Paper Report for: Wang_2015_J.Microbiol.Methods_113_27
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Title: An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440 Wang Y, Zhang C, Gong T, Zuo Z, Zhao F, Fan X, Yang C, Song C Ref: J Microbiol Methods, 113:27, 2015 : PubMed
A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHAMCL)-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P. mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHAMCL depolymerase) genes, and a 30kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P. mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHAMCL polymerase) loci of Pseudomonas putida KT-UPP simultaneously. Thus, we constructed a genetically stable and marker-free P. putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp-based counterselection system could be further adapted for P. mendocina NK-01 and P. putida KT2440 and used for genome reduction and metabolic pathway engineering.
        
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Wang Y, Zhang C, Gong T, Zuo Z, Zhao F, Fan X, Yang C, Song C (2015) An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440 J Microbiol Methods113: 27-33
Wang Y, Zhang C, Gong T, Zuo Z, Zhao F, Fan X, Yang C, Song C (2015) J Microbiol Methods113: 27-33