Geldanamycin产生菌生物合成相关基因的克隆与分析

Cloning and molecular analysis of geldanamycin biosynthetic genes from Streptomyces hygroscopicus

目的 从 geldanamycin产生菌吸水链霉菌 (S.hygroscopicus)中克隆生物合成基因 ,为阐明生物合成机制及改造其结构奠定基础。方法 以链霉菌 /大肠埃希氏菌穿梭 cosmids p KC5 0 5为载体构建了插入片段为 2 0～ 30 kb的 S.hygroscopicus总 DNA文库。以利福霉素中与 3-氨基 - 5 -羟基苯甲酸 (AHBA)合成相关基因为探针 ,经菌落杂交 ,Southern杂交筛选同源基因片段。测序结果与 Genbank进行同源性比较分析 ,并以attp- 噬菌体载体 KC5 15利用基因阻断实验对克隆的基因片段进行功能鉴定。结果 构建的基因文库含重组基因比率高、基因组覆盖率高 ,稳定性好。经同源基因探针杂交 ,从文库中筛选出 9个阳性 cosmids克隆p CGB2 0、p CGB2 6、p CGB2 8、p CGB2 9、p CGB36、p CGB45、p CGB49、p CGB6 3、p CGB75。测序分析表明 ,cosmidp CGB2 0中 Bam HI- Bam HI 8kb片段两端的不完整 ORF编码蛋白与竹桃霉素 (oleandomycin) PKS Module 5、Module 6 (一致性为 79% )和红霉内酯合成酶 ORF2 (一致性为 75 % )、ORF1(一致性为 73% )、ORF3(一致性为 70 % )高度同源 ;Bam HI- Bam HI3kb片段一端与编码类结核分枝杆菌的磷酸吡哆醛依赖的氨基转移酶家族中半胱氨酸脱硫酶的 DNA有同源性 ,该家族与 AHBA合成酶关系密切。

Geldanamycin is an antibiotic belongs to ansa group which has the interesting feature of antitumor and anti-protozoal activities. Recently we have found it also possesses a broad antivirus activity. To clone the geldanamycin biosynthetic genes from Streptomyces hygroscopicus may provide some clues to understanding the mechanism of geldanamycin biosynthesis and facilitate the further modification of its structure. A genomic library from the geldanamycin-producing strain was constructed from large (20~30kb) fragments of total DNA with pKC505, a E.coli/Streptomyces shuttle cosmid vector. The percentage of recombinant cosmids in constructed library was high and the coverage of genome was complete as about 99.99%. The library was maintained stable in E.coli during the storage. The gene probes which are essential for AHBA (3-amino-5-hydroxybenzoic acid) biosynthesiswere used for screening the homologous fragments from constructed library. The positive fragments were analyzed by enzyme digestion and sequenced, then compared with the database in Genbank. Positive cosmids pCGB20? pCGB36 ?pCGB45 ?pCGB49? pCGB63 pCGB75? pCGB26? pCGB28 and pCGB29 were obtained by colony and Southern blot. Sequence analysis revealed that the 8kb BamHI-BamHI fragment in pCGB20 was homologous with oleandomycin PKS Module 5, Module 6 (identity 79%) and erythronolide synthase ORF2 (identity 75%), ORF1(identity 73%), and 3kb BamHI-BamHI fragment in pCGB20 was homologous with a gene encoding probable cysteine desulfurase which is closely related with AHBA synthase. The relationship between cloned DNA and geldanamycin biosynthesis was identified with the integration-defective phage φC31 derivative vector KC515 by gene disruption. The BamHI-SacI 2.5kb in BamHI-BamHI 8kb fragment and the BamHI-SacI 1.5kb in BamHI-BamHI 3kb fragment were transfected into S.hygroscopicus. The products of disruptants were analyzed by bioautography--thin-layer chromatography (TLC). The result suggests that the cloned DNA sequences were likely involved in the geldanamycin biosynthesis of S.hygroscopicus.