公开(公告)号：EP1188838A1

公开(公告)日：2002-03-20

申请号：EP00936911.7

申请日：2000-06-13

申请人： Antibioticos, S.A.U.

发明人： VELASCO ALVAREZ, Javier ,  ADRIO FONDEVILA, José ,  MORENO VALLE, Miguel Angel ,  DIEZ GARCIA, Bruno ,  ESTEBAN MORALES, Manuel ,  BERNASCONI, Ermanno ,  BARREDO FUENTE, José Luis

IPC分类号： C12P35/00

CPC分类号： C12N9/0024 ,  C12N9/80 ,  C12P35/02

摘要： A method of obtaining 7-aminodesacetoxycephalosporanic acid (7-ADCA). The method is based on the use of desacetoxycephalosporin C (DAOC) produced directly by fermentation of Acremonium chrysogenum (also called Cephalosporium acremonium) and catalysed by the enzymatic activities D-amino-acid oxidase (DAO) and glutaryl-7-ACA acylase (GLA). The method of production of DAOC by fermentation of A. chrysogenum is based on inactivation of the cefEF gene and subsequent expression of the cefE gene giving rise to the strain A. chrysogenum △cefEF-cefE. The invention includes the following sections: (I) inactivation of the cefEF gene of A. chrysogenum, which codes for the enzymatic activities desacetoxycephalosporin C synthase (DAOCS or expandase) and desacetylcephalosporin C synthase (DACS or hydroxylase), (II) expression of the cefE gene, which codes for the enzymatic activity DAOCS, derived from an actinomycete in the transformant of A. chrysogenum inactivated in the cefEF gene, (III) development of a method of fermentation of the recombinant strain for the production of DAOC and (IV) enzymatic conversion of DAOC to 7-ADCA catalysed by the enzymes DAO and GLA. Alternatively, it would be possible to produce 7-ADCA directly by fermentation of A. chrysogenum by combined expression of the genes that code for DAO and GLA in the DAOC-producing strain A. chrysogenum △cefEF-cefE or by expression of a gene that codes for the enzyme cephalosporin acylase in A. chrysogenum △cefEF-cefE.

摘要翻译： 一种获得7-氨基去乙酸基头孢烷酸（7-ADCA）的方法。 该方法基于使用通过发酵产黄枝顶孢霉（也称为顶头孢霉）直接产生并由酶活性D-氨基酸氧化酶（DAO）和戊二酰-7-ACA酰基转移酶（GLA）催化的脱乙酸基头孢菌素C（DAOC） ）。 A.chrysogenum发酵生产DAOC的方法基于cefEF基因的失活和随后表达cefE基因，产生A.chrysogenum△cefEF-cefE菌株。 本发明包括以下部分：（I）失活A.chrysogenum的cefEF基因，其编码脱乙酰氧头孢菌素C合酶（DAOCS或扩环酶）和脱乙酰头孢菌素C合酶（DACS或羟化酶）的酶活性，（II）表达 （III）开发用于生产DAOC和（IV）的重组菌株的发酵方法，其中所述酶活性DAOCS编码源自在cefEF基因中失活的A.chrysogenum的转化体中的放线菌， 通过酶DAO和GLA催化将DAOC酶促转化为7-ADCA。 或者，可以通过在编码DAOC的菌株A.chrysogenum△cefEF-cefE中编码DAO和GLA的基因的组合表达或通过表达基因的表达而直接通过发酵A.chrysogenum来产生7-ADCA 编码A.chrysogenum△cefEF-cefE中的酶头孢菌素酰化酶。

公开(公告)号：EP1170369A1

公开(公告)日：2002-01-09

申请号：EP00914189.6

申请日：2000-04-07

申请人： Antibioticos, S.A.U.

发明人： VELASCO ALVAREZ, Javier ,  GUTIERREZ MARTIN, Santiago ,  CASQUEIRO BLANCO, Francisco Javier ,  CAMPOY GARCIA, Sonia ,  FIERRO FIERRO, Francisco ,  BARREDO FUENTE, José Luis ,  DIEZ GARCIA, Bruno ,  MARTIN MARTIN, Juan Francisco

IPC分类号： C12N15/55 ,  C12N9/18 ,  C12P35/00

CPC分类号： C12N9/18 ,  C12N9/58 ,  C12P35/00 ,  C12P35/02 ,  C12P35/06

摘要： An extracellular protease of Acremonium chrysogenum with CPC-acetylhydrolase activity and its use for the synthesis of deacetylated derivatives of cephalosporin C and inactivation of the gene for increasing the yield of cephalosporin. The method includes the preparation of an enzyme with CPC-acetylhydrolase activity (CPC-AH B) starting from culture media of the filamentous fungus A. chrysogenum C10 (ATCC 48272) and the biochemical characterization of the said enzyme. In addition, it describes the cloning and sequencing of a genomic DNA fragment and another of complementary DNA (cDNA) which contain the cah B gene that codes for the said enzyme. By using the gene in the cDNA form, the production of CPC-AH B enzymatic activity in Escherichia coli is described. Finally, a method is included for increasing the yield of cephalosporin in A. chrysogenum by inactivation of the cah B gene.

摘要翻译： 产黄枝顶孢的胞外蛋白酶与CPC-乙酰水解酶活性及其对头孢菌素C和基因失活的脱乙酰化衍生物的合成中的用途为提高头孢菌素的产率。 该方法包括用CPC-乙酰水解酶活性（CPC-AH B）从丝状真菌产黄青霉A. C10的培养基开始（ATCC 48272）和酶。所述的生化表征的酶的制备方法。 此外，它描述了克隆和基因组DNA片段的测序和另一个互补DNA（cDNA）其含有CAHB基因的酶做码在所述的。 通过使用中的cDNA形式的基因，生产在大肠杆菌中CPC-AH B酶促活性的描述。 最后，包括用于通过基因CAHB的失活增加头孢菌素的产率在产黄青霉A.一种方法。