基于宏基因组的未培养互营烃降解菌‘Candidatus Smithella cisternae’的生物信息学研究
发布时间：2019-06-20 19:18
【摘要】：通过厌氧微生物作用,将油藏残余油或重质原油降解并转化为甲烷(“油变气”)进行气态开采,是一项极具发展潜力的微生物采油新技术,这个过程需要通过细菌和古菌的互营代谢作用完成。我们小组通过多年努力,富集获得了正十六烷烃降解产甲烷菌系M82,利用稳定同位素探针技术,证实其中一类未培养Syntrophaceae是产甲烷条件下互营降解正十六烷烃的关键细菌类群。但是这类互营菌生长缓慢,一个培养周期需要数月甚数年的时间,其分离难度极大,迄今尚未见到互营烃降解菌的分离报道,这限制了人们开展互营烃降解菌的分子机理研究。本研究通过454高通量测序扩增细菌和古菌16S rRNA基因,发现中温正十六烷烃降解产甲烷菌系M82中的古菌主要属于甲烷袋状菌属(82%),互营菌科下的Smithella(55%)在细菌域中的丰度最高。利用高通量测序技术扩增菌系全基因组DNA,获得40 Gb的短片段序列,通过序列拼接获得144,759条scaffolds,N50 scaffold长度为4619 bp。其中片段丰度大于30000,GC摩尔含量约43%的高丰度片段聚集成团(cluster),根据序列相似性分析,发现这些序列大部分属于Proteobacteria,进一步通过16S rRNA基因相似性分析,发现这个cluster代表的是未培养互营烃降解菌Syntrophaceae(Clone HB1_11,GenBank No.of JX088314,相似性99.7%)。去掉该cluster低丰度以及分类水平上为古菌的序列后,该cluster中存在104个单拷贝基因(SCG),表明其基因组序列的完整度大于97%。基于四核苷酸多态性(TNF)的主成成分分析(PCA)显示聚类出来为一个物种。该基因组大小为2.75 Mb,GC含量为42.8%。该菌株代表一个新的未培养物种,命名为‘Candidatus Smithella cisternae’,菌株名定为M82_1。通过基因注释发现菌株M82-1有2815个ORFs,47个tRNA和3个rRNA。其中50%以上的基因能与KEGG、COG、GO和Pfam数据库中的序列相匹配。菌株M82_1具有编码延胡索酸还原酶、甲基丙二酰辅酶A变位酶和羧基转移酶的基因序列,完整的降解长链脂肪酸和丁酸的β-氧化途径的基因序列,表明该菌可能通过延胡索酸激活启动烷烃的降解。该菌编码代谢丙酸和乙酸的途径的基因不完整,以及羧酸循环、糖酵解和磷酸戊糖途径相关基因的不完整,这表明菌株M82_1可利用的底物范围有限。注释出Nif基因簇推测该菌可能具有固氮能力。我们获得未培养互营烷烃降解菌的基因组草图,并推测分析其遗传代谢潜力,这将为深入研究互营烃降解的分子机理研究提供基础。
[Abstract]:The degradation of residual oil or heavy crude oil into methane ("oil to gas") is a new microbial oil recovery technology with great potential for development through the degradation of residual oil or heavy crude oil into methane ("oil to gas"). This process needs to be completed by the mutual metabolism of bacteria and archaea. Through many years of efforts, our team has enriched the methanogenic strain M82 of n-hexadecane. By using stable isotopic probe technique, it is confirmed that one of the uncultured Syntrophaceae is the key bacterial group for the mutual degradation of n-hexadecane under methanogenic conditions. However, the growth of this kind of mutual bacteria is slow, and a culture cycle takes several months or several years, and its isolation is very difficult. Up to now, there have been no reports of the isolation of mutual hydrocarbon degradation bacteria, which limits the study of the molecular mechanism of mutual hydrocarbon degradation bacteria. In this study, the 16s rRNA gene of bacteria and archaea was amplified by high throughput sequencing. It was found that the archaea in methanogenic strain M82 were mainly belong to the genus methane bagged bacteria (82%), and the abundance of Smithella (55%) in the bacterial domain was the highest. The short fragment sequence of 40 Gb was obtained by amplification of the whole genome DNA, of the strain by high throughput sequencing technique, and 144759 scaffolds,N50 scaffold with a length of 4619 bp. were obtained by sequence splicing. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s rRNA gene, and it was found that this cluster represented 99.7% similarity of uncultured mutual hydrocarbon degradation bacteria Syntrophaceae (Clone HB1_11,GenBank No.of JX088314,). The abundance of fragments was more than 3000, and the high abundance fragments with GC molar content of about 43% were aggregated into clusters. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s DNA gene. After removing the sequence of archaea with low abundance and classification level of the cluster, there were 104 single copy gene (SCG), in the cluster, indicating that the integrity of the genome sequence was more than 97%. Principal component analysis (PCA) based on tetra-nucleotides polymorphism (TNF) showed that clustering came out as a species. The genome size was 2.75 Mb,GC and the content was 42.8%. The strain represents a new uncultured species named 'Candidatus Smithella cisternae', strain M82 鈮，
本文编号：2503465
[Abstract]:The degradation of residual oil or heavy crude oil into methane ("oil to gas") is a new microbial oil recovery technology with great potential for development through the degradation of residual oil or heavy crude oil into methane ("oil to gas"). This process needs to be completed by the mutual metabolism of bacteria and archaea. Through many years of efforts, our team has enriched the methanogenic strain M82 of n-hexadecane. By using stable isotopic probe technique, it is confirmed that one of the uncultured Syntrophaceae is the key bacterial group for the mutual degradation of n-hexadecane under methanogenic conditions. However, the growth of this kind of mutual bacteria is slow, and a culture cycle takes several months or several years, and its isolation is very difficult. Up to now, there have been no reports of the isolation of mutual hydrocarbon degradation bacteria, which limits the study of the molecular mechanism of mutual hydrocarbon degradation bacteria. In this study, the 16s rRNA gene of bacteria and archaea was amplified by high throughput sequencing. It was found that the archaea in methanogenic strain M82 were mainly belong to the genus methane bagged bacteria (82%), and the abundance of Smithella (55%) in the bacterial domain was the highest. The short fragment sequence of 40 Gb was obtained by amplification of the whole genome DNA, of the strain by high throughput sequencing technique, and 144759 scaffolds,N50 scaffold with a length of 4619 bp. were obtained by sequence splicing. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s rRNA gene, and it was found that this cluster represented 99.7% similarity of uncultured mutual hydrocarbon degradation bacteria Syntrophaceae (Clone HB1_11,GenBank No.of JX088314,). The abundance of fragments was more than 3000, and the high abundance fragments with GC molar content of about 43% were aggregated into clusters. According to the sequence similarity analysis, it was found that most of these sequences belonged to Proteobacteria, and further analyzed the similarity of 16s DNA gene. After removing the sequence of archaea with low abundance and classification level of the cluster, there were 104 single copy gene (SCG), in the cluster, indicating that the integrity of the genome sequence was more than 97%. Principal component analysis (PCA) based on tetra-nucleotides polymorphism (TNF) showed that clustering came out as a species. The genome size was 2.75 Mb,GC and the content was 42.8%. The strain represents a new uncultured species named 'Candidatus Smithella cisternae', strain M82 鈮，
本文编号：2503465
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