稀有放线菌Lechevalieria rhizosphaerae NEAU-A2的次级代谢产物研究及遗传操作系统的建立  

Secondary metabolites and genetic system of the rare actinobacteria Lechevalieria rhizosphaerae NEAU-A2

作　　者：余志银 颜一军[1] 刘重喜 黄建萍[1] 向文胜 黄胜雄[1] YUZhiyin;YAN Yijun;LIU Chongxi;HUANG Jianping;XIANG Wensheng;HUANG Shengxiong(State Key Laboratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany,Chinese Academy of Sciences,Kunming,Yunnan 650201,China;College of Life Science,Northeast Agricultural University,Harbin,Heilongjiang 150030,China)

机构地区：[1]中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室,云南昆明650201 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《微生物学通报》2021年第7期2318-2328,共11页Microbiology China

基　　金：国家自然科学基金(U1702285);中国科学院前沿交叉科学领域重点项目(QYZDB-SSW-SMC051);云南省基础研究计划项目(2019FJ007,2019FA034)。

摘　　要：【背景】菌株Lechevalieria rhizosphaerae NEAU-A2是一株新的稀有放线菌,其基因组中包含多个与次级代谢产物生物合成相关的基因簇,具有产生丰富代谢产物的潜力。【目的】对稀有放线菌L.rhizosphaerae NEAU-A2的次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并建立其遗传操作系统。【方法】应用硅胶柱色谱和高效液相色谱等方法对菌株NEAU-A2的次级代谢产物进行分离和纯化,整合质谱分析、核磁共振等方法进行结构解析。在对该菌株全基因组测序的基础上,以基因组序列中编码杂合的聚酮合酶-非核糖体肽合成酶(PolyketideSynthase-Nonribosomal Peptide Synthetase,PKS-NRPSs)基因1609为目标基因,利用PCR-Targeting介导的基因置换技术构建重组质粒,通过接合转移的方式导入野生菌株。【结果】从菌株NEAU-A2中分离鉴定了2个新的吲哚二聚化合物(1,2)和5个已知化合物N-乙酰色胺(3)、4-((2-(1H-Indol-3-Yl)Ethyl)Amino)-4-Oxobutanoic Acid(4)、Brevianamide F(5)、4S,7R-Germacra-1(10)E,5E-Diene-11-Ol(6)、1H-吡咯-2-羧酸(7)。接合子通过培养2代即可获得双交换突变菌株,PCR分析结果显示PKS-NRPS基因被成功中断。【结论】从稀有放线菌L.rhizosphaerae NEAU-A2中分离鉴定出7个化合物,并成功建立了该菌的遗传操作系统。[Background]Lechevalieria rhizosphaerae NEAU-A2 was identified as a novel rare actinobacteria,and genomic analysis shows that it contains a variety of biosynthetic gene clusters with the potential to generate multiple natural products.[Objective]Study on the secondary metabolites in L.rhizosphaerae NEAU-A2 to discover compounds with novel structures or unique biological activities,and further establish the genetic system of the strain.[Methods]The secondary metabolites were isolated and purified by using silica gel column chromatography,high performance liquid chromatography and other methods.The structures of compounds were elucidated by analysis of NMR and MS data.The mutant cosmid with the disruption of the gene 1609(a PKS-NRPS hybrid gene)was constructed using PCR-targeting method.Intergeneric conjugation was conducted for plasmid the transformation into wild type strain.[Results]Two new dimeric indole alkaloids(1–2),along with five known compounds,N-acetyltryptamine(3),4-((2-(1 H-indol-3-yl)ethyl)amino)-4-oxobutanoic acid(4),brevianamide F(5),4 S,7 R-germacra-1(10)E,5 E-diene-11-ol(6),1H-pyrrole-2-carboxylic acid(7),were isolated and identified from the fermentation broth of L.rhizosphaerae NEAU-A2.The double crossover recombinant strains were obtained after culturing two generations,and further verified by polymerase chain reaction(PCR).[Conclusion]Seven compounds were isolated and identified from strain NEAU-A2,and the genetic system of this novel rare actinobacteria L.rhizosphaerae NEAU-A2 was successfully established.

关 键 词：Lechevalieria rhizosphaerae NEAU-A2 代谢产物 分离鉴定 PCR-Targeting 接合转移 

分 类 号：Q933[生物学—微生物学]