棘白菌素类化合物生物合成基因簇的转录调控机制研究

Echinocandin class compounds, represented by pneumocandin B0(precursor of caspofungin, a new kind of antifungal agents),characterized by less likely to form side-effect and drug resistance, which also represent further development direction of new antibiotics with the characteristics of low toxicity, high safety and broad spectrum. Faced with the main bottlenecks of low yield of product and mass by-products during the large-scale production of pneumocandin B0, the transcriptional regulation mechanism of relative biosynthesis gene cluster is analyzed in this study in order to provide theoretical basis for the effectually operation of the cell factory. With the statistical thermodynamics method which is used to describe molecular motion in the chemical process, thermodynamic parameter of the transcription process is measured at the molecular level. The joint probability of RNA polymerase and promoter is explored and regulation methods of transcription factor to target gene is distinguished based on Boltzmann distribution probability. With the mechanism of pneumocandin B0 synthesis, the relationship between the environment and thermodynamic parameters is explored at the cellular level, which then guide the transcription regulation strategies of the gene cluster and regulate the synthesis process of pneumocandin B0.Finely, it is help to realize high-efficient and single-package production with lower cost of separation and purification and lay the theoretical foundation of the green production of secondary metabolite.

以纽莫康定B0（新型抗真菌药物卡泊芬净的前体）为代表的棘白菌素类化合物，具有毒副作用小，不易产生耐药性等特点，是未来新型低毒、安全、广谱性抗生素的发展方向。生产单位低、副产物多等问题是纽莫康定B0高效生物合成的瓶颈。本课题针对纽莫康定生产中代谢产物多样性的问题，以生物合成基因簇的转录体系为研究对象，旨在通过对相关结构基因和修饰基因表达的转录调控机制的解析，为细胞工厂的高效运作提供理论依据。采用化学过程中描述分子运动性能的统计热力学方法，在分子水平上测定表征基因簇转录过程的热力学参数，基于波尔兹曼分布的角度发掘RNA聚合酶和启动子的结合概率，判别转录因子对靶基因的调控方式，阐明纽莫康定合成的分子基础，进而在细胞水平上探索其与外界环境扰动的耦联关系，指导基因簇转录调控策略，精细定向调控纽莫康定B0的合成过程，以期实现高效单组分生产并降低其分离纯化成本，为次级代谢产物的低能耗和绿色生产奠定基础。

纽莫康定是真菌Glarea lozoyensis产生的棘白菌素类脂肽，通过非竞争性抑制细胞壁β-(1,3)-D-葡聚糖的合成，破坏真菌细胞壁的合成。其中，纽莫康定B0是重要抗真菌药物卡泊芬净的前体物。本课题以发酵法生产纽莫康定B0为研究对象，研究环境因素对底物吸收、产物合成及关键代谢物的影响。在本项目实施过程中，主要进行三方面工作：首先，在生理生化水平研究环境条件对底物吸收、代谢的作用机制，确定了纽莫康定B0合成的关键代谢物和关键代谢途径；其次，在分子水平上测定表征纽莫康定基因簇转录过程的热力学参数，解析环境因素对纽莫康定B0合成的影响；第三，在充分了解纽莫康定B0的合成途径和调控机制、系统分析细胞所处的环境对细胞合成纽莫康定B0的影响的基础上，优化发酵调控过程，实现G. lozoyenesis细胞中纽莫康定B0的高效合成。