Abstract
BackgroundClostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes 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, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect.ConclusionsThe method described in this study 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 and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents
Transformation efficiency of different industrially relevant solventogenic Clostridia In a previous study [10], we demonstrated that a restrictionless mutant of C. acetobutylicum could be transformed by electroporation with unmethylated pCons2.1 at very high efficiency (6 × 104 transformants/μg of unmethylated DNA)
The restrictionless, markerless generic method for genome modification in C. acetobutylicum and C. saccharobutylicum is a simple and useful tool for research groups involved in functional genomic studies and for further
Summary
Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The first family [7, 10] uses a replicative vector containing (i) a replacement cassette consisting of an antibiotic resistance gene (ThR) flanked by two FRT sequences, (ii) two sequences homologous to the selected regions around the target DNA sequence, and (iii) a counterselectable marker made either of the codon-optimized mazF toxin gene from Escherichia coli (under the control of a lactose-inducible promoter) or the upp gene [which encodes an uracil phosphoribosyl-transferase and leads to 5-fluorouracil (5-FU) toxicity] to allow the direct positive selection of double-crossover allelic exchange mutants After this first step, a second plasmid system expressing the FLP recombinase must be introduced, enabling efficient deployment of the FLP–FRT system to generate markerless deletion or integration mutants. A scar consisting of an FRT site remains at the target site, which can potentially act as a transcriptional terminator [15] or create a large chromosomal DNA deletion or inversion when several FRT sites are present on the chromosome [16, 17]
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