[Integration and expression of polyphosphate kinase gene in Pseudomonas putida].
In order to construct high accumulating-phosphate microorganism, the ppk gene from E. coli was inserted to the broad-host-range plasmid pBBR1MCS-2 to form plasmid pBBR1MCS-2-ppk. The complete ppk gene with promoter and terminator sequences from pBBR1MCS-2-ppk was then cloned and inserted to suicide plasmid pUTmini-Tn5 to form plasmid pUTmini-Tn5-ppk, which was transformed into Pseudomonas putida KT2440 by triparental conjugation. Finally, ppk gene was integrated into the chromosomal DNA of KT2440. The results of RT-PCR showed that the selected genetically engineered bacterium KT2440-PPK expressed ppk efficiently, while KT2440 as control expressed weakly. The results of artificial wastewater treatment showed after 1h inoculation, the concentration of poly-phosphate in KT2440-PPK came to the maximum approximately 3.05 mg/g, which was 15 times higher than that in KT2440 at the same experimental condition. And KT2440-PPK can remove more than 90% phosphate in artificial wastewater.
Du
Hong-wei
HW
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, China. hongwd@gmail.com
Wu
Jun
J
Xiao
Lin
L
Yang
Liu-yan
LY
Jiang
Li-juan
LJ
Wang
Xiao-lin
XL
D001673:Biodegradation, Environmental; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D016254:Mutagenesis, Insertional; D010758:Phosphorus / Q000302:isolation & purification* / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D014865:Waste Disposal, Fluid / Q000379:methods
D004269:DNA, Bacterial; D010758:Phosphorus; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase