Pleuromutilin Biosynthetic Studies

描述（由申请人提供）：由于许多微生物对目前可用的抗生素药物具有耐药性，因此在历史上的这一点上迫切需要对新抗生素进行研究。目前感兴趣的一个领域是截短侧耳素类抗菌剂。瑞他帕林是截短侧耳素的半合成衍生物，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，对瑞他帕林的耐药性可能会缓慢发展，这表明这类药物将成为现有抗生素的有用补充。我们研究的重点是发现截短侧耳素，真菌天然产物的前体瑞他帕林，是如何产生的真菌Clitopilus passeckerianus。在项目期间，我们的目标是发现负责截短侧耳素形成的生物合成基因簇，并表征负责形成截短侧耳素核心的特定二萜合酶和细胞色素P450酶。截短侧耳素在结构上是类异戊二烯天然产物的二萜家族的成员。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鉴定推定的基因簇，这将通过重组酶的生物化学表征进行验证。生物合成基因的知识最终应该使我们能够提高截短侧耳素生产的效率，为有前途的抗菌剂的半合成提供起始材料。此外，形成截短侧耳素骨架的新型二萜合酶的表征将增加我们对形成许多生物活性化合物的结构支架的这些不同酶的知识。 公共卫生相关性：截短侧耳素类抗生素，来源于真菌天然产物截短侧耳素，最近已被批准用于治疗人类细菌感染，包括对其他疗法有抗性的细菌感染。了解形成截短侧耳素所必需的真菌基因将允许合理的方法来改善这些抗生素的共同核心结构的生产，并将提供对自然如何形成这些重要化合物的更好理解。

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.