带有氯霉素抗性的分泌高产戊二酰－7－氨基头孢烷酸酰化酶的菌株
本发明提供了一种从假单胞菌中克隆并经基因工程改造得到的长为２．５ｋｂ的重组ＧＬ－７ＡＣＡ酰化酶基因及其表达元件和信号肽的序列。构建了该基因的高分泌表达重组质粒ｐＳｕｐｅｒＣＡ１Ｓ，该质粒转化至大肠杆菌菌株ＴＧ１中，得到的基因工程菌株ＴＧ１／ｐＳｕｐｅｒＣＡ１Ｓ在普通的ＬＢ培养基中，无需添加诱导剂即能高效分泌表达ＧＬ－７ＡＣＡ酰化酶，比活力可达到每克菌体１２０个单位。
发明专利
CN00125106.6
2000-09-08
CN1283697
2001-02-14
C12N15/55
中国科学院上海生物化学研究所
王恩多;李勇;郑勇刚;陈剑峰;姜卫红;赵国屏;杨蕴刘
200031上海市岳阳路320号
上海华东专利事务所
费开逵
上海;31
权利要求书1、一种重组戊二酰-7-氨基头胞烷酸酰化酶基因，其特征在于它编码GL-7ACA酰化酶的核苷酸序列以及相应的氨基酸序列如下：1 CTGGCCGAGCCGACCTCGACGCCGCAGGCGCCGATTGCGGCCTATAAACCGAGAAGCAATGAGATCCTG 691 LeuAlaGluProThrSerThrProGlnAlaProIleAlaAlaTyrLysProArgSerAsnGluIleLeu 2370 TGGGACGGCTACGGCGTCCCGCACATCTACGGGGTCGACGCGCCCTCAGCCTTCTACGGCTACGGCTGG 13824 TrpAspGlyTyrGlyValProHisIleTyrGlyValAspAlaProSerAlaPheTyrGlyTyrGlyTrp 46139 GCCCAGGCGCGCAGCCACGGCGACAATATCCTGCGCCTGTATGGAGAAGCGCGGGGCAAGGGGGCCGAA 20747 AlaGlnAlaArgSerHisGlyAspAsnIleLeuArgLeuTyrGlyGluAlaArgGlyLysGlyAlaGlu 69208 TACTGGGGCCCGGATGACGAACAGACGACCGTCTGGCGTCTGACCAACGGCGTGCCGGAGCGTGCCCAG 27670 TyrTrpGlyProAspAspGluGlnThrThrValTrpArgLeuThrAsnGlyValProGluArgAlaGln 138277 CAGTGGTATGCGCAGCAGTCGCCTGATTTCCGCGCCAACCTCGACGCCTTCGCGGCGGGCATCAACGCC 345139 GlnTrpTyrAlaGlnGlnSerProAspPheArgAlaAsnLeuAspAlaPheAlaAlaGlyIleAsnAla 207346 TATGCGCAGCAGAACCCCGACGACATCTCGCCCTGACTGCGGCAGGTGCTGCCGGTCGCCGGCGCCGAC 414208 TyrAlaGlnGlnAsnProAspAspIleSerProAspValArgGlnValLeuProValAlaGlyAlaAsp 276415 GTGGTGGCCCACGCCCATCGCCTGATGAACTTCCTCTATGTCGCGTCGCCCGGCCGCACCCTGGGCGAG 483277 ValValAlaHisAlaHisArgLeuMetAsnPheLeuTyrValAlaSerProGlyArgThrLeuGlyGlu 345484 GGCGACCCGCCGGACCTGGCCGATCAGGGATCCAACTCCTGGGCTGTGGCGCCGGCCAAGACGGCCAAT 552346 GlyAspProProAspLeuAlaAspGlnGlySerAsnSerTrpAlaValAlaProGlyLysThrAlaAsn 414553 GGGAACGCCCTGCTGCTGCAGAACCCGCACCTGTCCTGGACGACGGACTACTTCACCTACTACGAGGCG 621415 GlyAsnAlaLeuLeuLeuGlnAsnProHisLeuSerTrpThrThrAspTyrPheThrTyrTyrGluAla 483622 CATCTCTTCACGCCGGACTTCGAAATCTATGGCGCGACCCAGATCGGCCTGCCGGTCATCCGCTTCGCC 690484 HisLeuPheThrProAspPheGluIleTyrGlyAlaThrGlnIleGlyLeuProValIleArgpheAla 552691 TTCAATCAGCGGATGGGCATCACCAATACCGTCAACGGCATGGTGGGGGCCACCAACTATCGGCTGACG 759553 PheAsnGlnArgMetGlyIleThrAsnThrValAsnGlyMetValGlyAlaThrAsnTyrArgLeuThr 621760 CTTCAGGGCGACGGCTATCTGTATGACGGTCAGGTGCGGCCGTTCGAGCGGCGTCAGGCTTCGCATCGC 828622 LeuGlnGlyAspGlyTyrLeuTyrAspGlyGlnValArgProPheGluArgArgGlnAlaSerHisArg 690829 CTGCGTCAGGCGGACGGGTCGACGGTCGACAAGCCGTTGGAGATCCGTTCCAGCGTCCATGGCCCGGTC 897691 LeuArgGlnAlaAspGlySerThrValAspLysProLeuGluIleArgSerSerValHisGlyproVal 7591898 TTCGAGCGCGCGGACGGCACGGCCGTCGCCGTTCGGGTCGCCGGTCTGGATCGGCCGGGCATGCTCGAG 966760 PheGluArgAlaAspGlyThrAlaValAlaValArgValAlaGlyLeuAspArgProGlyMetLeuGlu 828967 CAGTATTTCGACATGATCACGGCGGACAGCTTCGACGACTACGAAGCCGCTATGGCGCGGATGCATGTG 1035829 GlnTyrPheAspMetIleThrAlaAspSerPheAspAspTyrGluAlaAlaMetAlaArgMetHisVal1036 CCGACCTTCAACATCGTCTACGCCGACCGCGAAGGGACCATCAACTACAGCTTCACGGCGTGGCGCCCA 1104ProThrPheAsnIleValTyrAlaAspArgGluGlyThrIleAsnTyrSerPheThrAlaTrpArgPro1105 AACGGGCCGAGGGCGACATCGCCTTCTGGCAGGGGCTCGGCCGGGCCATTCCTCGCGTTACTGTGGACT 1173AsnGlyProArgAlaThrSerProSerGlyArgGlySerAlaGlyProPheLeuAlaLeuLeuTrpThr1174 GAGACACACCCCTGGACGATCTGCCGCGCGTCACCAATCCGCCGGCCCGCTTCGTGCAGAACTCCAATG 1242GluThrHisProTrpThrIleCysArgAlaSerProIleArgArgProAlaSerCysArgThrProMet1243 ATCCGCCGTGGACGCCGACCTGGCCCGTCACCTACACGCCCAGGGACTTCCCCTCCTATCTGGCGCCCC 1311IleArgArgGlyArgArgProGlyProSerPROThrArgProGlyThrSerProProIleTrpArgPro1312 AGACGCGCACTCCCTGCGCGCTCAGCAAAGCGTGCGTCTGATGTCGAGAACGACGACCTGAGGCTGGAA 1380ArgArgAlaLeuProAlaArgSerAlaLysArgAlaSerAspValGluAsnAspAspLeuArgLeuGlu1381 CGCTTCATGGCGCTGCAGTTGAGCCACCGCGCCGTCATGGCCGACCGCACCTTGCCGGACCTGATCCCG 1449ArgPheMetAlaLeuGlnLeuSerHisArgAlaValMetAlaAspArgThrLeuProAspLeuIlePro1450 GCCGCCCTGATCGACCCCGATCCCGAGGTCCAGGCGGCGGCGCGCCTGCTGGCGGCGTGGGATCGTGAG 1518AlaAlaLeuIleAspProAspProGluValGlnAlaAlaAlaArgLeuLeuAlaAlaTrpAspArgGlu1519 TTCACCAGCGACAGCCGCGCCGCCCTGCTGTTCGAGGAATGGGCGCGTCTGTTCGCCGGCCAGAATTTC 1587PheThrSerAspSerArgAlaAlaLeuLeuPheGluGluTrpAlaArgLeuPheAlaGlyGlnAsnPhe1588 GCCGGCCAGGCGGGTTTCGCCACGCCCTGGTCGCTGGATAAGCCGGTCAGCACCCCCACGGCGTCCGCT 1656AlaGlyGlnAlaGlyPheAlaThrProTrpSerLeuAspLysProValSerThrProTyrGlyValArg1657 GACCCCAAGGCCGCCGTCGATCAACTGCGGACCGCCATCGCCAACACCAAGCGCAAATACGGCGCGATC 1725AspProLysAlaAlaValAspGlnLeuArgThrAlaIleAlaAsnThrLysArgLysTyrGlyAlaIle1726 GACCGCCCGTTCGGCGACGCCTCGCGCATGATCCTGAACGATGTGATTGTTCCGGGCGCCGCCGGCTAC 1794AspArgProPheGlyAspAlaSerArgMetIleLeuAsnAspValIleValProGlyAlaAlaGlyTyr1795 GGCAACCTGGGTTCCTTCCGGGTCTTCACCTGGTCCGATCCTGACGAAAACGGGGTTCGCACGCCCGTC 1863GlyAsnLeuGlySerPheArgValPheThrTrpSerAspProAspGluAsnGlyValArgThrProVal1864 CACGGCGAGACGTGGGTGGCGATGATCGAGTTCTCCACCCCGGTGCGGGCCTATGGCCTGATGAGCTAC 1932HisGlyGluThrTrpValAlaMetIleGluPheSerThrProValArgAlaTyrGlyLeuMetSerTyr21933 GGCAATTCTCGCCAGCCGGGCACGACGCACTACAGCGATCAGATCGAACGCGTGTCGCGGGCCGACTTC 2001GlyAsnSerArgGlnProGlyThrThrHisTyrSerAspGlnIleGluArgValSerArgAlaAspPhe2002 CGCGAGCTGTTGCTGCGGCGAGAGCAGGTCGAGGCCGCCGTCCAGGAACGCACGCCCTTCAACTTCAAG 2070ArgGluLeuLeuLeuArgArgGluGlnValGluAlaAlaValGlnGluArgThrProPheAsnpheLys2071 CCATGAAAGGCCTGACCATGACACGACGGATTGGGTATTCGGCGGGCGCGGCGTCTCTGGCGCTGATGG 2139ProTCGCCGCCTCTGGAGCGGCGGCGGGCGAGCCGGCCTTCACTTCGGTTCAGGTCGAGGGCTTTTCGGTACCGGGCGCGCTGTCGAAGGCCTGGGCCGACTTCGACAACGACGGCGACCTGGACCTGGCCGTCTCCTGGAAGAGCGGCGAAGTTCGCCTCTATCTCAACGACGGCGGCGCTTTCGTCAACATCGGCCCGGAGATGGGCTGCCTACCTCGGGCATCGAGATTCGCGGGTTGAGCTGGGGCGACTTCGACGGCGATGGGTTCGTCGAC