With High Ferrous Oxide Activity Of Thiobacillus Ferrooxidans Genetically Engineered Bacteria To Build

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.