Effects Of Sulfate On Desulfurization Metabolism Of Rhodococcus Sp. LY822, Cloning And Expression Of Its Desulfurization Genes

In this thesis, the effects of sulfate on desulfurization characteristics of Rhodococcus sp. LY822, and the cloning and expression of its dsz genes were investigated. The results preliminary clarified the biodesulfurization metabolism and verified the sulfur-starvation-induced mechanism of desulfurization enzyme formation in Rhodococcus sp. LY822.1. Effects of sulfate on Rhodococcus sp. LY822 growth and desulfurization were studied. Though Na₂SO₄ ranged from 0 to 0.4 mmol/L did not affect the growth of LY822, the desulfurization activity of LY822 was repressed. LY822 did not convert dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) in the presence of 0.03 mmol/L Na₂SO₄ in 48 hours. However, DBT was converted into 2-HBP when the concentration of Na₂SO₄ was below 0.01 mmol/L. Na₂SO₄ did not affect the desulfurization activity of LY822 cell-free extracts with desulfurization enzyme. The results indicated that the synthesis of desulfurization enzyme was repressed by sodium sulfate above 0.03 mmol/L, and was induced by sodium sulfate below 0.01 mmol/L. These data verified sulfur-starvation-induced mechanism of desulfurization enzyme.2. dsz promoter was cloned, herein dsz promoter-gfp report gene expression plasmid pBSG was constructed and transformed into the original bacteria (LY822). The results of laser scanning confocal microscopy and fluorescence spectrophotometer test showed that the fluorescence intensity of cell-free extracts without Na₂SO₄ from recombinant strains was three times of that with 0.1 mmol/L Na₂SO₄, veryfying the expression of dsz genes were repressed by sulfate. Western Blotting results showed that three specific bands of hybridization were generated in the medium with 0.2 mmol/L DBT and no specific band was obtained in the medium with Na₂SO₄, giving a further evidence of the repression of sulfate to desulfurization enzyme formation. Then taking total RNA of LY822 as templates for RT-PCR, cDNA fragments corresponding to dszA, dszB and dszC fragments were obtained in the medium with DBT, and no specific band was generated in the medium with Na₂SO₄. In conclusion, the regulation of repression to desulfurization enzyme expression occurred at the level of transcription.3. Desulfurization related genes of LY822 named dszA, dszB and dszC were separately cloned by PCR. Three expression plasmids, pETA, pETB and pETC, were constructed and transformed into E.coli BL21 strain. After the IPTG induction with them, dszA, dszB and dszC were expressed effectively in the recombinant E.coli BL21 strains. Sequences of the desulfurization genes were highly conservative. Desulfurization activity analysis showed that cell-free extracts of three recombinant strains could convert corresponding substrates, which proved that LY822 could specially break the C-S bond of DBT and convert it into 2-HBP by "4S" biodesulfurization pathway.4. DBT monooxygenase gene (dszC) was cloned from LY822 by PCR. The gene dszC expressed effectively after being inserted into plasmid pBSC and transformed into LY822-0 strain and Rhodococcus rhodochrous(ACCC NO. 10494). The recombinant strains could convert DBT into DBTO₂, and discharge the repression of sulfate.