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Background   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 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.    Results  The 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 Δ upp  (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.    Conclusions  The 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.

biotechnology for biofuels

An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors