title: Red recombinase assisted gene replacement in Klebsiella pneumoniae.
abstract: The Red recombinase system, the most convenient genetic tool applied in Escherichia coli and other bacteria, was introduced for gene replacement in Klebsiella pneumoniae. The novel K. pneumoniae gene replacement system comprised the Red and FLP recombinases expression vector pDK6-red and pDK6-flp, and linear DNA fragments which encompassed a selective marker gene with target gene flanking extensions; the latter were PCR amplified using a plasmid DNA template obtained by in vivo recombination in E. coli. In this study, dhak1 gene, encoding a subunit of dihydroxyacetone kinase II, was deleted markerlessly at a transformation ratio of 260 CFU/μg DNA, i.e., 1,000-fold higher than that achieved in the native way. Our studies provide an efficient method with detailed protocol to perform gene replacement in K. pneumoniae and has great potential to be developed as a routine genetic approach for this important industrial microorganism.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wei
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Jiping
authors.initials: J
authors.lastname: Shi
authors.forename: Jian
authors.initials: J
authors.lastname: Hao
mesh_terms: D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000378:metabolism; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial / Q000235:genetics*
chemical_list: D045522:Recombinases; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase