德氏假单胞R-8菌脱硫的“硫饥饿”诱导机理

Sulfur-starvation-induced Desulfurization Mechanism of Pseudomonas delafieldii R-8

以专一性脱硫菌德氏假单胞菌(Pseudomonas delafieldii)R-8为出发菌株,构建了含有重组质粒pRT-D的重组菌株R-8-D.在不同硫酸盐浓度条件下,研究了R-8和R-8-D脱硫的"硫饥饿"诱导机理,结果表明,在充足的Na2SO4(>0.023mmolL-1)条件下,R-8菌首先利用硫酸盐生长,脱硫酶的合成受阻遏,DBT不被利用,而R-8菌处于"硫饥饿"状态(Na2SO4浓度≤0.023mmolL-1)时,诱导了脱硫酶的合成,能利用DBT生长;Na2SO4在临界浓度以上时,R-8-D菌阻遏了脱硫基因的报告基因lacZ的表达,低于临界浓度时不抑制lacZ表达.本实验结果从细胞和分子水平上证实了R-8菌脱硫属"硫饥饿"诱导类型,并首次确定了脱硫微生物"硫饥饿"诱导的硫酸盐临界浓度为0.023mmolL-1,为构建高活性的、不受硫酸盐抑制的脱硫工程菌提供了理论依据和技术支持.

Pseudomonas delafieldii R-8 can convert dibenzothiophene into 2-hydroxybiphenyl and sulfate. The desulfurization operon of strain R-8 was cloned into expressing plasmid （pPR9TT）, and then reintroduced into strain R-8-0 to obtain engineering strain R-8-D. Strains R-8 and R-8-D were cultured in BSM medium with 0.2 mmol L^-1 DBT and different concentrations of sulfate, and sulfur-starvation-induced desulfurization mechanism of R-8 was studied. The results showed that strain R-8 cultured in sufficient Na2SO4 （〉 0.023 mmol L^-1） could grow and Na2SO4 be firstly used, but the synthesis of Dsz was repressed and DBT could not be used. On the contrary, DBT could be used and the synthesis of Dsz was induced at sulfur starvation （Na2SO4 concentration ≤ 0.023 mmol L^-1）. The expression of dsz reporter gene lacZ in R-8-D was inhibited above the critical concentration of Na2SO4, but it was not below the critical concentration. The results verified the sulfur-starvation-induced mechanism of desulfurization by strain R-8 at the cellular and molecular levels, and for the first time identified biodesulfurization sulfur-starvation induced by the critical concentration of sulfate was 0.023 mmol L^-1. 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. Fig 10, Tab 1, Ref 14