吸水链霉菌17997格尔德霉素部分生物合成基因簇的克隆和分析

Cloning and Analysis of Geldanamycin Partial Biosynthetic Gene Cluster of Streptomyces hygroscopicus 17997

吸水链霉菌17997(Streptomyceshy groscopicus17997)是我所从中国云南土壤中分离到的格尔德霉素(geldanamycin,GDM)产生菌,GDM具有良好的抗肿瘤和抗病毒活性,但其肝毒性和水溶性差的缺点限制了其在临床上的应用。为了实现对GDM结构的生物学改造,首先要获得GDM的生物合成基因。根据GDM后修饰基因——氨甲酰基转移酶基因(gdmN)的保守序列筛选S.hygroscopicus17997的柯斯质粒基因组文库,共获得6个阳性克隆,选择CT-4阳性柯斯质粒进行亚克隆和测序,又通过PCR延伸的方法获得了与CT4连锁的将近5kb的外源序列,共获得28.356kb的外源DNA序列,其中包含了13个可能阅读框架,通过同源比较证实该序列与S.hygroscopicusNRRL3602中的GDM生物合成基因有很高的同源性。为进一步研究GDM生物合成基因的功能,并通过组合生物学的方法改造GDM的结构奠定了基础。

A geldanamycin （GDM） producing strain, Streptornyces hygroscopicus 17997, was isolated from Yunnan China soil by our institute researchers, GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 （Heat Shock Protein 90） and alter its function. Hsp90 is a chaperone protein involved in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis. So it plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumor and antivirus bioactivity, but the hypato-toxicity and poor solubility in water limits its clinical use. Two CDM derivatives, 17-（ Allylamino）-17- demethoxygeldanamycin （17-AAG） and 17- dimethylamino- ethylamino-17-demethoxygeldanamycin （17-DMAG）, both showing lesser hepato-toxicity, are now in Phase Ⅱ and Phase I clinic trials. In order to accomplish the structure modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of possible post-PKS （ polyketides synthase） modification genes, the carbamoyhvansferase （CT） gene （gdmN） in GDM biosynthesis. The 732 bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT gene containing fragments were verified and localized by Southern blot. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28. 356kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. Based on BLAST analysis, this sequence contains 13 possible ORFs and their deduced functions are believed to be involved in GDM production. The ORF1 encoding products show homology （87 % ） with incomplete sixth module and complete seventh module of PKS, gdmA3, in S. hygroscopicus NRRL 3602. The ORF2-13 gene products are similar to gdmF（95%）, gdmM（88%）, gdmN（92%）, gdmn （92%）, I （93%）, J （90%）, K （93%）, G （96%）, gdm O （91%）,gdmP （93%） and two transcription regulation genes gdmRI （83%） and gdmRII （90%）. The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and to modify the structure of GDM through combinatorial biosynthesis.