title: An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440.
abstract: 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 (PHA(MCL))-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 PHA(MCL) depolymerase) genes, and a 30 kb 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 (PHA(MCL) 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.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Gong
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Zhenqiang
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Fengjie
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Fan
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China. Electronic address: yang_chao2008@hotmail.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China. Electronic address: songcj@nankai.edu.cn.
authors.forename: Cunjiang
authors.initials: C
authors.lastname: Song
mesh_terms: D043182:Carboxylesterase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D005472:Fluorouracil / Q000378:metabolism; D005822:Genetic Vectors; D016680:Genome, Bacterial; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D044203:Pseudomonas mendocina / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D011722:Pyrethrins / Q000378:metabolism
chemical_list: D011722:Pyrethrins; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043182:Carboxylesterase; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases; D005472:Fluorouracil
keywords: 5-Fluorouracil; Genome modification; Uracil phosphoribosyltransferase