珠江口沉积物来源链霉菌SCSIO 40020中bafilomycins的鉴定及其生物合成基因簇的异源表达  

Identification of bafilomycins and heterologous expression of its biosynthetic gene cluster from the Pearl River Estuary sediment-derived Streptomyces sp. SCSIO 40020

作　　者：翟诗岚 刘威 吴正超[1,3] 朱义广[1] 张文军[1] 张长生[1,2] 马亮 Shilan Zhai;Wei Liu;Zhengchao Wu;Yiguang Zhu;Wenjun Zhang;Changsheng Zhang;Liang Ma(Key Laboratory of Tropical Marine Bio-resources and Ecology,Guangdong Key Laboratory of Marine Materia Medica,RNAM Center for Marine Microbiology,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou 510301,Guangdong Province,China;University of Chinese Academy of Sciences,Beijing 100049,China;State Key Lboratory of Tropical Oceanography,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou 510301,Guangdong Province,China)

机构地区：[1]中国科学院南海海洋研究所,中国科学院热带海洋生物资源与生态重点实验室,中国科学院海洋微生物研究中心,广东省海洋药物重点实验室,广东广州510301 [2]中国科学院大学,北京100049 [3]中国科学院南海海洋所,热带海洋环境国家重点实验室,广东广州510301

出　　处：《微生物学报》2021年第12期3991-4005,共15页Acta Microbiologica Sinica

基　　金：广东省基础与应用基础研究基金(2021A1515010839);广州市科技计划(202102020513);国家自然科学基金(21907098,41706181);国家重点研发计划(2019YFC0312503);王宽诚教育基金(GJTD-2020-12)。

摘　　要：【目的】从珠江口沉积物来源的菌株SCSIO 40020中分离bafilomycins,并对其生物合成基因簇进行克隆和异源表达研究。【方法】通过分析菌株SCSIO 40020的16S rRNA基因序列并构建系统发育树以鉴定菌种,以柱层析法和制备色谱法对次级代谢产物进行分离纯化,借助波谱学手段完成单体化合物的结构鉴定,采用生物信息学分析定位bafilomycins的生物合成基因簇,通过筛选菌株SCSIO 40020基因组的细菌人工染色体文库和接合转移将bafilomycins生物合成基因簇导入3种链霉菌进行异源表达,利用高效液相色谱检测异源表达菌株的发酵产物。【结果】菌株SCSIO 40020被鉴定为链霉菌属菌株,从其发酵产物中分离鉴定了2个单体化合物bafilomycins A1和D。克隆了链霉菌SCSIO 40020中bafilomycins的生物合成基因簇并推导了其生物合成途径,在3种链霉菌中表达产生了bafilomycins。【结论】从珠江口环境中获得了一株产生bafilomycins的链霉菌SCSIO 40020,成功建立了该菌株次级代谢产物生物合成基因簇的异源表达体系,并首次在链霉菌Streptomyces lividans SBT18、Streptomyces coelicolor M1154和Streptomyces albus J1074中进行了表达,获得了bafilomycins,为后续bafilomycins结构类似物的生产和链霉菌SCSIO 40020中新结构活性化合物的挖掘奠定了基础。[Objective] To identify the macrolide antibiotic bafilomycins from the strain SCSIO 40020 isolated from the Pearl River Estuary sediment. To clone and heterologously express the biosynthetic gene cluster(BGC) of bafilomycins.[Methods] The strain SCSIO 40020 was identified based on phylogenetic analysis of its 16 S rRNA gene sequence. The generated compounds from SCSIO 40020 were purified via normal phase-column chromatography and semi-preparative chromatography. The chemical structures of the isolated compounds were subsequently elucidated by comprehensive spectroscopic analyses. The bafilomycins BGC was identified by using bioinformatics approach. Through screening the bacterial artificial chromosome(BAC) library of this strain, the BAC containing the bafilomycins BGC was introduced into three Streptomyces hosts by conjugation for expression. Afterwards, the fermentation culture of the recombinant strains were analyzed on high-performance liquid chromatography(HPLC). [Results] The strain SCSIO 40020 was identified as Streptomyces genus and two macrolide compounds bafilomycins A1 and D were isolated from the fermentation crude extract. The bafilomycins BGC was cloned and successfully expressed in three heterologous Streptomyces hosts. In addition, the biosynthetic pathway of bafilomycins was proposed. [Conclusion] The bafilomycin-producing strain Streptomyces sp. SCSIO 40020 was obtained from the Pearl River Estuary. The BAC heterologous expression system of this strain was successfully established. Moreover, the bafilomycins BGC was successfully expressed in Streptomyces lividans SBT18, Streptomyces coelicolor M1154 and Streptomyces albus J1074 for the first time, which laid solid foundation for the production of bafilomycin analogues and mining other interesting BGCs from Streptomyces sp. SCSIO 40020 in the further study.

关 键 词：链霉菌 bafilomycins 生物合成基因簇 异源表达 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]