This presentation will review METabolic EXplorer progress in designing optimal pathways towards cost effective production of n-butanol using Clostridium acetobutylicum. An overview of our intellectual property recently developed in this field will highlight the different strategies selected and underline the most effective one.
1- Development of a gene deletion/integration method without marker in C. acetobutylicum
We have developed an original method based on i) deletion and replacement of the target gene by an antibiotic resistance gene by a double crossover integration through homologous recombination of a replicative integrative plasmid, giving  segregationally highly stable mutants, ii) removing of the antibiotic resistance gene with the Flp recombinase system from Saccharomyces cerevisiae allowing the repeated use of the method for rapid construction of multiple, unmarked mutations in the same strain and iii) a C. acetobutylicum strain deleted for the upp gene, encoding uracil phosphoribosyl transferase, thus allowing the use of 5-Fluorouracil (5-FU) as a counter selectable marker and a positive selection of the double crossover integrants.
2- Metabolic engineering of C. acetobutylicum for the production of butanol at high yield
Using the method described above, we engineered the central metabolism of C. acetobutylicum to produce butanol at high yield. Results of batch and continuous cultures of the engineered strain will be presented.