Abstract
BackgroundClostridium 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.ResultsThe 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 (MLSR) 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.ConclusionsThe 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.
Highlights
Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of convert‐ ing various sugars and polysaccharides into solvents
The first part is a replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (ThR) flanked by two flippase recognition target (FRT) sequences, (3) two sequences homologous to the selected regions around the target DNA sequence, and (4) a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens to allow for the positive selection of double-crossover allelic exchange mutants
This method was successfully used by the Metabolic Explorer Company to develop and patent an industrial recombinant strain of C. acetobutylicum for n-butanol production. As this method has not been described in detail and to make it available to and usable by the scientific community, we report how this method was developed and its use to create a restriction-less, marker-less strain of C. acetobutylicum. We show that this strain lacking upp (CA_C2879, encoding the uracil–phosphoribosyltransferase), CA_C1502 encoding Cac824I and CA_C3535 encoding Cac824II can be transformed by non-methylated DNA at very high efficiency and can be used for rapid gene knock-in and knock-out using the upp/5-fluoro uracil (5-FU) counterselectable system for both functional genomic and metabolic engineering of C. acetobutylicum
Summary
Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of convert‐ ing various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). Clostridium acetobutylicum ATCC824 has been of interest in the postgenomic era due to the complete sequencing and annotation of its genome [1], supplying a wealth of information regarding its protein machinery This global knowledge has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering in order to develop industrial strains for the production of biofuels and bulk chemicals. To this end, several reverse genetic tools have been developed for C. acetobutylicum ATCC 824, including a gene inactivation system based on non-replicative [2, 3] and replicative plasmids [4,5,6,7] and the group II intron gene inactivation system [8, 9]. The second part is a plasmid system with inducible segregational instability, enabling efficient deployment of the FLP-FRT system to generate marker-less deletion or integration mutants
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