聚磷菌多聚磷酸盐激酶的分子生物学研究及高效除磷工程菌的构建
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摘要
水体富营养化是全球十大环境问题之一,氮磷尤其是磷的输入是引起水体富营养化的关键因素。为解决磷污染所带来的危害,在污水进入水体前进行有效处理,降低排放污水的磷浓度十分必要。强化生物除磷工艺(Enhancedbiological phosphorus removal,EBPR)是目前世界各国普遍使用的方法,该工艺具有污泥产生量少、不使用化学物质和运行经济等特点。EBPR系统中聚磷茵在磷的去除过程中起着至关重要的作用。为此,加大对聚磷菌的聚磷特性、聚磷相关基因及聚磷菌的工程化应用进行研究十分必要。
本文以高效聚磷菌聚磷机理研究为主线,以一株高效聚磷茵GM6为研究材料,一方面系统研究了ppk1编码的多聚磷酸盐激酶被敲除后对菌体生理生化性质的影响,另一方面通过强化表达ppk1编码的多聚磷酸盐激酶和PHA操纵元(phaC1-phaZ-phaC2)构建了两株高效除磷工程菌并结合本实验室已获得的phaZ突变株对EBPR系统中碳磷循环代谢进行研究。
1.ppk1突变对Pseudomonas putida GM6特性的影响及ppk2基因的克隆
ppk基因编码多聚磷酸盐激酶,主要负责多聚磷酸盐(Poly-P)的合成。大多数微生物体内编码PPK的基因都有ppk1和ppk2两类。通过同源重组敲除ppk1后,突变株在菌体形态,生理生化等方面发生显著变化:通过扫描电镜观察发现突变株较之原始菌株表面粗糙有凹凸不平感,且部分细胞一端或两端同时有凹陷,生长速度变慢,代时增加25%,生物膜形成能力下降50%,游动性与抗逆能力与原始菌株相比显著降低,这都可能是由于菌体多聚磷酸盐激酶活性的丧失,使细胞内Poly-P水平降低或是Poly-P链长组成发生变化,从而引发细胞功能的降低或缺失。而突变株与原始菌株相比在除磷能力上并没有显著变化,这可能是由于GM6中还存在着另一个编码多聚磷酸盐激酶的基因(ppk2)。根据种属间的同源性,利用快速染色体步移方法克隆了ppk2基因,并将其定向克隆到pET29a载体上进行表达,为进一步研究多聚磷酸盐激酶的功能打下了基础。
2.高效聚磷基因工程茵的构建
通过PCR方法从高效聚磷菌株GM6总DNA中扩增得到了ppk1、PHA及其自带的启动子,并定向克隆到pBBR1MCS-5载体上,构建了重组质粒pMEPE-PPK和pMEPE-PHA,在辅助质粒pRK2013的帮助下,通过三亲接合将pMEPE-PPK及pMEPE-PHA转移到原始菌株GM6中,获得的工程菌P.putidaGM6-PPK1和GM6-PHA。两株工程菌除磷能力较原始菌株GM6和对照菌株GM6-P5有了显著提高,而且在无抗性条件下质粒稳定,因此有着潜在的应用前景。通过模拟EBPR工艺,发现GM6-PPK1和GM6-PHA在厌氧/好氧交替的环境条件下强化表达不但提高了菌体好氧段的吸磷能力,而且厌氧段磷的释放和PHA的合成较之原始菌株也有显著增加,这说明ppk1和PHA的强化表达,不但增强了菌体的除磷能力,也在一定程度上提升了碳磷循环代谢的能力,使得菌体有更好的除磷和除COD的能力。
Eutrophication of water bodies is one of the ten serious global environmental problems. Nitrogen and phosphate, especially phosphate is the key contributor to eutrophication of water bodies. Discharge of untreated domestic sewage is one of the major sources of the phosphorus in the water bodies. In order to eliminate the phosphorus pollution, it is essential to treat the sewage to reduce its P concentration before it is discharged into rivers. Currently most large scale wastewater treatment plants use enhanced biological phosphorus removal (EBPR) process characterized by less sludge generation, free of chemicals and economical operation, which is extensively used in countries all over the world. PAOs play key roles in EBPR system to remove phosphorus. Therefore, it is quite necessary to characterize the regulation of related genes in PAOs.
This paper systematically studied the molecular mechanism of Poly-P accumulation in Pseudomonas putida GM6, the effects ppk1 mutation on the physiology of GM6. Two genetically engineered strains were constructed to improve the phosphate removal ability of GM6.
1. The effects of Poly-Phosphate kinase 1 (ppk1) mutation on Pseudomonas putida GM6 and Cloning of the Poly-Phosphate kinase 2 gene
The Poly-Phosphate kinase responsible for Poly-Phosphate (Poly-P) synthesis is encoded by ppk genes, which consists of ppk1 and ppk2. Pseudomonas putida GM6 is an efficient phosphate accumulating strain isolated from the enhanced biological phosphorus removal (EBPR) system in the activated sludge aerobic pond, and ppk1 was cloned and characterized from it previously. By Homologous recombination the ppk1 was knockout from the strain, characteristic of physiological and biochemical aspects of the mutant strain and the wild type GM6 was compared: the mutant grows much slower the generation time was elongated by 25%, while the biofilm formation ability and the motility decreased by 50%, resilience decreased significantly. Loss of Poly-Phosphate kinase activity might lead to the lower level of intracellular Poly-P concentration and the change of Poly-P chain length or composition, which may trigger reduction or loss of cell function and caused all above phenomenon. However, no significant difference was found between the mutant and the wild type GM6 in the removal of phosphate, this may be due to the existence of another ppk gene (ppk2 ) in the strain. Accordance to the homology of ppk2 between species ppk2 was cloned by rapid chromosome walking and expressed, this will lay the foundation for further study.
2. Construction of genetic engineered Poly-P accumulation strains
ppk1 gene and PHA operon with their promoter were amplified from the genomic DNA of GM6 by PCR. Recombinant plasmids pMEPE-PPK and pMEPE -PHA were constructed by ligating t ppk1 gene and PHA operon into broad host vector pBBR1MCS-5. With the help of plasmid pRK2013 , pMEPE-PPK and pMEPE -PHA were transferred into the strain GM6 to construct GM6-PPK1 and GM6-PHA. The Poly-Phosphate removal ability of GM6-PPK1 and GM6-PHA were increased compared with the original strain GM6. In order to stimulate the EBPR technique, the strains were grown under anaerobic and aerobic conditions. Results showed that GM6-PPK1 has stronger phosphate absorption ability in aerobic condition and it releases more phosphate and syntheses more PHA in anaerobic condition. The results show that the strengthened expression of ppk1 d PHA not only enhanced the dephosphate ability of the strain but improved the carbon and phosphate cycling metabolism, the above character made the strain a better one for dephosphate and remove COD.
本文以高效聚磷菌聚磷机理研究为主线,以一株高效聚磷茵GM6为研究材料,一方面系统研究了ppk1编码的多聚磷酸盐激酶被敲除后对菌体生理生化性质的影响,另一方面通过强化表达ppk1编码的多聚磷酸盐激酶和PHA操纵元(phaC1-phaZ-phaC2)构建了两株高效除磷工程菌并结合本实验室已获得的phaZ突变株对EBPR系统中碳磷循环代谢进行研究。
1.ppk1突变对Pseudomonas putida GM6特性的影响及ppk2基因的克隆
ppk基因编码多聚磷酸盐激酶,主要负责多聚磷酸盐(Poly-P)的合成。大多数微生物体内编码PPK的基因都有ppk1和ppk2两类。通过同源重组敲除ppk1后,突变株在菌体形态,生理生化等方面发生显著变化:通过扫描电镜观察发现突变株较之原始菌株表面粗糙有凹凸不平感,且部分细胞一端或两端同时有凹陷,生长速度变慢,代时增加25%,生物膜形成能力下降50%,游动性与抗逆能力与原始菌株相比显著降低,这都可能是由于菌体多聚磷酸盐激酶活性的丧失,使细胞内Poly-P水平降低或是Poly-P链长组成发生变化,从而引发细胞功能的降低或缺失。而突变株与原始菌株相比在除磷能力上并没有显著变化,这可能是由于GM6中还存在着另一个编码多聚磷酸盐激酶的基因(ppk2)。根据种属间的同源性,利用快速染色体步移方法克隆了ppk2基因,并将其定向克隆到pET29a载体上进行表达,为进一步研究多聚磷酸盐激酶的功能打下了基础。
2.高效聚磷基因工程茵的构建
通过PCR方法从高效聚磷菌株GM6总DNA中扩增得到了ppk1、PHA及其自带的启动子,并定向克隆到pBBR1MCS-5载体上,构建了重组质粒pMEPE-PPK和pMEPE-PHA,在辅助质粒pRK2013的帮助下,通过三亲接合将pMEPE-PPK及pMEPE-PHA转移到原始菌株GM6中,获得的工程菌P.putidaGM6-PPK1和GM6-PHA。两株工程菌除磷能力较原始菌株GM6和对照菌株GM6-P5有了显著提高,而且在无抗性条件下质粒稳定,因此有着潜在的应用前景。通过模拟EBPR工艺,发现GM6-PPK1和GM6-PHA在厌氧/好氧交替的环境条件下强化表达不但提高了菌体好氧段的吸磷能力,而且厌氧段磷的释放和PHA的合成较之原始菌株也有显著增加,这说明ppk1和PHA的强化表达,不但增强了菌体的除磷能力,也在一定程度上提升了碳磷循环代谢的能力,使得菌体有更好的除磷和除COD的能力。
Eutrophication of water bodies is one of the ten serious global environmental problems. Nitrogen and phosphate, especially phosphate is the key contributor to eutrophication of water bodies. Discharge of untreated domestic sewage is one of the major sources of the phosphorus in the water bodies. In order to eliminate the phosphorus pollution, it is essential to treat the sewage to reduce its P concentration before it is discharged into rivers. Currently most large scale wastewater treatment plants use enhanced biological phosphorus removal (EBPR) process characterized by less sludge generation, free of chemicals and economical operation, which is extensively used in countries all over the world. PAOs play key roles in EBPR system to remove phosphorus. Therefore, it is quite necessary to characterize the regulation of related genes in PAOs.
This paper systematically studied the molecular mechanism of Poly-P accumulation in Pseudomonas putida GM6, the effects ppk1 mutation on the physiology of GM6. Two genetically engineered strains were constructed to improve the phosphate removal ability of GM6.
1. The effects of Poly-Phosphate kinase 1 (ppk1) mutation on Pseudomonas putida GM6 and Cloning of the Poly-Phosphate kinase 2 gene
The Poly-Phosphate kinase responsible for Poly-Phosphate (Poly-P) synthesis is encoded by ppk genes, which consists of ppk1 and ppk2. Pseudomonas putida GM6 is an efficient phosphate accumulating strain isolated from the enhanced biological phosphorus removal (EBPR) system in the activated sludge aerobic pond, and ppk1 was cloned and characterized from it previously. By Homologous recombination the ppk1 was knockout from the strain, characteristic of physiological and biochemical aspects of the mutant strain and the wild type GM6 was compared: the mutant grows much slower the generation time was elongated by 25%, while the biofilm formation ability and the motility decreased by 50%, resilience decreased significantly. Loss of Poly-Phosphate kinase activity might lead to the lower level of intracellular Poly-P concentration and the change of Poly-P chain length or composition, which may trigger reduction or loss of cell function and caused all above phenomenon. However, no significant difference was found between the mutant and the wild type GM6 in the removal of phosphate, this may be due to the existence of another ppk gene (ppk2 ) in the strain. Accordance to the homology of ppk2 between species ppk2 was cloned by rapid chromosome walking and expressed, this will lay the foundation for further study.
2. Construction of genetic engineered Poly-P accumulation strains
ppk1 gene and PHA operon with their promoter were amplified from the genomic DNA of GM6 by PCR. Recombinant plasmids pMEPE-PPK and pMEPE -PHA were constructed by ligating t ppk1 gene and PHA operon into broad host vector pBBR1MCS-5. With the help of plasmid pRK2013 , pMEPE-PPK and pMEPE -PHA were transferred into the strain GM6 to construct GM6-PPK1 and GM6-PHA. The Poly-Phosphate removal ability of GM6-PPK1 and GM6-PHA were increased compared with the original strain GM6. In order to stimulate the EBPR technique, the strains were grown under anaerobic and aerobic conditions. Results showed that GM6-PPK1 has stronger phosphate absorption ability in aerobic condition and it releases more phosphate and syntheses more PHA in anaerobic condition. The results show that the strengthened expression of ppk1 d PHA not only enhanced the dephosphate ability of the strain but improved the carbon and phosphate cycling metabolism, the above character made the strain a better one for dephosphate and remove COD.
引文
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