【作者】 赫卫清；

【导师】 王以光；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2006， 博士

【摘要】 吸水链霉菌17997(Streptomyces hygroscopicus 17997)是我所从中国土壤中分离得到的格尔德霉素(geldanamycin，GDM)产生菌。GDM属于苯安莎类抗生素。最先发现GDM具有抗原虫和抗肿瘤作用，最近我所的研究发现GDM还具有良好的抗病毒活性。国外的研究表明GDM的这些生物学活性是因为它能特异性地抑制热休克蛋白90的ATP／ADP结构域，下调多种Hsp90的靶蛋白功能所致，包括酪氨酸激酶、类固醇激素受体和转录因子等。GDM作为Hsp90的特异性抑制剂，成为在抗癌和抗病毒领域中非常有潜力的药物。但因为GDM肝毒性比较大，水溶性不好，所以寻找其低毒可溶性好的衍生物成为当今研究的主要目标。GDM的一种衍生物17—烯丙氨基—17—脱甲氧基GDM(17-AAG)正在美国进行Ⅱ期临床实验。 已知安莎类抗生素的生物合成是以3—氨基—5—羟基苯甲酸(AHBA)为起始单位，由Ⅰ型聚酮合酶(PKS)催化顺序加上乙二酸，丙二酸或者甲氧基丙二酸等延伸单位，在酰胺合酶参与下形成聚酮体骨架，然后再进行后续的加工(post PKS modification)。在对GDM生物合成基因簇的研究中，本实验室在S．hygroscopicus 17997中曾发现了苯醌型和萘醌型两种AHBA生物合成基因和与之连锁的两个PKS基因簇，但其中未包括GDM的后修饰基因。本研究根据GDM的后修饰基因—氨甲酰基转移酶基因(gdmN)的保守序列设计PCR引物，在S．hygroscopicus 17997基因组中扩增出了732bp的基因片段，通过测序和同源比较，证实该基因片段与S．hygroscopicus NRRL 3602的gdmN有94％的同源性。利用该基因片段的引物，进行菌落PCR筛选S．hygroscopicus 17997的基因组文库，获得了6个阳性克隆，并利用732bp的PCR产物作为探针对阳性克隆进行Southern blot验证和定位。选择CT-4阳性柯斯质粒进行亚克隆测序，将测得的28.356kb DNA序列进行BLAST和ORF分析，证实与公布的S．hygroscopicus NRRL3602 GDM生物合成基因的部分序列基本一致。CT-4柯斯质粒克隆中外源序列可能包括12个基因：与gdmA3基因同源的PKS基因、酰胺合酶基因(gdmF)、单加氧酶基因(gdmM)、氨甲酰基转移酶基因(gdmN)、与甲氧基丙二酰—酰基携带蛋白合成相关的5个基因(gdmH、I、J、K和G)、LuxR家族的转录调节基因(gdmRⅡ)、氨基更多还原

【Abstract】 Geldanamycin (GDM), produced by the Streptomyces hygroscopicus 17997 which was isolated from China soil by our institute reseacher. GDM, belonging to the benzenenoid ansamycin, possess antitumor and antiprotozoon activity and recently, was discovered that it shows antivirus bioactivity. Extensive research have vertified that GDM can bind specifically to the ATP/ADP domain of heat shock protein 90 (Hsp90), thus down-regulate target proteins including tyrosine kinases, steroid receptors, transcription factors et al. As a specific inhibitor of Hsp90, GDM became a potential promising antitumor and antivirus medicine. To overcome the hypatotoxicity and poor solubility of GDM, the main research work was focused on finding its less toxic and more soluble derivatives. One of its derivatives, 17-AAG, is now in clinical trial (phase II) in United States. It was known that the biosynthesis of ansamycins involves the assembly 3-amino-5-hydroxybenzoic acid (AHBA) as starter unit, followed by the sequential addition of extender units such as acetate, malonate, and methoxymalonate to form a polyketide backbone catalyzed by type I polyketide synthsase (PKS), which then undergoes further downstream processing.During studies on the geldanamycin biosynthetic genes from Streptomyces hygroscopicus 17997, two possible separate AHBA biosynthetic gene clusters and two sets of PKS genes were found in our lab. But the involvement of these genes in the GDM biosynthesis was not identified, and the post-PKS modification genes were absent yet. In this study conserved sequences of one of possible post-PKS modification gene, the carbamoyltransferase gene (gdmN) in GDM biosynthesis were used to design the primers, by which a 732bp gene fragment was amplified from the Streptomyces hygroscopicus 17997 genome DNA. This gene fragment was sequenced and found it has identity of 94% with the gdmN of Streptomyces hygroscopicus NRRL 3602. Through the colony-PCR by the primers screening, six positive cosmid clones were identified from the Streptomyces hygroscopicus 17997 genomic library. The CT-4 cosmid was sub-cloned and sequenced. The 28.356kb foreign gene sequence of the CT-4 cosmid shows high identity with the known GDM biosynthesis gene cluster of Streptomyces hygroscopicus NRRL 3602 based on BLAST analysis. 12 possible ORFs were identified from the CT-4 cosmid including PKS gene更多还原