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合成酮内酯类3—脱氧—3—羰基—红霉内酯B糖多孢红霉菌M的构建
引用本文:张部昌,赵志虎,等.合成酮内酯类3—脱氧—3—羰基—红霉内酯B糖多孢红霉菌M的构建[J].生物工程学报,2002,18(2):198-203.
作者姓名:张部昌  赵志虎
作者单位:[1]安徽大学生命科学学院,合肥230039 [2]军事医学科学院生物工程研究所,北京100850
摘    要:大环内酯类抗生素基因工程是近年来研究的一个新领域,迄今已合成了100多种新的聚酮类化合物。以糖多孢红霉菌A226基因组DNA为模板,用重叠PCR方法扩增出去除KR6酶域DNA的约3.2kb DNA片段,克隆于pWHM3载体,构建了同源重组质粒pWHM2201。PEG介导原生质体转化法将pWHM2201转入糖多孢红霉菌A226,并整合于染色体红霉素合成基因位点。整合体在R3M斜面上生长两代后,制备原生质体涂R3M平皿。利用PCR鉴定筛选出8株KR6敲除的突变体糖多孢红霉菌M(1-8)。ZabsPec Fab质谱鉴定,证实糖多孢红霉菌M1合成了3-脱氧-3-羰基-红霉内酯B,一种新的酮内酯类化合物。

关 键 词:糖多孢红霉菌  3-脱氧-3-羰基-红霉内酯B  酮内酯类  原生质体转化  同源重组
文章编号:1000-3061(2002)02-0198-06
修稿时间:2001年8月15日

Construction of Saccharopolyspora erythraea M Synthesizing a Novel Ketolide 3-deoxy-3-oxo-erythronolide B
ZHANG Bu\|Chang , \ ZHAO Zhi\|Hu\\ WANG Yi\|Guang\\ MA Qing\|Jun.Construction of Saccharopolyspora erythraea M Synthesizing a Novel Ketolide 3-deoxy-3-oxo-erythronolide B[J].Chinese Journal of Biotechnology,2002,18(2):198-203.
Authors:ZHANG Bu\|Chang  \ ZHAO Zhi\|Hu\\ WANG Yi\|Guang\\ MA Qing\|Jun
Institution:Beijing Institute of Biotechnology, Beijing 100850, China.
Abstract:Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then.Using genomic DNA of \%S.erythraea\% A226 as a template,about 3.2kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier,which resulted in the construction of homologous recombinant plasmid pWHM2201.Plasmid pWHM2201 was introduced into protoplasts of \%S.erythraea\% A226 by PEG\|mediated transformation and integrated into the gene locus for erythromycin biosynthesis.After integrants grew for two generations on R3M media without Tsr,they were protoplasted and grown on R3M plates.By PCR identification,8 mutants without KR6 domain were selected out and named \%S.erythraea\% M(1 8).With the identification of mass spectrometry,it was proved that \%S.erythraea\% M1 synthesized a novel ketolide compound 3\|deoxy\|3\|oxo\|erythronolide B.
Keywords:Saccharopolyspora erythraea  \%3\|deoxy\|3\|oxo\|erythronolide B  ketolide  protoplast transformation  homologous recombination
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