具有高的亚铁氧化活性的氧化亚铁硫杆菌基因工程菌构建

【作者】 刘玮；

【导师】 林建强； 林建群；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans简称氧化亚铁硫杆菌)是一种在微生物冶金中起着重要作用细菌,也是目前研究的最多的浸矿细菌之一。氧化亚铁硫杆菌是一株极具代表性的极端嗜酸性严格自养细菌,它能在酸性条件将Fe2+氧化为Fe3+,或者将元素硫或还原性硫化物氧化为硫酸,并依靠这些无机物的氧化过程获得能量。该菌在细菌冶金、煤的脱硫、废水处理、污泥中重金属的浸出等方面发挥着重要作用。氧化亚铁硫杆菌能够酸性条件下将Fe2+氧化为Fe3+,而Fe3+作为一种强氧化剂,可以在酸性条件下将硫化矿物其他金属化合物氧化溶解,这是生物浸矿过程中至关重要的一步反应,因此,氧化亚铁硫杆菌氧化Fe2+的能力与其浸矿能力密切相关。对氧化亚铁硫杆菌Fe2+氧化能力的基因工程改造应基于对其氧化Fe2+途径的研究。嗜酸氧化亚铁硫杆菌氧化Fe2+主要是通过Fe2+电子传递系统。针对Fe2+电子传递系统,曾经提出过多种模型。目前具有最多实验证据支持也是最广为接受的电子传递途径模型为Fe2+→细胞色素蛋白Cyc2--+铜蓝蛋白Rusticyanin→细胞色素Cycl→aa3细胞色素c氧化酶→02。编码此电子传递链各组分蛋白的基因位于同一个操纵子内,该操纵子被命名为rus操纵子我们以广宿主范围IncQ族质粒pJRD215为基础,构建了含Ptac强启动子以及电子传递体蛋白编码基因的重组质粒pTRUS、pTCYC1以及pTCYC2,并将其成功导入了氧化亚铁硫杆菌ATCC19859,从而构建了氧化亚铁硫杆菌基因工程菌A.ferrooxidans(pTRUS)、A.ferrooxidans(pTCYC1)以及A.ferrooxidans(pTCYC2)。经Western-blotting验证,重组质粒上所携带的电子传递体编码基因在氧化亚铁硫杆菌中均得到了表达。为了检验以基因工程改良的方式来提高氧化亚铁硫杆菌Fe2+氧化活性的方式是否有效,我们检测了所构建的氧化亚铁硫杆菌基因工程菌的Fe2+氧化酶活性。实验结果表明,无论是休止细胞还是细胞提取液的检测结果,氧化亚铁硫杆菌基因工程菌A.ferrooxidans(pTRUS)、A.ferrooxidans(pTCYC1)的Fe2+氧化活性均有了一定程度的提高。在休止细胞中,氧化亚铁硫杆菌基因工程菌A.ferrooxidans(pTRUS)、A.ferrooxidans(pTCYC1)以及A.ferrooxidans(pTCYC2)的亚铁氧化酶活性为原始菌株ATCC19859相比,分别提高了19.85%、13.30%和7.68%。在细胞提取液中,相对于原始菌株氧化亚铁硫杆菌ATCC19859,基因工程菌A.ferrooxidans(pTRUS)和A.ferrooxidans(pTCYC1)的亚铁氧化活性分别提高了41.41%和38.57%；而基因工程菌A.ferrooxidans(pTCYC2)细胞提取液所测得的亚铁氧化酶活性与原始菌株区别不大。为了进一步探究基因工程菌Fe2+氧化活性的提高的原因,我们应用荧光定量PCR技术对基因工程中rus操纵子组成基因的表达水平进行了研究。结果表明,在基因工程菌4.ferrooxidans(pTRUS)和A.ferrooxidans(pTCYC1)中,除了质粒上所携带的目的基因的表达水平有提高以外,rus操纵子上的其它基因的表达水平也有一定的提高。通过对基因工程菌中rus操纵子基因表达水平的变化,初步对氧化亚铁硫杆菌Fe2+氧化电子传递系统限速步骤进行了推测,为氧化亚铁硫杆菌进一步的基因改良提供了理论指导。氧化亚铁硫杆菌在亚铁培养基中的Fe2+消耗情况及细菌生长情况检测结果显示,在Fe2+培养基中,氧化亚铁硫杆菌A.ferrooxidans(pTRUS)和A.ferrooxidans(pTCYC1)消耗Fe2+的速度相对于对照菌株有了明显的提高。尽管如此,基因工程菌与对照菌的生长曲线却并没有太大差异。更多还原

【Abstract】 Acidthiobacillus ferrooxidans, a gram-negative acidophilic chemolithoautotrophic bacterium, plays important role in bioleaching processes, is one of the thoroughly studied bacterium among the bioleaching microorganisms. It obtains energy mainly from the oxidation of ferrous iron (Fe2+) and reduced sulfur compounds. The biological oxidation of Fe2+ not simply served as an energy supply channel, the resulting product of ferric iron (Fe3+) is a powerful oxidant that can oxidize insoluble metallic sulfides to soluble form in acid media. Hence, the Fe2+ oxidizing activity of A. ferrooxidans strain will be directly related to its bioleaching activity.The electron chain oxidizing Fe2+ to Fe3+ in A. ferrooxidans has been studied in detail, and several models have been proposed. The currently accepted model proposed based on genetic and biochemical analyses suggests that electrons from ferrous iron oxdition first flow through the high-molecular weight c-type cytochrome Cyc2 to a periplasmic blue copper protein rusticyanin, and then electrons transport through the periplasmic dihemic c-type cytochrome Cyc1 and an aa3 type cytochrome oxidase towards O2 eventually. The proteins involved in the electron transfer chain described above are located in a single operon named rus operon.The wide-host-range plasmid pTRUS, pTCYC1 and pTCYC2 containing electron transfer proteins under the control of Ptac promoter was constructed and transferred into Acidithiobacillus ferrooxidans ATCC19859 using conjugation gene transfer method to generate the engineered strains of A. ferrooxidans(pTRUS), A. ferrooxidans(pTCYC1) and A. ferrooxidans(pTCYC2). Expression of the corresponding genes from plasmid pTRUS, pTCYCl and pTCYC2 in A. ferrooxidans was assessed by western blotting analysis. The results showed that the electron transfer proteins encoding genes fused with a 6×His tag gene was successfully expressed from plasmid pTRUS, pTCYCland pTCYC2 in A. ferrooxidans. Fe2+ oxidation activity of A. ferrooxidans(pTRUS), A. ferrooxidans(pTCYC1) and A. ferrooxidans(pTCYC2) was tested and compared with that of A. ferrooxidans ATCC 19859 and A. ferrooxidans (pJRD215). When tested with resting cells, the Fe2+ oxidation activities of Aferrooxidans(pTRUS), A. ferrooxidans(pTCYC1) and A. ferrooxidans(pTCYC2) increased by 19.85%、13.3% and 7.68% compared with A.ferrooxidans ATCC 19859. When tested with cell extracts, the Fe2+ oxidation activities of A. ferrooxidans(pTRUS) and A. ferrooxidans(pTCYC1) increased by 41.41% and 38.57% compared with A.ferrooxidans ATCC 19859, while the Fe2+ oxidation activity of A. ferrooxidans(pTCYC2) was similar to that of the A. ferrooxidans ATCC 19859.The levels of rus operon transcriptional products in A.ferrooxidans engineered strains were analyzed using real-time PCR. The results indicated that in A. ferrooxidans, the extra copies of electron transfer proteins encoding genes under the control of Ptac promoter significantly increased the mRNA levels of corresponding genes carried by introduced recombinant plasmids. In addition, the expression levels of other important genes in rus operon were also increased in A. ferrooxidans(pTRUS) and A. ferrooxidans(pTCYC1).In the batch cultures of A. ferrooxidans (pTRUS), A. ferrooxidans (pTCYC1) and A. ferrooxidans (pTCYC2), the decreases of Fe2+ concentrations were measured. A. ferrooxidans(pTRUS) and A. ferrooxidans(pTCYC1) oxidized Fe2+ more rapidly than the control of A. ferrooxidans ATCC 19859. In contrast, A. ferrooxidans(pTCYC2) had similar Fe2+ oxidizing curve with that of the control. The cell growths of A. ferrooxidans strains were determined and the results showed that the growth curves of the engineered strains had no remarkable difference compared with the control.更多还原