【作者】 徐建国；

【导师】 陶美凤；

【作者基本信息】 上海交通大学 ， 微生物学， 2017， 硕士

【摘要】 胶样链霉菌Sge12是从湖北省神农架原始森林分离的菌株,具有抗稻瘟病病原菌等抗真菌活性。围绕该菌株中抗真菌活性物质的挖掘及其生物合成,本研究进行了基因组测序与分析、抗真菌活性化合物分离纯化与结构鉴定、基因簇克隆与异源表达及其生物合成研究。首先,本论文对胶样链霉菌Sge12进行全基因组测序,anti SMASH预测分析发现基因组编码37个次级代谢基因簇。其中,并没有发现与已知抗真菌抗生素基因簇高度相似的基因簇,对活性化合物的鉴定及其基因簇的克隆形成挑战,但也暗示相应基因簇具有新颖性。同时,实验室前期构建了胶样链霉菌Sge12的基因组BAC文库,并用文库异源表达筛选技术获得7G7等三个具有抗真菌活性的BAC克隆。在此基础上,本论文通过末端测序和酶谱分析确定这些阳性BAC克隆之间共有69 kb重叠片段;将三个克隆转到变铅青链霉菌SBT18进行异源表达,其发酵产物具有相似的抗菌活性谱,暗示它们携带相同基因簇、可表达产生相同化合物。选取变铅青链霉菌SBT18/7G7进行大量固体发酵,根据活性追踪分离纯化化合物,化学结构解析表明活性化合物为脂黄霉素及其类似物NE-M2和NE-B1。随后,本研究通过大片段基因缺失和异源表达,将脂黄霉素基因簇定位在7G7中的21 kb片段上;再通过单基因敲除和异源表达,确定了脂黄霉素生物合成需要lxn A、lxn B、lxn C、lxn D、lxn E、lxn F、lxn G和lxn H等八个结构基因以及lxn R1和lxn R2两个正调控基因。进而通过LC-MS对各突变株进行代谢谱分析,发现lxn C缺失突变菌株积累了长链脂肪胺中间代谢产物。最后,根据基因敲除、生物信息学分析及突变株中间代谢产物的LC-MS分析结果,本研究推测了脂黄霉素的生物合成途径。综上,本研究克隆、定位并异源表达了脂黄霉素生物合成基因簇,并对其生物合成途径进行了分子遗传研究,为脂黄霉素高产菌株的构建及通过组合生物合成获得更多的抗真菌活性化合物的研究奠定基础。更多还原

【Abstract】 Streptomyces gelaticus Sge12,isolated from forest soil of Shengnongjia in Hubei Province,showed remarkable activities against pathogenic fungi such as rice blast fungus.Genome sequencing,chemical isolation and structure elucidation,cloning and heterologous expression of the target gene cluster,as well as biosynthesis study were conducted in this thesis in order to discover antifungal compounds produced by S.gelaticus Sge12 and to investigate their biosynthesis pathway.In the beginning,the genome of S.gelaticus Sge12 was sequenced,which revealed 37 secondary metabolite gene clusters according to analysis with anti SMASH.However no one of these gene clusters showed high similarity with known gene clusters for antifungal antibiotics,which might hinder the identification of the antifungal compound and cloning of their biosynthetic gene cluster.Nevertheless,it implied the novelty of the biosynthetic gene cluster for the unknown antifungal compound.A bacterial artificial chromosome（BAC）library of S.gelaticus Sge12 was constructed and screened via high throughput BAC Library EXpression and Analysis System（LEXAS）at the mean time by other in this laboratory,yielding three BAC clones（7G7 and others）able to produce antifungal activity in a heterologous expression host.In this thesis,the three BAC clones were confirmed belonging to one contig and sharing a ca.69 kb overlapping region through terminal sequencing and restricted digestion.Furthermore,heterologous expression of the three BAC clones in Streptomyces lividans SBT18 led to the production of antifungal activity with similar spectrum.These findings indicated that three BAC clones carried the same gene cluster producing the same bioactive compounds.Subsequently,S.lividans SBT18/7G7 was chosen to ferment and bioactive compounds were isolated using bioassay-guided method,leading to the identification of lipoxamycin and its analogs NE-M₂ and NE-B₁.Then,lipoxamycin gene cluster was located on a 21 kb region of7G7 by large-fragment deletion and heterologous expression.Eight structural genes lxn A、lxn B、lxn C、lxn D、lxn E、lxn F、lxn G and lxn H,as well as two positive regulatory genes lxn R1 and lxn R2 were determined to be essential for the production of lipoxamycin by single-gene in-frame deletion.A long-chain aliphatic amine intermediate was found to be accumulated in the lxn C mutant by metabolic profiling analysis based on LC-MS.Finally,the lipoxamycin biosynthetic pathway was proposed based on the results of gene deletion,bioinformatics analysis,and LC-MS analysis of the mutants.To sum up,the lipoxamycin biosynthetic gene cluster was successfully cloned,located and heterologously expressed in this work.The lipoxamycin biosynthesis pathway was also investigated through genetic manipulation.This study laid the foundation for the engineering of high-producing bacterial strains and the development of novel antifungals through combinatorial biosynthesis.更多还原