微生物次级代谢产物合成基因簇预测分析

微生物次级代谢产物及其生物微生物次级代谢产物及其生物 合成基因簇预测分析合成基因簇预测分析 antiSMASHantiSMASH antiSMASHantiSMASH 的优势与比较的优势与比较 antiSMASHantiSMASH 分析方法及流程分析方法及流程 次级代谢产物及其生物合成基因簇简介次级代谢产物及其生物合成基因簇简介 次级代谢产物的分类鉴定概貌次级代谢产物的分类鉴定概貌 主 要 内 容 2 微生物学发展史中四位重要人物微生物学发展史中四位重要人物 Louis Pasteur 法国人法国人 开创开创 了微生物技术了微生物技术 的新时代的新时代 Alexander Fleming 苏格兰苏格兰人人 发现青霉发现青霉 素及其治疗传染性疾素及其治疗传染性疾 病的功效病的功效 1945年获年获 得诺贝尔生理医学奖得诺贝尔生理医学奖 Selman worksman 美国人美国人 对土壤微生对土壤微生 物产生抗生素物质进物产生抗生素物质进 行了系统和开创性工行了系统和开创性工 作作 发现了抑制肺结发现了抑制肺结 核的链霉菌素核的链霉菌素 1952年年 获得诺贝尔生理医学获得诺贝尔生理医学 奖奖 Francisco Malpartida 西班牙人西班牙人 于于1984年年 第一个克隆了放线紫第一个克隆了放线紫 红素的完整合成基因红素的完整合成基因 簇簇 3 次级代谢产物 微生物生长到一定阶段才产生的化学结构十分复次级代谢产物 微生物生长到一定阶段才产生的化学结构十分复 杂 对该生物无明显生理功能 或并非是微生物生长和繁殖所必杂 对该生物无明显生理功能 或并非是微生物生长和繁殖所必 需的物质需的物质 主要来源 放线菌 真菌等主要来源 放线菌 真菌等 次级代谢产物简介次级代谢产物简介 ZwittermicinA 4 polyketides type I 聚酮聚酮 PK polyketides type II polyketides type III nonribosomal peptides 非核糖体肽非核糖体肽 NRP terpenes 萜烯 lantibiotics 羊毛硫抗生素 bacteriocins 细菌素细菌素 beta lactams 内酰胺 aminoglycosides aminocyclitols 氨基糖甙类 氨基环醇 aminocoumarins 基香豆素 siderophores 铁载体 ectoines 四氢嘧啶 butyrolactones 丁内酯 indoles 吲哚类 nucleosides 核苷 phosphoglycolipids 磷酸糖脂 melanins 黑素类 others 次级代谢产物简介次级代谢产物简介 5 抗生素 抗细菌 抗真菌 抗病毒 抗肿瘤等抗生素 抗细菌 抗真菌 抗病毒 抗肿瘤等 生理生理活性物质活性物质 酶抑制剂酶抑制剂 免疫抑制剂免疫抑制剂 受体拮抗剂受体拮抗剂 次级代谢产物简介次级代谢产物简介 6 7 次级代谢产物生物合成基因簇次级代谢产物生物合成基因簇 次级代谢产物的编码基因通常在基因组中成簇存在 编码次级代谢产物的编码基因通常在基因组中成簇存在 编码 具有多种功能的具有多种功能的复合酶复合酶 研究的最清楚为研究的最清楚为 NRPS nonribosomalpeptides synthetase PKS polyketides synthetase NRPS PKS hybrid 次级代谢产物生物合成基因簇次级代谢产物生物合成基因簇 和 复合酶的基因主要是由连续 和 复合酶的基因主要是由连续的的 模块模块 构成构成 每个基因模块的产物可催化多每个基因模块的产物可催化多 聚酮或多肽链的聚酮或多肽链的一轮延伸一轮延伸和和可能的修饰可能的修饰 一个基因可能含有多个一个基因可能含有多个模块模块 模块模块 非最小单位 下级单元 结构域 非最小单位 下级单元 结构域 一种一种次级代谢产物次级代谢产物的合成由多个基因共同编码的合成由多个基因共同编码 8 PKSPKS 9 McDaniel et al Proc Natl Acad Sci USA 96 1999 1846 1851 10 Brian M Kevany etal APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2009 1144 1155 NRPSNRPS PKSPKS PKSPKS PolyketidePolyketide synthasesynthase 必需结构域 必需结构域 酰基转移酶酰基转移酶 AcyltransferaseAcyltransferase 酮基合酶酮基合酶 ketosynthaseketosynthase 酰基载体蛋白酰基载体蛋白 AcylcarrierproteinAcylcarrierprotein 非非必需结构域 必需结构域 可能含有可能含有1 1 3 3个个修饰酮基修饰酮基的结构域的结构域 酮基还原酶酮基还原酶 脱水酶脱水酶 烯酰基还原酶烯酰基还原酶 ATACPKS 11 必需必需的结构域的结构域 腺苷酰化结构域腺苷酰化结构域 AndeylationAndeylation 缩合结构域缩合结构域 Condensation Condensation 肽酰载体蛋白肽酰载体蛋白结构域结构域 PeptidylcarrierproteinPeptidylcarrierprotein thiolationthiolationdomain domain 非必需结构域 差向异构化非必需结构域 差向异构化 Epimerization Epimerization L DL D 甲基化 甲基化 NmethylationNmethylation 氧化氧化 Oxidation Oxidation 等等 修饰结构域修饰结构域 NRPSNRPS nonribosomal peptide synthetasenonribosomal peptide synthetase APCPC 12 13 Genomics strategyGenomics strategy 基因型基因型to 表型表型 Traditional Traditional strategystrategy 表型表型 to 基因型基因型 All view genes background Large scale gene cluster searching Time saving No gene background Limited gene cluster searching Time consuming 次级合成基因簇的挖掘次级合成基因簇的挖掘 14 传统的天然产物分离通常是通过活性跟踪的策略 NATURE 417 2002 第一篇对第一篇对NRPS进行进行 预测的文章预测的文章 Microbiology 154 1555 1569 2008 J Antibiot 59 9 533 542 2006 J Antibiot 59 3 168 176 2006 次级合成基因簇的挖掘次级合成基因簇的挖掘 15 N Khaldi et al Fungal Genetics and Biology 47 2010 736 741 Gene clusters distributed in fungi genome gene clusters in the 27 sequenced fungal genomes predicted by SMURF 16 Core orthologous and species specifi c gene clusters in three fungi Gene clusters distributed in fungi genome 17 antiSMASH antibiotics Secondary Metabolite Analysis Shell V1 2 August 20th 2011 18 Based on profile hidden Markov models of genes that are specific for certain types of gene clusters antiSMASH is able to accurately identify the gene clusters encoding secondary metabolites of all known broad chemical classes antiSMASH not only detects the gene clusters but also offers detailed sequence analysis 19 NCBI BLAST HMMer3 Muscle 3 Glimmer 3 FastTree TreeGraph 2 Indigo depict PySVG JQuery SVG Analysis tools of antiSMASH 20 Pipeline for genomic analysis of secondary metabolites 21 Input files Genbank format or EMBL format annotated nucleotide file Fasta format the fasta should be single sequence FASTA files with one gene prediction by Glimmer3 prokaryotic data or GlimmerHMM eukaryotic data Transform the predicted results to EMBL format 22 Compound classDescriptionHMM nameSource NRPSCondensation domainCondensationPFAMPF00668 13 NRPSAdenylation domainAMP bindingPFAM PF00501 21 NRPSAdenylation domain with integrated oxidase A OXThis study PKSKetosynthase domainPKS KSSMART PKSAcyltransferasedomainPKS ATSMART PKS neg FabH fatty acid synthasefabHThis study PKSEnediyine ketosynthaseene KSYadavet al 2009 PKSModular ketosynthasemod KSYadav et al 2009 PKSType II PKS Chain length factort2clfThis study TerpeneTerpene synthaseTerpene synth CPFAM PF03936 9 TerpenePhytoene synthasephytoene synthThis study Detection of gene clusters Using HMMer3 tool http hmmer janelia org aligh Pfam source and other pHMMs library Filter negative and positive pHMMs Gene clusters are defi nedby locating clusters of signature gene pHMM hits spaced within 10 kb mutual dis tance 23 NRPS PKS domain architecture analysis Domains and subgroups are detected using another pHMMs library Conserved motifs are detected using the pHMMs in the CLUSEAN package 24 Predicted core structure PKS AT domain specifi cities are predicted using a 24 amino acid signature sequence of the active site as well as other pHMMs NRPS A domain specifi cities are predicted using both the signature sequence method and the support vector machines based method of NRPSPredictor2 25 analysis of secondary metabolism gene families smCOGs 26 analysis of secondary metabolism gene families smCOGs 27 Gene Cluster Blast Comparative Analysis Using BlastP align all gene clusters from NCBI nt database 15 February 2011 Homologous genes BLAST e value 25 of the sequence are given the same colors 28 Genome wide PFAM HMM BLAST analysis some regions important to secondary metabolism may have been missed in the gene cluster identification stage Prediction of potential unknown secondarymetabolite biosynthesis gene cluster types through modules of the CLUSEAN pipeline Require two database CLUSEAN sequence database Required files clusean faa PFAM A database Required files Pfam A full gz 29 Genome wide PFAM HMM BLAST analysis 30 From the resulting set of 484 cloned gene cluster GenBank fi les 473 97 7 were correctly identifi ed by antiSMASH and 468 96 7 were given exactly the same annotation by antiSMASH as by the articles describ ing their experimental characterization Accuracy of antiSMASH 31 Benchmark results on fi ve genome sequences Accuracy of antiSMASH 32 Comparison of different software tools for secondary metabolite biosynthesis analysis 33 antiSMASH predicted NRPS T1PKS gene clustersNP searcher predicted NRPS T1PKS gene clusters NP searcherTotal Of which also found by NP searcherTotal Of which also found by antiSMASH Pseudomonas fluorescens Pf 57666 Streptomyces griseus IFO 1335017131313 Kitasatospora setae NBRC 14216T12555 Salinispora tropica CNB 44011777 47313131 antiSMASH predicted T1PKS NRPS dmat gene clustersSMURF predicted T1PKS NRPS dmat gene clusters SMURFTotalAlso found by SMURFTotalAlso found by antiSMASH Penicillium chrysogenum45444544 Aspergillus fumigatus Af29329272928 74717472 Comparison of different software tools for secondary metabolite biosynthesis analysis 34 Operating system LINUX or Windows XP Vista 7 Internet Browser Firefox 3 x Internet Explorer 9 x Safari Google Chrome or Opera For using the genome wide HMM BLAST analysis RAM 2GB No of Processor cores 2 Usage of antiSMASH 35 antismash py options taxon Taxonomy of your input DNA sequence prokaryotic eukaryotic default p Specifying this is important because CLUSEAN modules cannot run on eukaryotic sequences cores Number of parallel CPUs to use for multiprocessing default all clusterblast Include ClusterBlast gene cluster comparison analysis default y smcogs Include smCOG analysis for functional predicti