硫酸盐对Rhodococcus sp. LY822脱硫代谢影响的研究及脱硫基因的克隆表达
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摘要
本论文以脱硫菌Rhodococcus sp．LY822为出发菌株，开展了硫酸盐对LY822菌脱硫代谢的影响、脱硫基因克隆表达及重组菌脱硫活性的研究，初步阐明了脱硫代谢机理，验证了脱硫酶生成的“硫饥饿”诱导机制。
1．研究了不同浓度的Na_2SO_4对Rhodococcus sp．LY822细胞生长和脱硫的影响。Na_2SO_4在0-0.4 mmol／L浓度范围内对LY822菌的生长没有影响，但对其生长细胞脱硫活性具有抑制作用；在有DBT和0.03 mmol／L以上的Na_2SO_4存在时，LY822菌生长细胞的粗酶液在脱硫反应48 h内没有2-HBP生成，而Na_2SO_4在0.01 mmol／L以下时，其脱硫反应能生成2-HBP；Na_2SO_4对含有脱硫酶的无细胞粗提液的脱硫活性无抑制作用。研究表明，0.03 mmol／L以上浓度的Na_2SO_4能阻遏脱硫酶的生成，而0.01 mmol／L以下浓度能诱导脱硫酶生成，从而验证了脱硫微生物脱硫酶生成的“硫饥饿”诱导机制。
2．克隆了LY822菌脱硫基因的启动子，构建了dsz启动子-gfp报告基因表达载体pBSG，并转化原始菌(LY822)，激光共聚焦显微镜和荧光分光光度计检测结果显示，无Na_2SO_4时菌体的荧光强度为存在0.1 mmol／L Na_2SO_4时的3倍，证实了脱硫基因的表达受硫酸盐的阻遏；Western Blotting结果显示，0.2 mmol／L DBT存在时下培养的LY822菌的全细胞蛋白有与DszA，DszB和DszC杂交的特异性条带生成，而0.2 mmol／L Na_2SO_4存在时则无特异性条带产生，进一步证明了脱硫酶的生成受到硫酸盐的阻遏；以LY822菌的总RNA为模板进行RT-PCR，0.2 mmol／L DBT存在时培养的LY822菌，可扩增得到与脱硫基因dszA，dszB和dszC大小相对应的cDNA片段；而0.2 mmol／L Na_2SO_4存在时培养的LY822菌，未扩增到特异性条带，表明硫酸盐对LY822菌脱硫基因表达的调控作用发生在转录水平上。
3．克隆了LY822菌的脱硫基因dszA，dszB和dszC，并在大肠杆菌BL21(DE3)中得到高效表达，与Rhodococcus erythropolis IGTS8相关基因的相似性分别为99.9％、100％和99.5％，证实了红球菌脱硫基因的高度保守性；3种重组大肠杆菌的无细胞粗酶液脱硫反应的代谢产物与已报道的“4S途径”中的相应代谢产物一致，证明LY822菌对DBT的降解符合专一性脱硫的“4S途径”；重组大肠杆菌无细胞粗提液表现出比原始菌(LY822)更高的脱硫活性。
4．构建了dszC基因的表达载体pBSC，转化至消除质粒的原始菌(LY822-0)和不具备脱硫特性的紫红红球菌(ACCC NO．10494)中，所获重组菌均具有脱硫反应活性，并不受硫酸盐影响。研究结果为构建不受硫酸盐阻遏的高效脱硫基因工程菌奠定了基础。
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 Na_2SO_4 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 Na_2SO_4 in 48 hours. However, DBT was converted into 2-HBP when the concentration of Na_2SO_4 was below 0.01 mmol/L. Na_2SO_4 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 Na_2SO_4 from recombinant strains was three times of that with 0.1 mmol/L Na_2SO_4, 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 Na_2SO_4, 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 Na_2SO_4. 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 DBTO_2, and discharge the repression of sulfate.
1．研究了不同浓度的Na_2SO_4对Rhodococcus sp．LY822细胞生长和脱硫的影响。Na_2SO_4在0-0.4 mmol／L浓度范围内对LY822菌的生长没有影响，但对其生长细胞脱硫活性具有抑制作用；在有DBT和0.03 mmol／L以上的Na_2SO_4存在时，LY822菌生长细胞的粗酶液在脱硫反应48 h内没有2-HBP生成，而Na_2SO_4在0.01 mmol／L以下时，其脱硫反应能生成2-HBP；Na_2SO_4对含有脱硫酶的无细胞粗提液的脱硫活性无抑制作用。研究表明，0.03 mmol／L以上浓度的Na_2SO_4能阻遏脱硫酶的生成，而0.01 mmol／L以下浓度能诱导脱硫酶生成，从而验证了脱硫微生物脱硫酶生成的“硫饥饿”诱导机制。
2．克隆了LY822菌脱硫基因的启动子，构建了dsz启动子-gfp报告基因表达载体pBSG，并转化原始菌(LY822)，激光共聚焦显微镜和荧光分光光度计检测结果显示，无Na_2SO_4时菌体的荧光强度为存在0.1 mmol／L Na_2SO_4时的3倍，证实了脱硫基因的表达受硫酸盐的阻遏；Western Blotting结果显示，0.2 mmol／L DBT存在时下培养的LY822菌的全细胞蛋白有与DszA，DszB和DszC杂交的特异性条带生成，而0.2 mmol／L Na_2SO_4存在时则无特异性条带产生，进一步证明了脱硫酶的生成受到硫酸盐的阻遏；以LY822菌的总RNA为模板进行RT-PCR，0.2 mmol／L DBT存在时培养的LY822菌，可扩增得到与脱硫基因dszA，dszB和dszC大小相对应的cDNA片段；而0.2 mmol／L Na_2SO_4存在时培养的LY822菌，未扩增到特异性条带，表明硫酸盐对LY822菌脱硫基因表达的调控作用发生在转录水平上。
3．克隆了LY822菌的脱硫基因dszA，dszB和dszC，并在大肠杆菌BL21(DE3)中得到高效表达，与Rhodococcus erythropolis IGTS8相关基因的相似性分别为99.9％、100％和99.5％，证实了红球菌脱硫基因的高度保守性；3种重组大肠杆菌的无细胞粗酶液脱硫反应的代谢产物与已报道的“4S途径”中的相应代谢产物一致，证明LY822菌对DBT的降解符合专一性脱硫的“4S途径”；重组大肠杆菌无细胞粗提液表现出比原始菌(LY822)更高的脱硫活性。
4．构建了dszC基因的表达载体pBSC，转化至消除质粒的原始菌(LY822-0)和不具备脱硫特性的紫红红球菌(ACCC NO．10494)中，所获重组菌均具有脱硫反应活性，并不受硫酸盐影响。研究结果为构建不受硫酸盐阻遏的高效脱硫基因工程菌奠定了基础。
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 Na_2SO_4 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 Na_2SO_4 in 48 hours. However, DBT was converted into 2-HBP when the concentration of Na_2SO_4 was below 0.01 mmol/L. Na_2SO_4 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 Na_2SO_4 from recombinant strains was three times of that with 0.1 mmol/L Na_2SO_4, 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 Na_2SO_4, 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 Na_2SO_4. 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 DBTO_2, and discharge the repression of sulfate.
引文
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