基于生物合成的海洋来源小单孢菌N160次级代谢产物挖掘

In the past decade, marine-derived actinomycetes have been documented as a significant resource for natural product discovery. Marine-derived actinomycetes produce various new secondary metabotites, and many of these metabolites possess biological activities such as antifungal, antibiotic, antiprotozoal, anti-flammatory and, antitumor activities et al. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites. The strain Micromonospora sp. SCSIO N160 was isolated from a sediment sample (E 116°17.754, N 22°41.083′) at a depth of 40m from the South China Sea and it was identified as a Micromonospora rosaria sp. on the basis of its 16S rRNA gene sequence. Three new fluostatins, IJK, together with a new natural product phenanthroviridone, and five known compounds, fluostatins CDEF, rabelomycin, were obtained from the fermentation broth of M. rosaria SCSIO N160 in our previous work. Fluostatins belong to the family of atypical angucyclines and contain a fluorenone chromophore with a unique 6/5/6/6 carbon ring skeleton. phenanthroviridone inhibited the growth of Staphylococcus aureus ATCC 29213, with MIC values of 1.0 and 0.25 μg/mL and had moderate activities toward SF-268 (IC50 0.09 μM) and MCF-7 (IC50 0.17 μM). We and others scientists have uncovered a uniform paradigm for their biosynthesis, suggesting that the 6/5/6/6 ring skeleton of fluostatins arose from a rabelomycin-like precursor by oxidative excision and subsequent ring rearrangement. Based on these, we intend to use the method of genetic manipulations to discover new natural products from the strain SCSIO N160. (1) Heterologous expression of the fluostatin biosynthetic gene cluster (fls) in Streptomyces coelicolor YF11, Streptomyces lividans TK64，Streptomyces albus, and Streptomyces sp. SCSIO 02999-47i, to activate biosynthetic potential for secondary metabolite production. (2) Build gene-inactivation mutants for new "non-natural" natural product from the corresponding gene-inactivation mutants, or activation of metabolic pathways to discover minor components. (3) chemoenzymatic transformation to generate a library of phenanthroviridone-like compounds. The development of new methodologies in genetic manipulations, bioinformatics, molecular genetics, and a better understanding of the biosynthetic principles that govern natural product assembly have driven the ongoing resurgence in natural product research. Natural products and their derivatives form the basis of many important drugs that have found wide spread use in the clinic, new secondary metabolites obtained from SCSIO N160 may form the basis for new drug leads. Besides, the structure of the novel compound bears a novel enzymatic mechanism. The project also provides a new basis for microbiologists to study the enzymatic mechanism, and provide inspiration of organic synthesis of small molecular compounds for organic chemists.

Micromonospora sp. SCSIO N160分离自南海海泥沉积物，前期从其次级代谢产物中分离得到生物合成机制独特的“非典型角环素类”化合物fluostatins I-K及活性显著的新天然产物Phenanthroviridone。基于对fluostatins生物合成的研究，拟：（1）将fluostatins基因簇在多个宿主中异源表达，充分激活其合成潜力。（2）构建突变株，建立“非天然”天然产物化合物库，或激活支路代谢途径，发掘微量组份。（3）以酶学方法结构衍生化，特别是定向合成、改造Phenanthroviridone。本项目将生物合成与菌株次级代谢相结合，期望充分激活其合成潜力，获得结构新颖的化合物，或通过体外酶反应，改造有潜力的小分子，为微生物药物提供先导化合物。同时，结构新颖的化合物蕴藏着新颖的酶学机制，本项目也为研究其酶学机制提供基础，为合成新的小分子化合物提供灵感。

Micromonospora rosaria SCSIO N160是从南海海泥沉积物中分离得到的小单孢菌，前期我们从Micromonospora rosaria SCSIO N160的次级代谢产物中或其异源表达菌株中发现新的fluostatins结构类似物及结构新颖的fluostatin二聚体；本项目中，通过扩大培养规模、异源表达、构建突变株等方法共获得40个化合物，其中22个为新化合物，2个新化合物具有较好的细胞毒活性，2个新化合物具有a-糖苷酶抑制活性。除此，以分离获得的化合物为基础，通过体外实验解析了floustatins异源二聚体或三聚体体外合成的非酶催化机制；结合基因敲除与回补初步确定了一个氧化酶的功能。通过蛋白晶体结构解析与突变阐明一个水解酶立体选择性的机制。在项目的支持下，新天然产物的获得、化合物合成机制的解析及酶催化机制的解析，为开发我国自主知识产权药物提供了支持。以上完成的研究内容已发表SCI论文7篇。申请专利3项。培养博士研究生1名，硕士研究生1名。