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

反转霉素A是反转链霉菌产生的一种聚酮类化合物，通过诱导破骨细胞特异性的凋亡来抑制骨吸收。为了获得新的红霉素A生物活性衍生物，对红霉素A生物合成基因簇进行了克隆。根据mRNA表达谱信息和基因组鸟枪测序数据，从反转霉菌基因组中获得了负责反转霉素A生物合成的聚酮生物合成基因簇。序列分析揭示了额外的聚酮起始模块，它结合了甲基丙二酰辅酶A。这表明红霉素A是通过添加丙酸单元进行生物合成的。有趣的是，从逆转链霉菌的次生代谢物中分离到了支持DNA序列信息的逆转霉素衍生物(逆转霉素A2)。在生物合成基因簇中，确定了催化反转霉素生物合成中间体C-18羟基化的P450。由于该酶是产生反转霉素A衍生物的目标蛋白质，因此晶体图形分析目前正在进行中。通过生化分析和基因敲除，对该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.