Pleuromutilin Biosynthetic Studies

描述(由申请人提供)：在历史的这一点上，迫切需要研究新的抗生素，因为许多微生物已经对目前可用的抗生素药物产生了抗药性。目前感兴趣的一个领域是切割胸膜的抗菌剂。瑞帕帕蛋白是一种半合成的切割胸膜蛋白的衍生物，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，对瑞帕蛋白的抗药性可能会缓慢发展，这表明这类药物将是目前抗生素的有用补充。我们的研究重点是发现Plitopilus passeckerianus是如何产生重组蛋白的真菌天然产物--截胸菌素的。在项目期间，我们的目标是发现负责形成截胸素的生物合成基因簇，并鉴定负责形成截胸素核心的特定二萜合成酶和细胞色素P450酶。从结构上讲，截胸素是异戊二烯类天然产物中二萜类化合物家族的成员。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识别假定的基因簇，这将通过重组酶的生化特征得到验证。对生物合成基因的了解最终将使我们能够提高截胸素的生产效率，为有希望的抗菌剂的半合成提供起始材料。此外，对形成截胸素骨架的新型二萜合成酶的表征将增加我们对这些不同的酶的知识，这些酶构成了许多生物活性化合物的结构支架。 公共卫生相关性：从真菌天然产物截胸素中提取的截胸素类抗生素，最近已被批准用于治疗人类细菌感染，包括那些对其他疗法具有抗药性的感染。了解形成截胸素所需的真菌基因将有助于采取合理的方法来改进这些抗生素的共同核心结构的生产，并有助于更好地了解自然界如何形成这些重要的化合物。

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.