Genetic engineering of reveromycin A biosynthetic gene cluster and creation of its derivatives

回复霉素A是由回复菌链霉菌产生的聚酮化合物，通过特异性诱导破骨细胞凋亡来抑制骨吸收。为了获得新的具有生物活性的回复霉素A衍生物，克隆了回复霉素A生物合成基因簇。根据mRNA表达模式和基因组鸟枪测序数据，从S. reveromyceticus基因组。序列分析揭示了另外的聚酮起始模块，其并入甲基丙二酰辅酶A。这表明，回复霉素A是生物合成的额外的丙酸单位。有趣的是，支持DNA序列信息的回复霉素衍生物（回复霉素A2）从S.回复菌在生物合成基因簇中，鉴定了催化还原霉素生物合成中间体的C-18羟基化的P450。由于该酶是一个目标蛋白，以创造逆转录霉素A衍生物，晶体图形分析，现在正在进行中。通过生化分析和基因敲除，对簇中未知基因的功能进行了研究。

Reveromycin A, which is a polyketide compound produced by Streptomyces reveromyceticus, inhibits bone resorption by inducing apoptosis specifically in osteoclasts. To obtain new bioactive derivatives of reveromycin A, the cloning of reveromycin A biosynthetic gene cluster was conducted. Based on the information from mRNA expression pattern and genome shotgun sequencing data, the polyketide biosynthetic gene cluster responsible for reveromycin A biosynthesis was obtained from S. reveromyceticus genome. The sequence analysis revealed the additional polyketide starter module that incorporates methylmalonyl CoA. This suggests that reveromycin A is biosynthesized with additional propionate unit. Interestingly, the reveromycin derivative (reveromycin A2) supporting the DNA sequence information was isolated from the secondary metabolites from S. reveromyceticus. In the biosynthetic gene cluster, P450 which catalyzes the C-18 hydroxylation of reveromycin biosynthetic intermediate was identified. Because the enzyme is a target protein to create reveromycin A derivatives, the crystal graphic analysis is now in progress. The function of unknown genes in the cluster is also investigated through biochemical analysis and gene knock out.