Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture.

Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate](int) and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium.

Dusséaux S ，Croux C ，Soucaille P ，Meynial-Salles I ... -  《-》

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation.

Lee J ，Jang YS ，Choi SJ ，Im JA ，Song H ，Cho JH ，Seung do Y ，Papoutsakis ET ，Bennett GN ，Lee SY ... -  《-》

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol-butanol-ethanol fuel mixture.

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone-butanol-ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol-butanol-ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab-scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot-scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab-scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production.

Jang YS ，Malaviya A ，Lee J ，Im JA ，Lee SY ，Lee J ，Eom MH ，Cho JH ，Seung do Y ... -  《-》

Chromosomal integration of the pSOL1 megaplasmid of Clostridium acetobutylicum for continuous and stable advanced biofuels production.

Biofuel production by Clostridium acetobutylicum is compromised by strain degeneration due to loss of its pSOL1 megaplasmid. Here we used engineering biology to stably integrate pSOL1 into the chromosome together with a synthetic isopropanol pathway. In a membrane bioreactor continuously fed with glucose mineral medium, the final strain produced advanced biofuels, n-butanol and isopropanol, at high yield (0.31 g g-1), titre (15.4 g l-1) and productivity (15.5 g l-1 h-1) without degeneration.

Ehsaan M ，Yoo M ，Kuit W ，Foulquier C ，Soucaille P ，Minton NP ... -  《Nature Microbiology》

Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum.

Jang YS ，Lee JY ，Lee J ，Park JH ，Im JA ，Eom MH ，Lee J ，Lee SH ，Song H ，Cho JH ，Seung do Y ，Lee SY ... -  《mBio》