产诺加霉素链霉菌中失活snogA基因的研究

诺加霉素是重要的蒽环类抗肿瘤抗生素，由黑胡桃链霉菌ATCC27451发酵产生。本研究从诺加霉素产生茵中克隆得到560bp的氨基甲基化酶（snogA）编码基因片段，并将其插入基因整合型质粒pKCll39的多克隆位点，构建得到基因中断质粒pLMX-3-58。通过接合转移和同源重组，构建得到氨基甲基化酶编码基因被中断的重组菌株删-3-59。基因重组突变株基因型验证结果表明，中断质粒以正确方式整合入基因组，将氨基甲基化酶编码基因中断。发酵验证结果表明，重组茵株发酵产物中不含有诺加霉素。本研究表明snogA基因在诺加霉素生物合成途径中是必需的。这为进一步阐明诺加霉素生物合成途径和组合生物合成改造诺加霉素提供了参考。

Inactivation of snogA gene in nogalamycin producing strain

Streptomyces nogalater ATCC27451 was a producer of nogalamycin, one kind of the anthracycline antitumor antibiotics. In this research, the partial sequence （560 bp） of snogA coding amino methylase was cloned and the 560 bp sequence in the multiple cloning site of the integrative plasmid pKC1139 was inserted to construct the recombinant plasmid pLMX-3-58. The pLMX-3-58 was recombined into the chromosome of the nogalamycin producing-strain by the conjugation and the single crossover homogenous recombination event to produce the snogA inactivation mutation, m~X-3-59 was obtained. The genotype checking result of mLMX-3-59 showed that the pLMX-3-58 was reeombined in the fight site of the nogalamycin producing-strain chromosome. According to the verification by fermentation, it demonstrated that the strain rnLMX-3-59 was characterized by the absence of synthesis of nogalamycin. This research indicated that the snogA gene was essential for the nogalamycin biosynthesis, which provided the reference for further illustrating the biosynthetic pathway of nogalamycin and the combinatorial biosynthesis for modifying nogalamycin.