Study On The Construction Of Engineering Acidiothiobacillus Thiooxidans And Its Metabolism Of Organic Compounds

Acidithiobacillus thiooxidans is a kind of chemolithotroph. It lacks several key enzyme activity involved in the heterotrophic pathway, such as the phosphofructokinase in Embden-Meyerhof pathway (EMP) and the a-oxoglutarate dehydrogenase in tricarboxylic acid cycle (TCA). Without special energy substrate it cannot utilize organic compounds to grow quickly, therefore the characteristic of its slow growth rate and cell yield not only increases the difficulty of its theoretical study, but also limits its application in the process of bioleaching, desufurizing of the coal and depleting the sufur in waste water.etc. To increase its ability of assimilating organic compounds, this bacteria could be constructed by introducing foreign genes encoding the key enzymes of heterotrophic pathway in order to utilize organic compounds for better growth.In the former study, two genes encoding the enzymes mentioned above were cloned from Escherichia coli, and then were transferred into the Acidithiobacillus thiooxidans separately. The ability of assimilating glucose of the engineering bacteria containing pfkA was improved to some extent, while the a-ketoglutarate dehydrogenase complex is composed of three subunits encoded by sucA, sucB and lpdA, respectively, and their expression was low in the engineering strain. In this study the expression of these genes in Acidithiobacillus thiooxidans 19377 was first detected by RT-PCR, SDS-PAGE, Western-blotting and enzyme activity detection. This time, all results indicated that the three genes encoding the a-ketoglutarate dehydrogenase complex could be translated into active protein. Meanwhile the promoter of sucA was substituted by the tac promoter, and the recombinant plasmid pJRD215-tac-sucAB-lpdA was constructed, which was then transferred into Acidithiobacillus thiooxidans 19377 by conjugation. Enzyme activity indicated that the tac promoter could indeed enhance the expression level fora-ketoglutarate dehydrogenase. Furthermore, engineering bacteria A. thiooxidans 19377(pJRD215 -pfkA-sucAB-lpdA) was constructed similarly.To measure whether the engineering bacteria constructed above could utilize organic matters better, several experiments were brought into effect. First, glucose and several organic acids in TCA, such as pyruvate, citrate, a-ketoglutarate, succinate and oxalacetate, were selected to be added into the culture separately to study their influences on the wild type Acidithiobacillus thiooxidans 19377. And several conclusions listed as follows were obtained:a) Organic compounds of low concentrations stimulated the growth as well as increased the cell yield of the bacteria with special substrate existed, while with the increasing of the concentration of the organic compounds, the growth of the bacteria was inhibited, its lag phase was prolonged obviously. b) Different organic compounds selected exhibited inhibitory effect on the wild type bacteria in different degrees. Citrate was observed to extend the lag phase of the bacteria slightly at a concentration of 100 mM, whereas pyruvate and oxaloacetate were inhibitory between 10-4 and 10-3 mM. c) The measured concentrations of organic compounds decreased with time, indicating uptake or assimilation by the cells, while the mechanism of the organic compounds entering into the cells was not clear. d) The cells could grow well in Starkey-S0 inorganic salt clture with the glucose concentration of 5 g/L, and the cell dry weight increased by 0.089 mg with 1 mg glucose consumption. It was speculated there was other glucose metabolism pathways, such as hexose monophosphate pathway (HMP) in bacteria. Besides, the increase of the cell yield rate indicted the glucose participated in the energy metabolism. And we also obtain the primitive inhibitory concentrations of different organic compounds selected. Based on this, glucose, pyruvate and a-ketoglutarate were chosen to study the growth characteristic of the engineering bacteria constructed previously. Compared to the wild type bacteria, the engineering bacteria grew better on the organic compounds, and the glucose contributed to the energy production partly. As for the ability of utilizing organic compounds, the bacteria containing sole enzyme gene was better than the one containing the two enzyme genes, one of the possible reasons of this may be that the increase of the recombinant plasmid aggravates the burden of the cells, causing inhibitory effect on the normal metabolism of the cells, the other may be that the genes interfered with each other during the expression process. This work provides experimental basis on clarifying the relationships of autotrophs and heterotrophs, as well as method of constructing engineering bacteria of better characteristic further.