Molecular Biology Research Of Polyphosphate Kinase In Polyphosphate Accumulating Organisms And Construction Of Genetical Engineered Polyphosphate Accumulation Strains

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 geneThe 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 strainsppk1 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.