【作者】 于子超；

【导师】 陈秀兰； 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 假交替单胞菌广泛分布于海洋环境中,并且仅分布于海洋环境中。目前已从深海以及极地等众多海洋环境分离到假交替单胞菌。研究表明假交替单胞菌的适应机制和存活策略具有多样性和有效性,这使得它们能够广泛的生存于各种海洋环境中。假交替单胞菌是研究海洋微生物适应海洋特殊生境的重要模式菌,对假交替单胞菌环境适应机制的深入研究将有助于揭示生命适应不同海洋环境的能力。而且,由于其能产生很多活性物质和胞外酶类,假交替单胞菌也被认为是具有重要应用价值的一类细菌,本文首先以北极海冰假交替单胞菌为研究对象,首次从北极海冰假交替单胞菌中分离到丝状噬菌体,进而研究了丝状噬菌体对宿主菌在生长、耐受性及运动性等方面的影响及分子机制,揭示了丝状噬菌体介导的假交替单胞菌适应海冰环境的机制。然后建立了深海假交替单胞菌Pseudoalteromonas sp. SM9913的高效接合转移体系,并在此基础上建立了Ps. sp.SM9913基因敲除体系,为深入研究假交替单胞菌适应深海环境的遗传机制奠定基础；建立了北极海冰假交替单胞菌Ps. sp. SM20429的接合转移体系及适冷异源表达体系,成功表达了在大肠杆菌表达体系中无法成熟表达的适冷金属蛋白酶,为深入研究该酶的催化机制及实现适冷酶的大量表达奠定基础。一、丝状噬菌体介导的假交替单胞菌适应海冰环境的机制研究表明,假交替单胞菌广泛地分布在北极海冰中,是北极海冰中的优势菌株。然而对这类菌株如何适应海冰环境还没有深入的研究。有研究表明海冰中的噬菌体可以通过裂解宿主细菌从而调控微生物群落的结构以适应海冰环境。然而作为不裂解宿主的丝状噬菌体,是否在海冰环境中存在,若存在又是如何调控微生物群落还不得而知。本文在实验室前期从北极海冰Ps. sp. BSi20327中分离的质粒pSM327的基础上,通过对其序列的详细分析,认为pSM327应是丝状噬菌体在宿主胞内的复制形式(RF)。从Ps. sp. BSi20327的培养液中提取并观察到了丝状噬菌体。并进而研究了丝状噬菌体的基本性质和在北极海冰假交替单胞菌中的分布。深入研究了其对宿主产生的影响及机制。取得了以下结果：(1)北极海冰假交替单胞菌丝状噬菌体f327的发现、基本性质及其分布的研究从Ps. sp. BSi20327的胞外提取并观察到了丝状噬菌体,将其命名为f327。f327呈细丝状,长约1.5μm,宽约14nm。详细分析了其基因组结构,f327基因组为单链DNA,全长包含6114个核苷酸,存在9个ORF。这些ORF编码的蛋白在序列相似性和排列的顺序上都与已发现的丝状噬菌体很相似。按编码的蛋白的功能划分,可将这9个ORF划分成四个模块：复制模块、结构模块、组装模块和调控模块。利用PCR扩增的方法,检测了f327在北极海冰假交替单胞菌中的分布情况,结果表明36%的菌株含有f327或其高度同源的丝状噬菌体。通过构建进化树以及DNA-DNA杂交分析,发现含丝状噬菌体的宿主属于假交替单胞菌的同一个种,说明f327或其高度同源的噬菌体广泛地分别于同一个种的假交替单胞菌中。如此广泛的分布说明f327可能会对宿主假交替单胞菌群落产生一定的影响,进而调控假交替单胞菌群落适应海冰环境的变化。(2)丝状噬菌体f327的生态学功能及其影响宿主假交替单胞菌的分子机制研究为了研究f327对宿主的影响,通过高温培养的方法消除了f327在宿主胞内的复制形式RF327,将无RF327的菌株命名为Ps. sp. BSi20327A。在Ps. sp. BSi20327A中检测不到单链的丝状噬菌体的存在,其噬菌体基因的转录水平仅为Ps. sp. BSi20327的3%-7%。通过比较Ps. sp. BSi20327与Ps. sp. BSi20327A在生长速率、群落密度、耐受性和运动性方面的不同,发现f327可以减慢宿主的生长速率、降低宿主群落密度、削弱宿主对高盐和H202的耐受性,但是可以增强宿主的运动能力。对Ps. sp. BSi20327与Ps. sp. BSi20327A在不同盐度培养下的转录组分析,揭示了f327影响宿主的分子机制。当在3%的NaCl浓度下,f327存在的宿主Ps.sp. BSi20327中除了噬菌体的基因表达上调外,宿主染色体上的噬菌体休克蛋白基因(Psp)、与鞭毛组装相关的基因和细菌趋化性基因表达上调。而噬菌体的表达组装以及鞭毛的组装都是耗能的过程,所以这两个过程耗能的增多导致Ps. sp. BSi20327在3%的NaCl下生长速度减慢。在8%的NaCl浓度下除了上述基因在Ps. sp. BSi20327中表达上调外,与表达TCA循环中关键酶有关的基因在Ps. sp. BSi20327中表达下调,从而导致了Ps. sp. BSi20327产能的减少。此外转录延伸因子基因和核糖体蛋白基因在Ps. sp. BSi20327表达下调,这使得Ps. sp.BSi20327中基因转录和翻译的速率降低。因此在8%的NaCl下f327大量表达不仅消耗更多的能量而且还影响宿主能量的产生和基因的转录与翻译,从而导致在8%的NaCl下Ps. sp. BSi20327的生长速率大幅降低。细菌通常会通过从外界吸收或自身合成相容性溶质(包括谷氨酸、脯氨酸及糖类等)来抵抗外界的高渗环境,当NaCl浓度由3%升高到8%时,与氨基酸运输有关的基因以及参与谷氨酸和脯氨酸合成的基因在Ps. sp. BSi20327和Ps. sp. BS120327A中都上调表达,但是Ps. sp. BSi20327中上调表达的幅度更大,尤其是在8%的NaCl下的表达量要高于在Ps. sp. BSi20327A中的表达量。因为这些基因与相容性溶质的积累和抵抗外界高渗环境有关,所以由以上结果可以推断,8%的NaCl对Ps. sp.BSi20327产生的高渗压力要比对Ps. sp. BSi20327A产生的压力大。因此Ps. sp. BSi20327需要积累更多的相容性溶质来抵抗外界的高渗环境。这从另一个侧面说明f327的大量表达导致了Ps. sp. BSi20327耐盐性的降低。而f327的存在使得宿主中与鞭毛合成相关的基因以及趋化性基因的表达上调,使得宿主的运动性提高。根据以上结果,我们提出了f327调控假交替单胞菌宿主群落以适应不同季节海冰环境变化的模型：在冬季,由于低温和极夜,海冰中的细菌面对着高盐和缺乏的营养。,f327通过降低宿主生长速度和高盐耐受性而减小宿主细菌群落的规模从而减少营养的消耗；与此同时,f327可以提高宿主的运动能力,帮助宿主更快的到达适合其生长的环境;在夏季,随着温度的升高、海冰的融化,虽然海冰中的盐度降低,但是由于极昼长时间的日照,海冰中的细菌面对着高浓度的H2O2和丰富的营养。f327通过减低宿主群落密度和H2O2的耐受性,避免宿主群落在营养物质非常丰富时过度地繁殖,从而对生态系统产生破坏。因此,虽然丝状噬菌体不裂解宿主,但是可以温和地调控宿主群落,使其适应北极海冰环境的季节性变化。我们的研究结果首次揭示了海冰丝状噬菌体的生态功能及其介导的假交替单胞菌适应海冰环境的机制。二、Pseudoalteromonas sp. SM9913基因敲除体系的建立深海细菌Ps. sp. SM9913分离自1815m的深海沉积物,是一株适冷菌。通过对Ps. sp. SM9913的全基因组测序,从基因水平上发现它含有其它环境中的假交替单胞菌没有的一些特性,可能与其适应深海沉积物环境有关。然而由于遗传操作工具的缺乏,极大地限制了研究该菌适应深海环境的遗传机制。虽然实验室前期工作建立了假交替单胞菌电转化体系,但由于其转化Ps. sp. SM9913的效率极低,无法进行下游的遗传操作。为此本文首先建立了Ps. sp. SM9913高效率转化体系,并在此基础上建立Ps. sp. SM9913基因敲除体系,实现在Ps. sp. SM9913中进行无标记基因敲除。取得以下结果：(1) Ps. sp. SM9913接合转移体系的建立检测了Ps. sp. SM9913对不同抗生素的敏感性,发现其对红霉素具有较高的敏感性,因此将红霉素抗性基因作为在Ps. sp. SM9913中进行遗传操作的筛选标记。实验室前期从北极海冰Ps. sp. BSi20429中筛选到一个内源质粒pSM429,并对其功能区进行了研究。在此基础上,以pGEM-T easy载体为骨架,先后连入pSM429上与复制有关的最小功能区、接合转移起始序列和红霉素抗性基因,构建成能进行接合转移的Pseudoalteromonas-Escherichia coli穿梭载体pOriT-4EM。利用pOriT-4EM,以E. coli ET12567(pUZ8002)为供体菌,通过优化接合转移条件,建立了Ps. sp. SM9913高效接合转移体系,其接合转移效率为1.8×10-3。为在Ps. sp. SM9913中进行遗传操作奠定基础。(2) Ps. sp. SM9913基因敲除体系的建立以Ps. sp. SM9913多糖合成基因簇中的糖基转移酶基因epsT为靶基因,构建基因敲除体系。选择epsT基因上下游各700-800bp的DNA片段作为同源臂,以红霉素抗性基因作为正向筛选标记,以果聚糖蔗糖酶基因(sacB)为负向筛选标记,通过改造pOriT-4EM,构建了进行epsT基因敲除的自杀载体pMT。利用接合转移将pMT转入Ps. sp. SM9913中,经过两次同源重组,筛选到了epsT基因的缺失突变株,该突变株的胞外多糖产量比野生菌下降了73%左右。在epsT基因敲除过程中,没有在Ps. sp. SM9913染色体上引入任何筛选标记,因此可以利用相同的筛选标记在Ps. sp. SM9913中进行多次基因敲除。并利用该方法敲除了Ps. sp. SM9913的多个基因。这为深入研究深海微生物的环境适应机制提供了技术手段。三、假交替单胞菌适冷异源表达体系的建立随着适冷微生物的不断发现和研究,由其产生的适冷酶因其巨大的研究价值和应用潜力而引起越来越多的关注。然而由于适冷酶的热稳定性差,在高温表达时容易发生错误折叠而形成没有活性的蛋白,所以利用目前最常用的中温大肠杆菌表达体系表达适冷酶时常常得不到有活性的成熟酶,仅以包涵体的形式存在。虽然可以通过降低大肠杆菌的生长温度来提高适冷酶的表达效率,但这大大延长了表达所用的时间。因此发展以适冷菌为宿主的表达体系,将对适冷酶的理论研究和实际应用具有重要意义。实验室前期工作将北极海冰细菌Ps. sp. BSi20429中的质粒pSM429消除,得到了质粒消除菌株Ps. sp. SM20429。并以Ps. sp.SM20429为宿主,利用大肠杆菌lac启动子初步构建了一个表达体系,但其表达温度为25-30℃,无法实现蛋白的适冷表达。因此本文通过构建报告载体筛选低温下的强启动子,并进一步构建表达载体,以Ps. sp. SM20429为宿主进行适冷酶的低温表达。取得以下结果：(1) Ps. sp. SM20429接合转移体系和报告载体的构建及低温启动子的筛选用氯霉素抗性基因替换了pOriT-4EM的红霉素抗性基因,获得质粒pOriT-4CM。在构建的Ps. sp. SM9913接合转移体系的基础上,以Ps. sp. SM20429为受体菌,建立了Ps. sp. SM20429接合转移体系,其接合转移效率约为4×10-3。将红霉素抗性基因和来自北极海冰Ps. sp. BSw20308的适冷纤维素酶基因作为报告基因,利用Ps. sp. BSi20429的冷休克蛋白、热休克蛋白和木聚糖酶基因的启动子,在pOriT-4CM的基础上构建了一系列报告载体。将报告载体转入Ps. sp. SM20429,通过检测转化子的红霉素抗性发现只有含冷休克蛋白基因和木聚糖酶基因启动子的报告载体表达了红霉素抗性基因。通过纤维素酶的活性的比较,发现冷休克蛋白基因和木聚糖酶基因启动子都能够调控纤维素酶基因在5-15℃表达,而木聚糖酶基因的启动子在10-15℃具有更高的强度,更适合表达载体的构建。因此选择木聚糖酶启动子,进一步完善构建适冷表达体系。(2)适冷表达体系的建立和适冷酶的表达实验室前期研究表明,蛋白酶pseudoalterin是南海沉积物细菌Ps. sp. CF6-2所产的主要胞外蛋白酶,该蛋白酶是一个新型的M23家族的蛋白酶,为适冷酶,其最适酶活温度为25℃,并且具有很差的热稳定性。该酶具有很高的弹性蛋白降解活性,是一个弹性蛋白酶。由于该酶的成熟过程不是自成熟,其成熟过程需要其它酶的参与,该酶无法在大肠杆菌中异源表达得到成熟酶的形式。以pOriT-4CM为骨架,连入木聚糖酶基因的启动子,并在其下游分别连入多克隆位点和His-tag,构建表达载体pEV。通过克隆pseudoalterin的基因到pEV中木聚糖酶基因启动子的下游,构建了pseudoalterin的表达载体。将该表达载体转入Ps. sp. SM20429中,利用燕麦木聚糖作为诱导剂,通过优化诱导剂的浓度和诱导温度,表达出了成熟的pseudoalterin,并利用其携带的His-tag,通过亲和层析纯化了pseudoalterin,并进行了N端测序,其序列与野生菌所产的pseudoalterin一致。表达量达到16-20U/ml,比活252±13U/mg。利用构建的适冷表达系统,还成功表达并纯化了增强型绿色荧光蛋白(EGFP)和两个来自Ps. sp. SM9913的与胞外多糖合成相关的适冷酶,N-乙酰葡糖胺差向异构酶和N-乙酰-D-甘露糖胺脱氢酶。该表达体系的建立为深入研究某些适冷酶的催化机制以及适冷酶的大量制备奠定了基础。更多还原

【Abstract】 Members of the Pseudoalteromonas genus are typical marine bacteria. So far, all of the reported species of the Pseudoalteromonas genus are isolated from the marine environment, including deep sea and polar zones. Many studies suggest that Pseudoalteromonas strains have developed multiple adaptation mechanisms for the extreme environments, which make these strains widely distribute in various marine environment. Pseudoalteromonas strains are important models for the investigation of the mechanisms by which marine microorganisms are adapted to the marine environments. A further study of the environmental adaptation mechanisms of Pseudoalteromonas will be helpful to our understanding of how microorganisms are adapted to and evolve in the marine environments. Many Pseudoalteromonas strains can secrete bioactive substances and extracellular enzymes and thus may have potential applications in industry. In the thesis, Pseudoalteromonas strains from Arctic sea ice were studied to gain insights into their environment adaptation mechanism. Firstly, a filamentous phage was isolated from Arctic sea ice Pseudoalteromonas and its effects on the growth, stress tolerance and motility of the host were studied and a filamentous phage-mediated mechanism for the survival of Pseudoalteromonas in sea ice was proposed. Next, a conjugal transfer system with high efficiency was constructed in Ps. sp. SM9913and based on this conjugal transfer system, a gene knockout system was constructed in Ps. sp. SM9913. Lastly, a conjugal transfer system for Arctic sea ice Pseudoalteromonas sp. SM20429was constructed and a cold-adapted expression system was developed using Ps. sp. SM20429as the host. With this expression system, a cold-adapted metalloprotease that cannot be maturely expressed in Escherichia coli expression system was successfully expressed and purified using the cold-adapted expression system.Ⅰ. Environmental adaptation mechanisms of Pseudoalteromonas mediated by filamentous phage in sea icePseudoalteromonas is one of the predominant culturable bacterial groups within the Arctic sea ice ecosystem. Many studies show that the host mortality and adjusting of microbial community structure in polar areas are regulated by lytic viruses. However, the ecological role of filamentous phages that cannot split the host in sea ice is still unknown. In the dissertation, sequence analysis of plasmid pSM327, a plasmid previously isolated from Arctic sea ice strain Pseudoalteromonas sp. BSi20327, indicated that, pSM327may be the replicative form (RF) of a filamentous phage. In the following study, a filamentous phage was isolated from Ps. sp BSi20327culture. The basic characteristatics of the filamentous phage and their distribution in the Arctic sea-ice Pseudoalteromonas were studied. Furthermore, the ecological role of this filamentous phage was revealed.(1) Discovery of a filamentous phage from Arctic sea ice Pseudoalteromonas and its basic characteristics and distributionA filamentous phage named f327was extracted from the culture of Ps. sp. BSi20327and observed by electron microscope. f327exhibits a filament-like structure, with14nm in width and approximately1.5μm in length, agreeing well with the morphological characteristics shared by filamentous phages. The genomic type of O27is single-stranded DNA (ssDNA) and its genome contains6114nucleotides. Genome of f327is composed of9open reading frames (ORFs). Sequence analysis reveals that each pSM327ORF shows the greatest homology to the ORFs at the corresponding positions in the well-characterized filamentous phages. The genes of f327can be classified into4functional modules, replication, structure, assembly and regulation. The distribution of this filamentous phage in Arctic sea ice Pseudoalteromonas strains was investigated by PCR amplification. The results indicated that36%of the Pseudoalteromonas strains (19/53) contain f327or f327-like genes. Phylogenetic tree based on the rpoD gene and DNA-DNA hybridization analysis indicated that all these phage-containing strains belong to the same species of Pseudoalteromonas.(2) Ecological role of the filamentous phage and the molecular mechanism of f327affecting the hostTo investigate the role of f327in the sea ice ecosystem, an RF327-cured derivative of Ps. sp. BSi20327was obtained and named Ps. sp. BSi20327A. No filamentous phage ssDNA was detected in Ps. sp. BSi20327A and comparative transcriptomic analysis showed that the transcription level of RF327in Ps. sp. BSi20327A is3%-7%of those in Ps. sp. BSi20327. Comparison of Ps. sp. BSi20327and Ps. sp. BSi20327A showed that f327can reduce the host growth rate, lower the cell density of the host community, impair the host tolerance against NaCl and H2O2and increase the motility of the host.To determine the mechanism underlying how f327affects its host BSi20327, the whole transcriptomes of BSi20327and BSi20327A grown in different concentrations of NaCl were sequenced and compared. The results showed that, at3%NaCl, only genes encoded by phage f327and related to phage shock, flagellar assembly and bacterial chemotaxis were up-regulated in Ps. sp. BSi20327. As the processes of phage and flagella assembly are energy-intensive, the reduction in the growth rate of Ps. sp. BSi20327in3%NaCl may be mainly due to these two energy-intensive processes. At8%NaCl, in addition to those up-regulated at3%NaCl, genes encoding key enzymes in the TCA cycle are down-regulated in Ps. sp. BSi20327, which would result in a reduction in energy production. Genes related to transcription elongation factor and ribosome assembly are also down-regulated in Ps. sp. BSi20327, which likely decreases the rate of gene transcription and translation. Therefore, at8%NaCl, over-expression of f327in Ps. sp. BSi20327not only consumes energy but also affects energy production and gene transcription and translation of the host, which will severely reduce the host growth rate. Bacteria usually respond to hyperosmolarity by accumulating compatible solutes, such as some amino acids (glutamate and proline) and sugars, by absorbing or synthesizing them. Based on transcriptome analysis, genes involved in amino acid transport and glutamate and proline synthesis, are up-regulated in both Ps. sp. BSi20327and Ps. sp. BSi20327A in3-8%NaCl. However, these genes are up-regulated to a greater extent in Ps. sp. BSi20327compared to Ps. sp. BSi20327A, especially at8%NaCl. Because these genes are related to the accumulation of compatible solutes to cope with the hyperosmotic environment, this result suggests that8%NaCl is more stressful to Ps. sp. BSi20327than that to Ps. sp. BSi20327A. Therefore, Ps. sp. BSi20327needs to accumulate more compatible solutes to resist the hyperosmolarity. Due to existence of O27, genes related to flagella assembly and bacterial chemotaxis are up-regulated in Ps. sp. BSi20327, which enhance the motility of the host.At last, the model of how the filamentous phage regulates the host community to improve the survival of the host in Arctic sea ice was proposed. In winter, the bacteria in sea ice are confronted with high salinity and nutrient deficiency due to the low temperatures and long polar nights. The reduction in the growth rate and NaCl tolerance of Ps. sp. BSi20327caused by f327would diminish the community scale of Ps. sp. BSi20327and thereby reduce nutrient consumption; in the meantime, motility enhancement caused by f327can help Ps. sp. BSi20327access environments suitable for growth. These regulatory effects of phage f327on the host cells are helpful for the survival of the host community during the long nutrient-deficient polar night winter days. In summer, although the salinity in the sea ice decreases as temperature increases, the sea ice bacteria are faced with rich nutrients and a high concentration of H2O2due to the polar day and intense sunlight. Owing to the presence of f327, the H2O2tolerance of Ps. sp. BSi20327decreases, which would lead to a high mortality rate of Ps. sp. BSi20327due to the high concentration of H2O2. In addition, the presence of f327also reduces the cell density of the Ps. sp. BSi20327community. These mechanisms may help avoid the over-blooming of BSi20327during the months of round-the-clock sunlight and the nutrient-rich summer.II. Construction of the gene knockout system in Pseudoalteromonas sp. SM9913Pseudoalteromonas sp. SM9913, isolated from the deep-sea sediment at1855m, is a psychrophilic strain. The genome of Ps. sp. SM9913has been sequenced and some specific features of how Ps. sp. SM9913adapts to the deep-sea environment have been revealed, which are different from the Pseudoalteromonas living in other environments. However, due to the lack of a genetic system in Ps. sp. SM9913, it is difficult to verify the predicted features by experiments. Therefore, it is necessary to develop a genetic system for the reverse genetics in Ps. sp. SM9913to investigate the genetic mechanism of how the bacterium adapts to the deep-sea sedimentary environment. Although an electroporation genetic transformation system was constructed in Ps. sp. SM9913in our previous work, due to its very low transformation efficiency, the downstream genetic manipulation cannot be performed. In the dissertation, a conjugal transfer system with high efficiency was constructed. Based on this conjugal transfer system, a gene knockout system was constructed.(1) Construction of the conjugal transfer systemThe antibiotic sensitivity of Pseudoalteromonas sp. SM9913was characterized and the erythromycin resistance gene was chosen as the selection marker. In the previous work, pSM429(a plasmid from the sea-ice bacterium Pseudoalteromonas sp. BSi20429) was screened and its functional region was studied. The minimal region for replication of pSM429, the conjugative transfer initiation origin and erythromycin resistance gene were introduced into the bone vector pGEM-T easy vector, respectively. The resultant shuttle plasmid, pOriT-4EM, can replicate in both Pseudoalteromonas and Escherichia coli. Afterwards, using E. coli ET12567(pUZ8002) as the donor, pOriT-4EM was transferred into Ps. sp. SM9913by conjugation. A conjugal transfer system with high efficiency (1.8x10-3) was constructed after optimizing the mating conditions, which lays the foundation of genetic manipulation in this strain.(2) Construction of the gene knockout system in Ps. sp. SM9913The epsT gene which encodes the UDP-glucose lipid carrier transferase was selected as the target gene for gene inactivation by in-frame deletion. Two homologous fragments of about800bp were selected from the flanking sequences of epsT gene. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and levansucrase (sacB) gene was the counter-selection marker. The epsT gene was in-frame deleted with a two-step integration-segregation strategy after transferring the suicide vector pMT into Ps. sp. SM9913. The obtained△epsT mutant showed approximately73%decrease in the yield of exopolysaccharides. No selection marker was introduced in the chromosome, which makes it possible to knockout several genes in the same host. Thus, a system for gene knockout in Ps. sp. SM9913was successfully constructed, which is helpful for further investigating the molecular mechanism of Pseudoalteromonas adapted to the deep-sea environment.III. Construction of the cold-adapted heterologous expression system for PseudoalteromonasAlong with the discovery and study of cold adapted microbes, the cold-adapted enzymes secreted by them have drawn much attention due to their great values in fundamental research and potentials in application. However, expression of genes of cold-adapted enzymes in the mostly used mesophilic E. colt host often results in the enzymatically inactive inclusion bodies. The reason for the inactivity is misfolding of the polypeptide chains when they are expressed in a common E. coli strain at37℃. Although lowering the induction temperature may improve the expression of some cold-adapted proteins, the expression time has to be prolonged at low temperatures. Therefore, development of the expression system which uses cold-adapted microbe as the host will be helpful for both the fundamental research and industrial applications of the cold-adapted enzymes. In our previous work, an expression system was preliminarily developed using Ps. sp. SM20429(a plasmid-cured strain of Ps. sp. BSi20429) as the host, but proteins must be expressed at25-30℃in that system. In the dissertation, strong promoter of Ps. sp. BSi20429was screened by construction of the reporter plasmid, and expression vector was constructed. Three cold-adapted enzymes were successfully expressed in Ps. sp. BSi20429at low temperatures.(1) Construction of the conjugal transfer system and screening of the strong promoter at low temperatures by construction of reporter plasmidsThe erythromycin resistance gene in the shuttle vector pOriT-4EM was replaced by the chloromycetin resistance gene and the resultant plasmid was named as pOriT-4CM. Based on the Ps. sp. SM9913conjugal transfer system, the Ps. sp. SM20429transfer system was constructed using Ps. sp. SM20429as the recipient strain and an efficiency of4x10-3was obtained. A series of reporter plasmids were constructed using the promoter of the xylanase gene, cold shock protein gene (CspA) and heat shock protein gene (dnaK) from Ps. sp. BSi20429and the erythromycin resistance gene and the gene encoding the catalytic domain and the signal peptide of the cold-adapted cellulase Cel308from the Arctic sea ice strain Ps. sp. BSw20308as the reporter gene. The reporter plasmids were transferred into Ps. sp. SM20429by conjugation. The erythromycin resistance and cellulase activity were tested to compare the strength of the promoters. The results showed that the erythromycin resistance gene was expressed under the control of the promoter of the xylanase gene and CspA gene. The cellulase was also expressed under the control of these two promoters at5-15℃, but the promoter of the xylanase gene is stronger than the CspA promoter at10-15℃. Therefore, the promoter of the xylanase gene was chosen for construction of the protein expression system in SM20429at low temperatures. (2) Construction of a cold-adapted expression system and recombinant expression of cold-adapted enzymesPseudoalterin, a novel M23A subfamily protease secreted by Psendoalteromonas sp. CF6-2from deep-sea sediment, is a cold-adapted enzyme with low optimal temperature (25℃) and high thermolability. However, because it cannot mature by autoprocess, pseudoalterin is unable to be expressed as a mature form in E. coli, which severely impedes the further study of its structure and function.A DNA fragment containing the promoter of the xylanase gene, multiple cloning sites and6repeated CAC (for His tag) was synthesized and introduced into the bone vector pOriT-4CM to construct the expression vector, pEV. Pseudoalterin gene was cloned into pEV to construct the expression plasmid for pseudoalterin. After transferring the expression plasmid into Ps. sp. SM20429, pseudoalterin was expressed using spelt xylan as the inducer. By optimizing the inducer concentration and induction temperature, active pseudoalterin was produced and purified from the culture by Ni affinity chromatography. N-terminal sequencing indicated that the recombinant pseudoalterin has the same N-terminal sequence as the native enzyme purified from the culture of the wild strain. The yield of the recombinant pseudoalterin was16-20U/ml with the specific activity of252+13U/mg. To detect the applicability of the expression system, the enhanced green fluorescent protein (EGFP) and another two cold-adapted proteins from Pseudoalteromonas sp. SM9913(UDP-N-acetylglucosamine2-epimerase and UDP-N-acetyl-mannosamine dehydrogenase) were expressed and purified. The development of this expression system is helpful for the further study of pseudoalterin and can be used for the expression of other cold-adapted proteins that are intractable for the E. coli expression system.更多还原