English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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  Cac 824I restriction endonuclease encoding gene ( CA_C1502 ) and the  upp  gene ( CA_C2879 ) in the  C. acetobutylicum  ATCC824 chromosome. The resulting  C. acetobutylicum  Δ cac1502 Δ upp  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 ( Cac 824II) 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.

biotechnology for biofuels

Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum