【作者】 李富超；

【导师】 刘兆普；

【作者基本信息】 南京农业大学 ， 应用海洋生物学， 2011， 博士

【摘要】 生境复杂的海洋链霉菌相对于陆生链霉菌,更能耐受高盐、高压、低温、缺氧、寡营养等特殊生存条件,海洋生态环境的多样性、特殊性和复杂性赋予海洋链霉菌种类、遗传和生态功能的多样性,而这些正是海洋链霉菌次级代谢产物多样性的物质基础,海洋链霉菌继陆生链霉菌后成为新颖活性物质的重要来源。链霉菌Streptomyces griseoaurantiacus M045分离自青岛胶州湾海洋沉积物中,可以产生手霉素A、中尼霉素A和B等抗生素,具有良好的生物活性,中尼霉素对乳腺癌及黑色素瘤具有显著的抑制活性(IC50= 2.41μg/mL),对农作物病原真菌的抗菌效果明显,优于武夷菌素水剂、克菌康、宝丽安及百菌清等商品农药,因此具有良好的开发潜力。本研究利用454 GS FLX测序仪,结合Illumina Solexa测序技术,完成了链霉菌S. griseoaurantiacus M045的全基因组测序,其为线性基因组,7,712,377 bp,GC含量72.73%。该基因组中,预测得到6839个开放阅读框(ORFs),614个预测蛋白是其特有的,发现660个paralog蛋白家族,4416个预测蛋白可以归到COG数据库,确定有234个蛋白属于分泌蛋白,1352个蛋白属于跨膜蛋白,106个蛋白是脂蛋白。链霉菌M045具有完整的Sec分泌系统,及部分Tat分泌途径,其有助于各种初级和次级代谢产物的合成及分泌。通过系统发育分析,在链霉菌M045基因组中发现了不同于陆生链霉菌的切割脂蛋白信号肽的Ⅱ型信号肽酶和转肽酶的基因,而与同样来自海洋环境的Salinispora专性海洋放线菌具有一定的同源,可能与海洋环境适应相关。链霉菌M045的基因组提供了大量与环境适应相关的功能基因,具有完整的螯铁蛋白pyochelin的生物合成途径,推测是为了适应海洋缺铁环境。在链霉菌M045基因组上发现了完整的编码气囊蛋白的基因簇,探讨了气囊蛋白对于链霉菌适应水生环境的机制。通过基因组岛预测分析,发现了18个基因组岛,编码了许多环境适应及次级代谢的相关基因,推测链霉菌在进化过程中,通过基因组岛可以把次级代谢途径和环境适应结合起来。链霉菌M045基因组上分布着大量与次级代谢相关的基因,本论文解析了手霉素A和中尼霉素A的生物合成基因簇,包括32个编码基因,其中有两个序列很短,为未定功能蛋白基因。ChiA1/A2/A3三个功能基因编码蛋白参与了核心C7N结构的合成和转运；ChiB1/B2/B3/B4/B5/B6/B7/B8/B9参与了upper聚酮链(B)的合成和延伸；ChiCl/C2/C3/C4/C5C6/B7/B8/B9参与了lower聚酮链(C)的合成和延伸；ChiD1/D2/D3参与了C5N结构单元的合成和连接；ChiE1/E2/E3/E4是合成过程中的氧化还原酶,其中推测ChiE4催化了氯的取代和C链的重排,从而由手霉素A生成中尼霉素A; ChiM1/M2是转运和分泌蛋白,用于药物的转运；ChiR1/R2/R3是转录调控因子。对C5N结构单元合成的关键酶编码基因ChiD2和C7N结构单元合成的关键酶编码基因ChiA1,构建了基因敲除载体,通过大肠杆菌-链霉菌间的接合转移,获得了敲除掉ChiD2基因的链霉菌M045突变株,不再合成手霉素A和中尼霉素A、B,初步验证了ChiD2基因的功能。本研究完成了第一个海洋来源的链霉菌基因组草图,它提供了一个丰富的“基因pool",为我们进一步进行比较基因组学、海洋链霉菌的环境适应性进化、基因簇功能研究及抗生素的组合生物合成奠定了基础。更多还原

【Abstract】 Specific environmental conditions can stimulate marine microorganisms to produce novel metabolites, and marine streptomycetes are important resources of bioactive compounds. Streptomyces griseoaurantiacus M045 isolated from sediment from Jiaozhou Bay in China produces manumycin A and chinikomycins antibiotics. Chinikomycins are the first discovered truly natural manumycin antibiotics with a para orientation of the side chains (p-aminobenzoic acid core component, pABA). Chinikomycins show moderate antitumor activity and inhibition of plant pathogenic fungi. Herein we present a draft genome sequence of S. griseoaurantiacus M045; it is the first sequenced streptomycete derived from the marine environment.The nucleotide sequence was determined using a 454 GS FLX sequencer. A total of 607,480 reads including up to 276,865,258 bp were obtained, which represented a 36-fold coverage of the genome. Assembly was performed using the GS de novo Assembler Software; it produced 60 contigs with a total size of 7,709,283 bp. In addition,570-fold coverage of pair-end sequences (2×120 bp) produced by Illumina Solexa technology was mapped to the genome sequence to promote sequence quality and fill in gaps. Finally, we obtained the S. griseoaurantiacus M045 draft genome of 7,712,377 bp distributed in 46 contigs with a GC content of 72.73%.Putative protein-coding sequences were predicted using Glimmer and GeneMark software. Functional annotation was based on the BLASTP with KEGG and NR databases. tRNA genes were directly predicted with tRNAscan-SE v1.23. The signal peptide cleavage sites, transmembrane topologies, and lipoproteins were predicted using SignalP3.0 with hidden Markov models, ConPredⅡ, and LipoP1.0, respectively. Genomic islands were predicted on the IslandViewer web service using the IslandPath-DIMOB and SIGI-HMM methods.The draft genome consists of one linear chromosome with 6 rRNA operons,65 tRNA genes, and 6839 protein-coding genes (CDSs). Among the CDSs,4416 proteins could be assigned to COG families. Biological functions could be assigned to 4966 (72.6%) of the predicted proteins, whereas 1259 CDSs (18.4%) are homologous to conserved proteins of unknown function in other organisms. The remaining 614 hypothetical proteins (9%) have no match to any known proteins in the databases. At least 660 multigene (paralog) families containing 2693 predicted proteins were identified. Two-component regulatory systems, which detect and respond to changes in the marine environment, are widely distributed on the chromosome.As for the subcellular localization of the proteins,234 proteins were identified as secreted proteins,1352 proteins as transmembrane proteins, and 106 proteins as lipoproteins. In addition,18 genomic islands occur in the genome; the genomic islands link secondary metabolism to functional and environmental adaptation in marine S. griseoaurantiacus M045.S. griseoaurantiacus M045 contains general secretory pathway and partial twin-arginine translocation pathway. The sortase and lipoprotein signal peptidase of S. griseoaurantiacus M045 are homologous with Salini.spora respectively, but not found in S. coelicolor A3(2), S. avermitilis MA-4680 and S. griseus IFO13350. S. griseoaurantiacus M045 has abundant functional genes for environmental adaptation, whole biosynthesis pathway of pyochelin for marine iron-limited environment and encoding gas vesicle synthesis protein for seawater adaptation.Genome analysis revealed a number of genes related to biosynthesis of secondary metabolites, including some involved in NRPS-PKS I fusion, PKS II and PKS III pathways. Manumycin A contains a 3-amino-4-hydroxybenzoic acid (3,4-AHBA) core component, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone (C5N) moiety. The gene cluster for manumycin A contains 32 genes, including 3-amino-4-hydroxybenzoic acid (3,4-AHBA) synthase,3,4-AHBA carrier protein,3-oxoacyl-(acyl carrier protein) synthase, and 5-aminolevulinate synthase. Chinikomycins contain a pABA core component, but share a partial gene cluster with manumycin A that consists of two triene polyketides and a 2-amino-3-hydroxycyclopent-2-enone biosynthetic pathway.S. griseoaurantiacus M045 is the first marine streptomycete for which a genome sequence has been reported. It is considered that this work provides insight into the functional adaptation and combinatorial biosynthesis of bioactive molecules produced by marine streptomycetes.更多还原