title: Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum.
abstract: BACKGROUND
  Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.
  
  
  RESULTS
  The method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLS (R) ) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the "marker-less" genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate.
  
  
  CONCLUSIONS
  The restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Croux
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.
authors.forename: Ngoc-Phuong-Thao
authors.initials: NP
authors.lastname: Nguyen
authors.affiliation: College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea.
authors.forename: Jieun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Metabolic Explorer, Saint-Beauzire, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Raynaud
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Saint-Prix
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.
authors.forename: Maria
authors.initials: M
authors.lastname: Gonzalez-Pajuelo
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Meynial-Salles
authors.affiliation: LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France.|Metabolic Explorer, Saint-Beauzire, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Soucaille
keywords: 5-FU; Cac824I; Clostridium acetobutylicum; FLP; FRT; Gene deletion; Gene replacement; upp; upp gene
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