【作者】 周宏；

【导师】 张先恩； 陈冠军； 刘巍峰；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 随着石油、煤炭等非可再生资源的逐渐枯竭,新的可再生能源的开发已经成为人们迫切的需求,以纤维素为主要成分的植物生物质是地球上最丰富的可再生资源。然而由于纤维素的结构,使其难以被高效降解。结晶纤维素的有效降解是实现木质纤维素高效转化的关键之一。自然界中多种生物具有纤维素降解能力,除了以游离纤维素酶系模式降解纤维素的真菌和以纤维小体模式降解纤维素的厌氧细菌外,某些好氧噬纤维菌如Cytophaga hutchinsonii同样具有极强的结晶纤维素降解能力。随着C. hutchinsonii基因组测序的完成,发现该菌基因组中不具有编码外切纤维素酶的基因,且内切纤维素酶缺少纤维素结合结构域,同时也没有编码典型运动器官的各个基因。因此可以认为,与已经阐明的微生物纤维素降解模式不同,C.hutchinsonii可能具有独特的纤维素水解代谢机制。初步研究结果表明,C. hutchinsonii对结晶纤维素降解呈现底物接触依赖性,大部分纤维素酶活定位于细胞表面,且小分子纤维寡糖的吸收利用可能构成了噬纤维菌整个纤维素降解过程的一个重要环节。C. hutchinsonii可以在固体介质表面快速滑动,gldJ滑动基因的失活导致菌体滑动性丧失,同时无法利用纤维素,说明运动也很可能与纤维素降解之间存在某种联系。由于纤维素酶历来难于进行有活性的异源表达,为了有效的寻找纤维素降解过程中参与的相关基因,建立高效的遗传操作体系就成为了深入开展实验的前提。本论文探索了C. hutchinsonii各种遗传操作手段,初步完善了其遗传操作体系,并且在此基础上进行了大量的转座子筛选工作,获得了一些有价值的与纤维素利用相关的突变株；同时,通过与亲缘关系较近的菌株的生物信息学分析比较,推测了C. hutchinsonii可能的纤维寡糖转运模型,并对其进行了研究。本论文的主要研究内容及创新性结果如下：一、优化了C. hutchinsonii固体培养条件的优化及其遗传操作体系,使得C.hutchinsonii的遗传操作更易于进行。针对已初步建立的C. hutchinsonii遗传操作体系的研究,首先优化了培养条件。对野生型菌株不同抗生素的耐受程度进行了摸索,发现红霉素、氯霉素、四环素等均可作为C. hutchinsonii的抗性筛选标记。通过比较接合和电转化两种基因转移方式,发现电转化具有更高的转移效率和更稳定的遗传性,并成功将复制型及转座质粒转入C. hutchinsonii中,验证了能在该菌中发挥作用的抗性筛选基因并确定了合适的筛选浓度。在此基础上构建了氯霉素抗性的回补质粒,找到可用于蛋白体内表达的系列启动子序列,包括一些组成型或未知功能启动子（Pgap,P1276,P1284）和阿拉伯糖诱导型启动子（ParaB）,能够启动GFP在C. hutchinsonii体内的表达。构建的C. hutchinsonii遗传操作体系为其各种内源或外源蛋白的表达,甚至是特定条件下表达提供了可能。二、构建了纤维素酶基因chu₁107、chu₁280、chu₁335插入失活突变株,证明单一纤维素酶基因失活突变并不影响菌株的纤维素利用生长能力。为了研究C. hutchinsonii独特的纤维素酶系中是否存在某个关键性的酶组分,我们构建了三个纤维素酶基因（基因编号分别为chu₁107、chu₁280和chu1335）各自的插入失活突变株。研究发现失活突变株Chin1107, Chin1280和Chin1335在葡萄糖上的生长与野生型差别不大,而且在以纤维素为碳源的液体培养基以及滤纸等不溶性碳源的固体平板上的生长也与野生型基本相同。这一结果说明单一纤维素酶的缺失,并不会严重影响C. hutchinsonii对纤维素的利用。不同碳源培养野生型菌体,细胞分级分离提取各组分蛋白后,发现纤维二糖培养的可溶性组分中存在一条稳定的诱导条带,经质谱鉴定为BglX,是位于周质空间的β-葡萄糖苷酶,且该蛋白编码基因在纤维素条件下转录上调。该葡萄糖苷酶可能在纤维二糖甚至纤维素的利用中具有重要作用。三、转座诱变获得一株固体培养基上纤维素利用缺陷的突变株,同时发现与野生型菌株相比,其菌体扩散能力下降,外膜蛋白组分发生变化。经转座诱变筛选到一纤维素利用缺陷突变株ChT68,经鉴定该突变株中转座子的插入位置为chu₁719,编码一个位置功能的蛋白。ChT68具有较为特殊的纤维素利用表型,即在以纤维素为碳源的固体培养基中生长明显弱于野生型,而液体培养基中则无差异。利用单插入失活的方法定点插入失活了chu₁719,获得了突变株Chin1719。突变株Chin1719的生长表型与转座突变株ChT68一致；硬琼脂平板表面扩散能力一定程度缺失,显微镜下观察到边缘整齐,与野生型差别明显；突变株膜蛋白的CMCase酶活比野生型降低约40%,提取的外膜蛋白吸附纤维素后,SDS-PAGE结果表明突变株与野生型的吸附蛋白存在明显差异,差异蛋白包括GldJ、GldN、CHU₀007、CHU₃434和CHU3732。由此说明,CHU1719可能在纤维素利用、运动性或蛋白分泌等方面发挥一定的作用。四、对基因组中注释的两个susC-like基因分别失活,突变株对纤维素的利用基本不发生变化；转座子诱变鉴定到的一个TonB-dependent receptors编码基因chu1276,与上下游基因构成一个Sus-like系统,参与纤维素的降解。分析显示,C. hutchinsonii基因组中存在类似Bacteroides thetaiotaomicron中淀粉降解利用系统（starch utilization system, Sus）中的两个重要蛋白组分SusC和SusD。对C. hutchinsonii的两个susC-like基因进行插入失活后,证明单独的susC-like基因对于C. hutchinsonii降解纤维素不是必需的。通过转座子诱变,获得插入位点分别为chu₁276、chu₁277和chu1278的转座突变株T127,T423,C-34,突变株的纤维素降解能力丧失。此外,T127除了和T423一样在纤维二糖碳源上生长延迟外,对低浓度葡萄糖的利用也具有明显延迟,但经过低浓度葡萄糖预培养后这种延迟现象消失。进一步分析发现基因chu₁276及其上下游的基因座位符合Sus-like系统中多糖利用座位（polysaccharide utilization loci, PUL）的组成。基因簇上游预测的双组分调控系统编码基因chu₁265/chu₁266的失活突变株Chin1265和Chin1266在纤维素碳源上的生长与野生型没有显著差异。CHU1276预测为外膜蛋白,其C端预测含有DNA结合结构域,可以响应低浓度葡萄糖信号的诱导,推测可能参与纤维素水解产物的结合过程,同时该SusC-like蛋白自身还起到转录因子的作用,调控下游基因甚至其他纤维素利用相关基因的表达。CHU1278具有明显的脂蛋白特征,它可能定位于外膜可能作为SusD-like蛋白参与纤维素水解产物的结合过程。五、通过高通量转录组测序,从全基因组水平上考察了野生型和chu₁276失活突变株在不同碳源上的转录差异,从而系统分析了C. hutchinsonii对不同碳源的响应,以及chu1276失活对各相关基因转录表达的影响。分别提取同样诱导条件下野生型和chu1276失活突变株T127的总RNA,分离mRNA后进行全序列测定,对测序得到的数据进行分析归类后,得到了以下结果：以葡萄糖或纤维素为碳源时,相比于无碳源条件,既有表现为上调也有表现为下调的基因,其中未知功能蛋白编码基因占很大一部分；通过GO和Pathway显著性富集分析,并没有发现参与碳水化合物降解和代谢的相关过程；野生型中,相比葡萄糖碳源,内切纤维素酶可以被无碳源诱导表达,并不是纤维素特异性诱导,而p-葡萄糖苷酶则为纤维素特异性诱导；chu₁276～chu₁279的表达为纤维素特异性诱导,且在突变株中其表达均下调,该基因座位在C. hutchinsonii降解纤维素的过程中可能发挥重要作用。更多还原

【Abstract】 With oil, coal and other non-renewable resources gradually depleting, exploring renewable energy utilization has become an urgent need. As the main component of plant biomass, cellulose is the most abundant renewable resource on earth. Degradation of crystalline cellulose is the key factor for lignocellulosic biomass conversion. In addition to the free cellulase system of fungi and complexed cellulosome of anaerobic bacteria, some aerobic bacteria such as Cytophaga hutchinsonii have a strong ability to degrade crystalline cellulose in a different manner. Genome sequences of C. hutchinsonii suggested that it do not have cellobiohydrolases and endoglucanases lack the cellulose binding domain, genes encoding typical motility organelles are also absent.Preliminary results indicated that C. hutchinsonii degrade crystalline cellulose depending on its contact to the subsrate, and the cellulase activities mostly located in the cell surface. Besides, the uptake and utilization of cello-oligosaccharide may constitute an important step of the cellulose degradation process. C. hutchinsonii can glide quickly along the surface of solid medium. Inactivation of the gliding motility gene gldJ led to the loss of cell motility as well as the ability of cellulose degradation. It was inferred that there may be a link between cellulose degradation and cell motility. Since cellulases are always difficult to be heterologously expressed with activities, it’s urgent to establish an efficient genetic manipulation system so that genes involved in cellulose degradation can be efficiently identified. This thesis explored the genetic manipulation tools of C.hutchinsoni and initially established the genetic manipulation system; on the basis of a lot of transposon screening, some cellulose-utilization deficient mutants were gained; according to the bioinformatic analysis, a hypothetical model of cellooligosaccharides transport by C. hutchinsonii was proposed. The main contents and results are as follows:1. Cultivation of C. hutchinsonii on solid medium was optimized and genetic manipulation system was developed preliminarily.In order to establish the genetic manipulation system of C. hutchinsonii, cultivation of strains on solid culture was been promoted at first and we found that the appropriate agar concentration for colony formation was0.6%. Tolerance of different antibiotics for wild-type strain was explored and results indicated that erythromycin, chloramphenicol or tetracycline can be used as resistance selection markers for C. hutchinsonii. A variety of genetic transformation methods had been tried. The efficiency of electroporation was found relatively higher and more stable than that of conjunction. Then a replicating plasmid carrying the replication origin from C. hutchinsonii genome and a transpon plasmid were successfully transported into C. hutchinsonii. It was verified that these resistance gene can work in the strain and the appropriate screening concentrations were determined. Then a complement plamid with chloramphenicol resistance gene was constructed. Some promoters which could promote GFP expression in C. hutchinsonii can be used to express many other proteins in vivo in future.2. Inactivation mutant Chin1107, Chin1280and Chinl335were constructed by targeted gene insertion and it was indicated that inactivation of any one of cellulase genes may not decrease the ability of cellulose degradation of C. hutchinsonii.A single cellulase may play different roles in different bacteria. As is known, C. hutchinsonii has a unique cellulose degradation mechnism, wherein in order to study whether a critical component of the noncomplexed cellulase system is existed, we constructed insertion inactivation mutant strains of three cellulase genes, respectively. The growth of Chin1107, Chin1280and Chin1335were similar to wild type strain, no matter glucose or avicel as the sole carbon source in liquid medium. The difference was also not apparent when cultivated on filter paper plate. Perhaps the lack of a single cellulase did not seriously affect the cellulose utilization of C. hutchinsonii. Outer membrane, inner membrane and cytoplasmic proteins were seperated from wild-type cells incubated in PY10supplied with different carbon sources. Among all the proteins, one was induced in cellobiose compared to glucose. By mass spectrometry, it was identified as Bg1X which is located in the periplasmic and annotated as β-glucosidase. The transcriptional of bglx gene was upregulated in cellulose cultures. The β-glucosidase Bg1X may play an important role in utilization of cellobiose and cellulose. 3. A mutant deficient in cellulose degradation was obtained by transposon mutagenesis screening. The ability of colony spreading and components of outer membrane proteins were both different between the mutant and wild type strains.A transposon mutant ChT68was obtained with the phenotype of nomal growth in liquid medium but weaker growth on solid medium with cellulose as the sole carbon source compared to the wild type. It was identified that the insertion position of transposon was chu₁719encoding a hypothetical protein. Targeted gene inactivation of chu₁719was achieved by electroporation of suicide vector pLYIN1719. The inactivation muant Chin1719exhibited same phenotypes with ChT68. Colony spread of Chin1719was deficient on hard agar plate. CMCase activity of membrane proteins was reduced by about40%than the wild type and cellulose adhesion proteins from outer membrane proteins of Chin1719and WT were significant different. Differential proteins included G1dJ, G1dN, CHU₀007, CHU₃434and CHU₃732. These results suggested that CHU₁719may play a role in cellulose utilization, spreading and protein secretion.4. Inactivation of chu₀546and chu₀553which were annotated as susC-like genes did not influence cellulose utilization of mutant strains. However, a predicted TonB-dependent receptor-encoding gene, chu₁276, was considered to play an important role in cellulose utilization by constituting the sus-like system with its downstream genes.As is known, a cell envelope-associated multiprotein system named Sus （starch utilization system） enables Bacteroides thetaiotaomicron to bind and degrade starch, of which SusC and SusD are essential components. Chin0546and Chin0553, of which susC-like genes chu₀546and chu₀553was inactivated respectively, grew normally on glucose and cellulse. T127, T423and C-34, the transpons mutants of chu₁276, chu₁277and chu₁278could not degrate cellulose. A quite long lag phase can be observed when T127was cultivated on liquid medium with0.1%glucose or cellobiose as the sole carbon source. Upstream genes chu₁265/chu₁266was also inactivated, but no differential phenotypes between mutants and WT were detected. CHU₁276was located in outer membrane and could respond to low concentration glucose. As a DNA-binding module was also predicted at its C-terminal, we speculated that CHU₁276was not only a SusC-like protein but also a transcription regulator. The transcription of some cellulases was down regulated in T127correspondingly. CHU₁278should be located to lipid bilayer of outer membrane to function normally.5. The gene expression differentiation was evaluated by transcriptome analysis between wild-type and inactivated mutant of chu₁276on different carbon sources. Several genes including those respond to cellulase inducing signals, involved in metabolic pathway and signal transduction were identified. These results supplied us with more details about the transcriptional regulation of cellulase in C. hutchinsonii.Total RNA of wild-type and T127were extracted from similarly induction conditions with different carbon sources. mRNA sequence were separated and sequenced. The data were analyzed and classified giving the following results:Compared to the non-carbon source condition, there were up-regulated genes and down-regulated genes both when cultured on glucose and cellulose. Among them, genes encoding hypothetical proteins accounting for a large part. Through GO and Pathway significant enrichment analysis, no processes involved in carbohydrate metabolism were figured out. In wild type strain, the most endoglucanases can be induced in non-carbon condition, but not specifically cellulose induced; while3β-glucosidases were specifically induced by cellulose. The expression of chu₁276～chu₁279was cellulose-specific induced, and their transcription levels in T127were down-regulated. So it is inferred that the chu₁276～chu₁279loci may play an important role in the cellulose degradation of C. hutchinsonii.更多还原