Desulfurization Metabolic Regulation And Construction Of Constitutive Engineered Strain For Desulfurization Of Pseudomonas Delafieldii R-8

Pseudomonas delafieldii R-8 is able to desulfurize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway. We carried out the desulfurization metabolic regulation of strain R-8 and constitutive expressing dsz egineered strain for BDS in this paper by molecular biology and bio-informatics. These results will further reveal the mechanism of desulfurization metabolic regulation, and helpful to construct the highly activity desulfurization engineering bacteria.Firstly the sulfur-starvation-induced desulfurization mechanism of strain R-8 was studied which cultured in BSM medium with 0.2 mmol/L DBT and different concentrations of sulfate. The results showed that organic sulfur DBT could be used as sulfur source by strain R-8 and the desulfurization activity was induced when DBT as the sole sulfur source and the initial concentration of Na₂SO₄ less than or equal to 0.023 mmol/L in BSM. On the contrary, cultured in more than 0.023 mmol/L initial concentration of Na₂SO₄ could normally grow, but the desulfurization activity was inhibited. The results verified the mechanism of desulfurization by strain R-8 was sulfur-starvation- -induced at the cellular level. Then the dsz operon of strain R-8 was cloned into the expressing plasmid (pPR9TT) to construct recombinant plasmid pPR-D, and then pPR-D reintroduced into strain R-8-0 (strain R-8 lost plasmid containing the dsz gene cluster) to obtain engineering strain R-8-D by electrotransformation. The mechanism of desulfurization by biodesulfurization bacteria also was furtherly verified sulfur-starvation-induced at the molecular level. The expression of lacZ reporter gene in R-8-D was inhibited above the critical concentration (0.023 mmol/L) of Na₂SO₄, but it was not below the critical concentration. These results also confirmed the expression of dsz expression were repressed by sulfate.Secondly the dsz promoter serial deletion fragments by PCR from strain R-8 were cloned. These fragments were cloned into the promoter detection of expression vector pPR9TT to construct recombinant plasmids p-pPR, then p-pPR reintroduced into strain R-8 to obtain engineering strains R-8-P by electrotransformation, the expression of reporter gene lacZ in R-8-P were determined induced by DBT, so we can preliminary identify the dsz core promoter regulation and function region. The results showed that the desulfurization gene transcription start site upstream 300 bp promoter sequences with 100% activity, 150 bp to 42%, less than 75 bp for 0, compared with the normal dsz promoter activity in strain R-8. These results proved that dsz promoter core function region was located upstream 300 bp, which was shorter 85 nucleotides than Rhodococcus erythropolis IGTS8 reported 385 bp. Then we forecasted 300 bp promoter regulation region by BPROM, the results showed that -10 region with the sequence AGCCATGAT, -25 region of CTGCTT, andρD17 binding site is at -21 to -28.Finnally a constitutive engineering strain for desulfurization was constructed that called strain R-8-C which relieving the sulfate repression on the basis of those results we got. The dszABC in strain R-8 was cloned into expression vector pPR9TT with gap promoter to build a constitutive expression plasmid pgap-dsz-9TT, then reintroduced into strain R-8-0 to obtain strain R-8-C by electrotransformation. Strain R-8-C can convert dibenzothiophene into 2-hydroxybiphenyl in BSM medium with Na₂SO₄ concentration more than 0.1 mmol/L, compared with the wild type strain R-8 (DBT as the sole source of sulfur), still had 93% desulfurization activity that was not inhibited and repressed by sulfate. We also verified Dsz was expressed in strain R-8-C by the whole-cell protein SDS-PAGE electrophoresis and western blotting in the high concentration of sulfate in the presence of BSM. These results are theoretically and technically helpful for the construction of highly active and reliable desulphurization engineering strains that will not be inhibited by sulfate.