分泌型高产戊二酰-7-氨基头孢烷酸酰化酶的新菌株
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pETCA1S,该质粒转化至大肠杆菌菌株BL21(DE3),得到的基因工程菌株BL21(DE3)/pETCA1S在普通的TB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82个单位。
发明专利
CN00125105.8
2000-09-08
CN1283696
2001-02-14
C12N15/55
中国科学院上海生物化学研究所
王恩多;郑勇刚;陈剑峰;姜卫红;赵国屏
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 GGCGACCCGCCGGACCTGGCCGATCAGGGATCCAACTCCTGGGCTGTGGCGCCGGGCAAGACGGCCAAT 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