Studies On The Expression Of E. Coli α-Ketoglutarate Dehydrogenase Complex Genes In The Chemolithotroph Acidithiobacillus Thiooxidans To Facilitate Its Metabolism Of Organic Compounds

Acidithiobacillus 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. It has been used widely in metal leaching from mineral ores, in the desulfurization of coal, and in the treatment of sulfur containing waste water and garbage. However, the slow growth rate and the low cell yield of this organism and its sensitivity to heavy metals have limited its further use. Enzymological research revealed that A. thiooxidans is deficient in some key enzymes of the EMP pathway and Krebs cycle, such as phosphofructokinase, andα-ketoglutarate dehydrogenase. So, these organisms could not respire organic substance adequately and obtain energy from them.α-ketoglutarate dehydrogenase complex is an important enzyme of the Krebs cycle. The purpose of this study is to introduce the cloned sucAB and lpdA genes for this enzyme into A. thiooxidans and to construct new A. thiooxidans gene engineering strain with the expressedα-ketoglutarate dehydrogenase activities and better growth rates on account of metabolism of organic compounds.In the former study, the two sucAB and lpdA gene fragments coding forα-ketoglutarate dehydrogenase complex, which contain its own promoter and ribosome binding site, had been amplified separately by the method of PCR using the genomic DNA of E. coli K12 as the template, and cloned together into plasmid pUC18. In this study, the sucAB and lpdA genes were first subcloned into the wide-host-range plasmid pJRD215 to construct the recombinant plasmid pJRD215-sucAB-lpdA. The agarose gel electrophoretic pattern of recombinant plasmid pJRD215-sucAB-lpdA digested by EcoR I and Hind III showed that pJRD215-sucAB-lpdA carried the heterogeneous 4.8 kb sucAB and 1.8 kb lpdA gene fragments. The specific active ofα-ketoglutarate dehydrogenase expressed by the sucAB and lpdA genes in recombinant plasmid pJRD215-sucAB-lpdA could be detected and the corresponding proteins were verified by the SDS-PAGE. By the way, both the genes for Km^r and Sm^r in pJRD215-sucAB-lpdA could be expressed in E. coli cells.The recombinant plasmid pJRD215- sucAB-lpdA was then transformed into E. coli SM10. Using E. coli SM10 (pJRD215-sucAB-lpdA) as the donors and wild type A. thiooxidans ATCC19377 as the recipients, the recombinant plasmid pJRD215-sucAB-lpdA could be mobilized into A. thiooxidans strains with the aid of tra gene on the genomic DNA of E. coli SM10. The transfer frequency of plasmid pJRD215-sucAB-lpdA from E. coli SM10 to A. thiooxidans was 2.4×10^-6 using Km as the selective marker. The presence of pJRD215-sucAB-lpdA in A. thiooxidans transconjugants were verified by plasmid isolation and transconjugants colony PCR.The specific activity ofα-ketoglutarate dehydrogenase was not detectable in the wild type A. thiooxidans ATCC19377, which revealed the natural deficient of this enzyme in A. thiooxidans strains. However, it was detectable in the gene engineering A. thiooxidans ATCC19377 harboring plasmid pJRD215- sucAB-lpdA, although at a very low level. The expression of sucAB and lpdA genes in A. thiooxidans might suggest that the promoter sequences of E. coli could be correctly recognized by the RNA polymerase of autotrophic bacterium. And these studies also implied that the gene expression system might be similarin E. coli and A. thiooxidans, though there were great physiological differences between them.The expression of sucAB and lpdA genes in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) was also detected at the level of transcription and translation. Agarose gel electrophoresis of RT-PCR products confirmed the transcriptions of sucB and lpdA genes. However, the enzyme activities measured in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) were unexpectedly low whether at the protein level or at the enzymatic activity level. This might indicate that there are some differences in the gene expression and control system between A. thiooxidans and E. coli. The stability of plasmid pJRD215-sucAB-lpdA in A. thiooxidans ATCC19377 was determined by checking for km and Sm resistance. About 71% of A. thiooxidans cells carried the recombinant plasmids after being cultured for 5 generations without selective pressure, which showed that pJRD215-sucAB-lpdA was maintained consistently in A thiooxidans.The growth of A. thiooxidans ATCC19377 (pJRD215- sucAB-lpdA) was also studied by adding different concentration of glucose, organic acids (α-ketoglutarate acid, citrate acid and pyruvate acid) in Starkey-S⁰ liquid medium. Glucose caused some stimulation on the cell growth of gene engineering strain, butα-ketoglutarate acid and citrate acid showed no effect on the growth of both the wild type and gene engineering strains under the studied concentration level. Pyruvate acid could inhibit the growth of both the wild type and gene engineering strains, but the inhibition could be partially relieved by the gene engineering strains. Although glucose could facilitate the cell growth of A. thiooxidans, but only under the supply of inorganic sulfur, which might indicate the absence of electron transfer system to produce ATP by the metabolism of organic compounds. So, only limited organic compounds could be metabolized by A. thiooxidans.In conclusion, the genes forα-ketoglutarate dehydrogenase complex were introduced into A. thiooxidans for the first time, and the sucAB and lpdA genes could be expressed in A. thiooxidans under its own promoter. This indicated that the heterotrophic complicated genes for the enzymes (α-ketoglutarate dehydrogenase complex) in the central metabolic pathway could be expressed and assembled in the extremely acidophilic, obligately chemolithoautotrophic A. thiooxidans. Though possessing low enzymatic activities, the constructed gene engineering strains could metabolize glucose more efficient and mitigate the inhibition caused by the metabolic intermediates such as pyruvate acid, and the faster growth strains constructed would be more useful for the application. Our work will not only open a way to investigate the phylogenetic relationship between heterotrophic and autotrophic bateria, but also provide a new insight into how to improve the growth rate and leaching performance of the obligately autotrophic bacteria.