In late 2012, A new discovery should make the alternative fuel butanol more attractive to the biofuel industry.

Scientist Hao Feng has found a way around the bottleneck that has frustrated producers in the past and could significantly reduce the cost of the energy involved in making it as well. His team was able to isolate the butanol molecules during the fermentation process so it does not kill the organisms and produces 100% or more butanol. After the fermentation process, they used a process called cloud point separation to recover the butanol which used 4 times less energy.[17] Also in late 2012, utilizing systems metabolic engineering, a Korean research team at the formally the Korea Advanced Institute of Science and Technology (KAIST) has succeeded in demonstrating an optimized process to increase butanol production by generating an engineered bacterium. Professor Sang Yup Lee at the Department of Chemical and Biomolecular Engineering, KAIST, Dr. Do Young Seung at GS Caltex, a large oil refining company in Korea, and Dr. Yu-Sin Jang at BioFuelChem, a startup butanol company in Korea, applied a systems metabolic engineering approach to improve the production of butanol through enhancing the performance of Clostridium acetobutylicum, one of the best known butanol-producing bacteria. In addition, the downstream process was optimized and an in situ recovery process was integrated to achieve higher butanol titer, yield, and productivity. The combination of systems metabolic engineering and bioprocess optimization resulted in the development of a process capable of producing more than 585 g of butanol from 1.8 kg of glucose, which allows the production of this important industrial solvent and advanced biofuel to be cost competitive.[18] The anaerobic bacteria C. pasteurianum, C. acetobutylicum, and other Clostridium species have metabolic pathways that convert glycerol to butanol through fermentation. However, the production of butanol from glycerol by fermentation in C. Pasteurianum is low. To counter this, a group of researchers used chemical mutagenesis to create a hyper butanol producing strain. The best mutant strain in this study "MBEL_GLY2" produced 10.8 grams butanol per 80 gram glycerol fed to the bacteria. This is an improvement compared to the 7.6 g butanol produced by the native bacteria.[14] Many organisms have the capacity to produce butanol utilizing an acetyl-CoA dependent pathway. The main problem with this pathway is the first reaction involving the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. This reaction is thermodynamically unfavorable due to the positive Gibbs free energy associated with it (dG = 6.8 kcal/mol).[19] Some experimentation has been done that involves increasing the carbon storage through the organism by utilizing carbon dioxide flow through photosynthetic organisms. To follow in this path of research, scientists have attempted to engineer reaction pathways that can enable photosynthetic organisms (like blue-green algae) to produce butanol more efficiently.[20] A study done by Ethan I. Lan and James C. Liao attempted to utilize the ATP produced during photosynthesis in blue-green algae to work around the thermodynamically unfavorable acetylCoA condensation to acetoacetyl-CoA. The native system was re-engineered to have acetyl-CoA react with ATP and CO2 to form an intermediate, malonyl-CoA. Malonyl-CoA then reacts with another acetyl-CoA to form the desired acetoacetyl-CoA. The energy release from ATP hydrolysis (dG = -7.3 kcal/mol) makes this pathway significantly more favorable than standard condensation. Because blue-green algae generate NADPH during photosynthesis, it can be

assumed that the cofactor environment is NADPH rich. Therefore, the native reaction pathway was further engineered to use NADPH rather than the standard NADH. All of these adjustments led to a 4-fold increase in butanol production, showing the importance of ATP and cofactor driving forces as a design principle in pathway engineering