格尔德霉素生物合成基因功能的验证

格尔德霉素(Geldanamycin, Gdm)作为热休克蛋白90的特异性抑制剂, 是非常有前景的抗肿瘤和抗病毒的药物,我们已从吸水链霉菌17997(Streptomyces hygroscopicus 17997)的基因文库中获得了Gdm大部分生物合成基因。为了研究主要基因的功能, 选择了聚酮合酶基因(Polyketide synthase gene, pks)的第六模块、单加氧酶基因(Mono-oxygenase gene, gdmM)和氨甲酰基转移酶基因(Carbamoyltransferase gene, gdmN)3个基因作为靶点分别进行基因阻断, 获得了基因同源双交换的阻断变株△pks、△gdmM和△gdmN。经HPLC检测证实这些基因的阻断变株均不产生Gdm, 基因回复实验排除了基因阻断所可能造成的极性效应对其它基因表达的影响, 说明所克隆的pks、gdmM和gdmN基因确实是Gdm生物合成所必须的基因。

Roles of Geldanamycin BiosyntheticGenes?in Streptomyces hygroscopicus 17997

Geldanamycin (Gdm), an inhibitor of heat shock protein 90 (Hsp90), shows antitumor and antivirus bioactivity. Most Geldanamycin biosynthetic genes have been cloned from the genome library of Streptomyces hygroscopicus 17997. In this report, polyketide synthase (pks) gene, mono-oxygenase (gdmM) gene and carbamoyltransferase gene (gdmN) were subjected to inactivation. Three gene disrupted mutants (deltapks, deltagdmM and deltagdmN) were obtained by double crossover. No Geldanamycin production was detected in three mutant strains cultured in fermentation broth. Gene complementation experiments excluded the possible polar effect of gene disruption on other genes. These results confirmed that pks, gdmM and gdmN genes were essential for Geldanamycin biosynthesis.