An efficient method for markerless mutant generation by allelic exchange in and using suicide vectors.

BACKGROUND

and 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 and combines an improved electroporation method with the use of (i) restrictionless Δ (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 (from ) 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 and . Among the edited genes, a mutation in the gene that abolished solvent formation in was introduced in 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.