Pleuromutilin Biosynthetic Studies

描述（由申请人提供）：由于许多微生物已经对目前可用的抗生素药物产生耐药性，因此在历史上这一点上迫切需要研究新的抗生素。目前感兴趣的一个领域是胸膜残素类抗菌剂。Retapamulin是一种半合成的胸膜残素衍生物，于2007年被批准用于治疗皮肤感染，包括葡萄球菌感染。研究表明，对retapamulin的耐药性可能会缓慢发展，这表明这类药物将是对现有抗生素的有益补充。我们的研究重点是发现复盖霉素的真菌天然产物前体——胸膜残素是如何由传塞克氏梭菌（Clitopilus passkerianus）产生的。在项目期间，我们的目标是发现负责形成胸膜残蛋白的生物合成基因簇，并表征形成胸膜残蛋白核心的特定二萜合成酶和细胞色素P450酶。从结构上讲，胸膜残素是类异戊二烯天然产物二萜家族的一员。利用下一代测序技术（Illumina测序）的全基因组测序将提供足够质量的序列，以使用生物信息学方法识别假定的基因簇，这将通过重组酶的生化表征进行验证。生物合成基因的知识最终将使我们能够提高胸膜残蛋白的生产效率，为有前途的抗菌剂的半合成提供起始材料。此外，表征形成胸膜残蛋白骨架的新型二萜合成酶将增加我们对这些形成许多生物活性化合物结构支架的不同酶的认识。

DESCRIPTION (provided by applicant): Research on new antibiotics is critically needed at this point in history as many microorganisms have become resistant to the currently available antibiotic drugs. One area of current interest is the pleuromutilin class of antibacterial agents. Retapamulin, a semisynthetic derivative of pleuromutilin, was approved in 2007 to treat skin infections, including Staph infections. Research suggests that resistance to retapamulin is likely to develop slowly, which suggests that this class of drugs will be a useful addition to current antibiotics. The focus of our research is to discover how pleuromutilin, the fungal natural product precursor to retapamulin, is produced by the fungus Clitopilus passeckerianus. Within the project period, we aim to discover the biosynthetic gene cluster responsible for the formation of pleuromutilin and to characterize the specific diterpene synthase and cytochrome P450 enzymes responsible for forming the pleuromutilin core. Structurally, pleuromutilin is a member of the diterpene family of isoprenoid natural products. Full genome sequencing utilizing next generation sequencing technologies (Illumina sequencing) will provide sequence of sufficient quality to identify the putative gene cluster using a bioinformatics approach, which will be verified by biochemical characterization of the recombinant enzymes. Knowledge of the biosynthetic genes should eventually allow us to increase the efficiency of pleuromutilin production, providing the starting material for the semi-synthesis of promising antibacterial agents. Additionally, characterization of the novel diterpene synthase that forms the pleuromutilin skeleton will add to our knowledge of these diverse enzymes that form the structural scaffolds to many bioactive compounds. PUBLIC HEALTH RELEVANCE: The pleuromutilin class of antibiotics, derived from the fungal natural product pleuromutilin, has recently been approved for the treatment of human bacterial infections, including those resistant to other therapies. An understanding of the fungal genes necessary to form pleuromutilin will allow for rational approaches to improve the production of the common core structure of these antibiotics and will provide a greater understanding of how nature forms these important compounds.