Pseudomonas delafieldii R-8脱硫代谢调控研究及组成型脱硫工程菌的构建
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摘要
本文以脱硫菌Pseudomonas delafieldii R-8为研究对象,采用分子生物学和生物信息学等研究方法,开展了R-8菌脱硫代谢调控及组成型脱硫工程菌构建的研究,旨在进一步阐明脱硫代谢调控机理,为构建高活性的脱硫工程菌奠定理论基础。
在添加了不同浓度硫酸盐的BSM培养基培养条件下,对R-8菌脱硫代谢的“硫饥饿”诱导机理进行了研究。实验结果表明以有机硫DBT为唯一硫源和添加的Na_2SO_4初始浓度小于或等于0.023 mmol/L时,能降解DBT,表现出正常的脱硫活性;而在起始浓度大于0.023 mmol/L时,R-8菌能正常生长,但脱硫活性受到抑制。该结果从细胞水平上验证了R-8菌脱硫代谢为“硫饥饿”诱导类型;通过构建融合脱硫操纵子pPR9TT穿梭表达重组质粒pRT-D,电转化消除脱硫质粒的R-8-0菌,获得了重组菌R-8-D,以R-8-D菌为研究材料,进一步从分子水平上验证了脱硫菌脱硫代谢“硫饥饿”诱导机制。当Na_2SO_4在R-8菌脱硫代谢“硫饥饿”诱导的临界浓度(0.023 mmol/L)以上时,报告基因lacZ不表达,而在低于临界浓度条件下,LacZ被诱导表达,同时还证实了脱硫基因的表达受硫酸盐的阻遏。
采用PCR的方法,从R-8菌中克隆了脱硫基因启动子系列缺失片段,利用启动子探测型表达载体pPR9TT分别构建了含启动子缺失片段的系列重组质粒p-pPR,转化原始菌R-8获得了系列重组菌R-8-P,通过测定各重组菌中报告基因lacZ的表达量,对R-8菌脱硫基因启动子调控功能区进行了初步鉴定。实验结果表明,与正常脱硫基因启动子活性相比,脱硫基因转录起始位点上游300 bp启动子序列具100℅活性,150 bp为42%,小于75 bp活性为0。研究结果证明了脱硫基因启动子核心功能区位于上游300 bp序列内,与已报道的红平红球菌(Rhodococcus erythropolis)IGTS 8全长为385 bp的脱硫基因启动子相比,启动子全长缩短了85个核苷酸。采用生物信息学软件BPROM对R-8菌脱硫基因启动子功能区DNA序列进行预测,分析结果表明-10区序列为AGCCATGAT,-35区序列为CTGCTT,在-21～-28位点有一个ρD17蛋白结合位点。
在上述研究结果基础上,为了获得不受硫酸盐阻遏脱硫作用的脱硫工程菌,将R-8菌中的脱硫基因dszABC和组成型gap基因启动子克隆到表达载体pPR9TT中,获得的重组质粒pRT-C转化R-8-0菌,得到了组成型工程菌R-8-C。R-8-C菌在Na_2SO_4浓度大于或等于0.1 mmol/L条件下,与原始菌R-8在以DBT为唯一硫源的培养条件下相比,其脱硫活性达93%,证明了该工程菌脱硫作用几乎不受硫酸盐阻遏。通过R-8-C菌的全细胞SDS-PAGE电泳和无细胞提取液western blotting再次验证了该工程菌能在较高硫酸盐浓度(0.1 mmol/L)存在条件下表达出脱硫酶蛋白。这些结果为阐明脱硫代谢调控机理,进一步研究脱硫基因调控机制及构建高活性的脱硫工程菌奠定了理论基础和技术支持。
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 Na_2SO_4 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 Na_2SO_4 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 Na_2SO_4, 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 Na_2SO_4 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.
在添加了不同浓度硫酸盐的BSM培养基培养条件下,对R-8菌脱硫代谢的“硫饥饿”诱导机理进行了研究。实验结果表明以有机硫DBT为唯一硫源和添加的Na_2SO_4初始浓度小于或等于0.023 mmol/L时,能降解DBT,表现出正常的脱硫活性;而在起始浓度大于0.023 mmol/L时,R-8菌能正常生长,但脱硫活性受到抑制。该结果从细胞水平上验证了R-8菌脱硫代谢为“硫饥饿”诱导类型;通过构建融合脱硫操纵子pPR9TT穿梭表达重组质粒pRT-D,电转化消除脱硫质粒的R-8-0菌,获得了重组菌R-8-D,以R-8-D菌为研究材料,进一步从分子水平上验证了脱硫菌脱硫代谢“硫饥饿”诱导机制。当Na_2SO_4在R-8菌脱硫代谢“硫饥饿”诱导的临界浓度(0.023 mmol/L)以上时,报告基因lacZ不表达,而在低于临界浓度条件下,LacZ被诱导表达,同时还证实了脱硫基因的表达受硫酸盐的阻遏。
采用PCR的方法,从R-8菌中克隆了脱硫基因启动子系列缺失片段,利用启动子探测型表达载体pPR9TT分别构建了含启动子缺失片段的系列重组质粒p-pPR,转化原始菌R-8获得了系列重组菌R-8-P,通过测定各重组菌中报告基因lacZ的表达量,对R-8菌脱硫基因启动子调控功能区进行了初步鉴定。实验结果表明,与正常脱硫基因启动子活性相比,脱硫基因转录起始位点上游300 bp启动子序列具100℅活性,150 bp为42%,小于75 bp活性为0。研究结果证明了脱硫基因启动子核心功能区位于上游300 bp序列内,与已报道的红平红球菌(Rhodococcus erythropolis)IGTS 8全长为385 bp的脱硫基因启动子相比,启动子全长缩短了85个核苷酸。采用生物信息学软件BPROM对R-8菌脱硫基因启动子功能区DNA序列进行预测,分析结果表明-10区序列为AGCCATGAT,-35区序列为CTGCTT,在-21～-28位点有一个ρD17蛋白结合位点。
在上述研究结果基础上,为了获得不受硫酸盐阻遏脱硫作用的脱硫工程菌,将R-8菌中的脱硫基因dszABC和组成型gap基因启动子克隆到表达载体pPR9TT中,获得的重组质粒pRT-C转化R-8-0菌,得到了组成型工程菌R-8-C。R-8-C菌在Na_2SO_4浓度大于或等于0.1 mmol/L条件下,与原始菌R-8在以DBT为唯一硫源的培养条件下相比,其脱硫活性达93%,证明了该工程菌脱硫作用几乎不受硫酸盐阻遏。通过R-8-C菌的全细胞SDS-PAGE电泳和无细胞提取液western blotting再次验证了该工程菌能在较高硫酸盐浓度(0.1 mmol/L)存在条件下表达出脱硫酶蛋白。这些结果为阐明脱硫代谢调控机理,进一步研究脱硫基因调控机制及构建高活性的脱硫工程菌奠定了理论基础和技术支持。
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 Na_2SO_4 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 Na_2SO_4 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 Na_2SO_4, 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 Na_2SO_4 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.
引文
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