Bioconversion of Methane to Butanol By Metabolic Engineering of Methanosarcina Acetivorans and Escherichia coli

Microbial bioconversion has been widely studied for the production of biofuels, an alternative to current fuel technologies. One target substrate for these processes is natural gas, a major energy resource primarily composed of methane, which can be converted to liquid alcohols, a more attractive fuel source. A scheme that converts methane to liquid fuel using bioconversion would combine two critical attributes for competitive fuel production: a higher energy density product and a highly specific and efficient process. A two-step process is being developed for the conversion of methane to butanol that involves two organisms, the anaerobic archaeon Methanosarcina acetivorans and the eubacterium Escherichia coli. Methane consumption and methanol production will be accomplished by an engineered M. acetivorans strain, while second conversion of methanol to butanol will be accomplished by an engineered E. coli strain. Strain development of both organisms is guided by modern metabolic engineering technologies including ¹³C metabolic flux analysis (¹³C-MFA). So far, a network model for M. acetivorans was developed and validated and the dynamics of methane metabolism were elucidated. Additionally, methanol consumption and utilization by E. coli was engineered and confirmed with ¹³C-tracers. The use of a co-substrate with methanol was studied as well, which may be more economically viable than fermentation of methanol alone. To this end, flux analysis was performed on E. coli under various conditions using glucose and xylose as carbon sources to identify optimal conditions and co-substrates for methanol consumption. The simultaneous co-utilization of these sugars with methanol is also studied through ¹³C-flux analysis. Further optimization of the process consists of using ¹³C-MFA to determine fluxes of the engineered strains and drive carbon flow towards the desired product. Not only will the development of these engineered organisms produce an economical form of fuel production, but it will also demonstrate the capabilities of bioconversion and pave the way for further opportunities towards alternative fuels.