论文标题:氧化硫硫杆菌基因工程菌的构建及其葡萄糖代谢途径的研究
Construction of Bioengineering Bacteria Tt-12(pSDK-1) and the Study of It"s Glucose Metabolism Pathways
论文作者
论文导师 包振民；胡景杰,论文学位 硕士,论文专业 生态学
论文单位 中国海洋大学,点击次数 84,论文页数 74页File Size1385K
2006-04-01论文网 http://www.lw23.com/lunwen_49235957/
Thiobacillus thiooxidans; conjugative transfer; NMR; metabolism pathway
氧化硫硫杆菌是细菌冶金的一种重要细菌,是一种具有代表性的极端嗜酸性严格自养细菌,它能氧化元素硫和还原性的硫化合物,以CO_2作为主要碳源而生长,是一种严格好氧的专性自养化能无机营养细菌。 氧化硫硫杆菌缺乏EMP、ED途径以及TCA循环中的关键酶—磷酸果糖激酶、6-磷酸葡萄糖酸脱水酶、特征性酶2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶以及α-酮戊二酸脱氢酶等不能以有机质作为能源和碳源。只能通过固定CO_2获得碳源,从无机物的氧化获得能源,而无机物的氧化还原电位较高,可供这些细菌生长利用的能量很少,所以这类细菌往往生长缓慢,代时长,细胞得率低。这些因素直接影响了该菌的实际应用。这就需要用遗传学的方法改良这些菌种,使之适合于实际应用的要求。本研究的主要内容如下: 第一部分内容为利用接合转移的方法构建能够利用葡萄糖的氧化硫硫杆菌基因工程菌。pJRD215质粒载体及携带外源磷酸果糖激酶的重组质粒pSDK-1具有广泛寄主范围,可以在转移性质粒的诱动下进行转移。E.coli SM10菌株染色体上整合有RP4质粒的tra基因,也可推动含有mob位点的非转移性质粒进行接合转移。以SM10(pSDK-1)为供体菌,野生型氧化硫硫杆菌Tt-12作为受体菌进行了接合转移,获得了含有外源磷酸果糖激酶基因的氧化硫硫杆菌基因工程菌Tt-12(pSDK-1)。我们对质粒的稳定性进行了测定,结果表明,在无选择压力条件下连续传代50次,重组质粒pSDK-1在氧化硫硫杆菌Tt-12中的保存率仍可达到74%,说明质粒在氧化硫硫杆菌中比较稳定。对所构建的氧化硫硫杆菌基因工程菌在含有葡萄糖的Starkey-S0液体培养基中的生长状况以及工程菌对葡萄糖的利用情况等方面进行了研究。结果表明,对照野生型氧化硫硫杆菌菌株不能利用培养液中的葡萄糖进行生长,而氧化硫硫杆菌基因工程菌Tt-12(pSDK-1)却可利用葡萄糖,并随着葡萄糖浓度的增加其生长量逐渐增加。 为了研究外源基因的导入对氧化硫硫杆菌的一些主要代谢途径究竟有什么样的
Thiobacillus thiooxidans is a gram-negative, extremely acidophilic obligately autotrophic bacterium, which can obtain energy from the chemolithotrophic oxidation of inorganic sulphur and its compounds and use this energy to support autotrophic growth on carbon dioxide. Thiobacillus thiooxidans is of great importance in bioming. Enzymological research revealed that Thiobacillus thiooxidans is deficient in some key enzymes of the EMP, ED pathways and Krebs cycle, such as phosphofructokinase, 6-phosphogluconate dehydrase, andα-ketoglutarate dehydrogenase activities. So, these organisms could not respire organic substance and obtain energy from them and can only obtain energy from the chemolithotrophic oxidation of inorganic sulphur and its compounds and use this energy to support autotrophic growth on carbon dioxide. Therefore, the slow growth rate and the low cell yield of this organism and its sensitivity to heavy metals has limited its further use. Using E. coli SM10 (pSDK-1) as the donors and T. thiooxidans Tt-12 as the recipients, the plasmid pSDK-1 could be mobilized into T. thiooxidans strains with the aid of tra gene on the genomic DNA of E. coli SM10. The stability of plasmid pSDK-1 in Tt-12 was tested. About 74% of T. thiooxidans cells carried the recombinant plasmids after being cultured for 50 generations without selective pressure. Glucose caused a significant stimulation on the cell growth of the transconjugants, but had no effect on the wild type of T. thiooxidans. Since the fixation of CO2 has a high energy requirement, synthesis of a part of the cell material from glucose instead of CO2 should have an energy sparing effect, which should lead to an increase in cell yield. Result showed that the concentration of glucose in the growth medium did not change for Tt-12 strain but decreased gradually for Tt-12(pSDK-1) transconjugants during the cultivation, however the level of glucose decreased very slowly. 13C NMR and 31P NMR is used to study the metabolism pathways of glucose in Tt-12(pSDK-1). Result showed that the activity of the phosphofructokinase-1 was
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