潜在NMDA受体拮抗剂分子Stachybocin A生物合成研究

Stachybocin A, a dimeric meroterpenoid biogenetically originated from polyketide-terpenoid hybrid pathway, is consisted of two same phenylspirodrimane units connected by a C5 alkyl chain. The pharmacological evaluation showed that stachybocin A had significant antagonistic effects on the N-methyl-D-aspartic (NMDA) receptor, which indicated it as a promising lead compound for the treatment of inflammatory pain. However, its low yield and difficult accessibility by chemical synthesis restrict the further research and development as a drug lead/candidate. Thus, revealing the biosynthetic pathway of stachybocin A would provide a theoretical basis for solving such problem. On the basis of stachybocin A-yielding strain Stachybotrys chartarum CGMCC 3.5365 and its whole genome sequencing, the biosynthetic pathway will be elucidated by targeted gene deletion, isolation and structural identification of intermediates in the mutants, and heterologous expression and functional validation of the responsible enzymes. Especially, the enzymatic mechanism of the two key reactions for the cyclization of terpene unit in the monomer and coupling reaction of two monomers to dimer will be deciphered. This study will lay the foundation for the efficient biosynthesis of stachybocin A and its analogues, and pave the way for drug discovery. Therefore, the project is of theoretical significance and practical value.

Stachybocin A为聚酮-异戊二烯混源杂萜二聚体，是由两个完全相同的苯基螺环补身烷单聚体分子通过C5烷基链连接而成。药理学研究发现，stachybocin A对N-甲基-D-天冬氨酸（NMDA）受体具有很强的拮抗活性，是一个很有前景的治疗炎症性疼痛的药物先导化合物。然而其天然产量低，化学合成困难，药源难以保证，研究其生物合成则有可能为解决这一难题提供理论依据。本研究拟在stachybocin A生产菌株Stachybotrys chartarum CGMCC 3.5365全基因组测序基础上，通过基因敲除、生物合成中间体分离鉴定、基因外源表达及功能鉴定等技术手段解析stachybocin A生物合成途径，重点阐明单聚体萜类单元环合及二聚体形成两步关键反应的酶学机制，为stachybocin A及其类似物的高效生物合成及进一步新药研制奠定基础，具有重要理论意义及应用价值。

Stachybocin A为苯基螺环补身烷杂萜二聚体，是一个很有前景的治疗炎症性疼痛的药物先导化合物。然而其来源受限，药源难以保证，研究其生物合成途径并进行高效生物合成，可解决这一难题，亦可基于其生物合成途径人为构建合成模块，进而定向规模化制备或通过组合生物合成获得结构多样、成药性更强的衍生物。.Stachybocin A生产菌株Stachybotrys chartarum中富含多种Stachybocin A结构类似物，对其次级代谢产物进行深入研究，从分离鉴定的结构信息合理推导Stachybocin A生物合成途径，并从中获得生物合成中间体，为体外酶催化验证基因功能提供底物。此外，基于生物信息学分析及基因组挖掘策略，我们对S. chartarum进行全基因组测序，通过对目标化合物Stachybocin A化学结构剖析推测其生物合成基因应含有聚酮合酶、异戊烯基转移酶、依赖黄素单加氧酶、萜类环合酶、氧化还原酶等，进一步通过基因敲除、生物合成中间体结构鉴定、基因外源表达及催化功能鉴定等技术手段，初步阐明Stachybocin A生物合成途径。研究发现参与Stachybocin A生物合成基因不成簇且催化二聚体形成为自发反应。.该研究为Stachybocin A及其类似物的高效生物合成提供理论及实验依据，并为药物先导化合物/候选药物发现及其进一步开发利用奠定基础，具有重要理论意义及应用价值。