 (de) | 2004-12-15 |
CA2221518A1 (en) | 1996-12-27 |
KR19990022760A (ko) | 1999-03-25 |
HK1010087A1 (en) | 1999-06-11 |
US6171814B1 (en) | 2001-01-09 |
DE69633921D1 (de) | 2004-12-30 |
EP0832247B1 (en) | 2004-11-24 |
JPH11507546A (ja) | 1999-07-06 |
JP3996640B2 (ja) | 2007-10-24 |
KR100439081B1 (ko) | 2004-07-16 |
KR100468932B1 (ko) | 2005-02-02 |
EP0832247A1 (en) | 1998-04-01 |
ES2233969T3 (es) | 2005-06-16 |
DE69633921T2 (de) | 2005-11-10 |
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