Engineering of an environmental isolate of bacillus megaterium for biochemical production under supercritical CO2
July 14-18, 2019
Continuous processing is a mainstay for chemical production but is far less common for biochemical processes. The increase in productivity and corresponding decrease in costs make continuous processing an intriguing option for bulk chemicals where price is a major consideration. Among the various challenges of continuous bioprocessing are the risks of contamination and the toxicity of the target products. Supercritical carbon dioxide (scCO2) may provide a means to address both of these issues. scCO2 is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones and aldehydes. scCO2 is also known for its broad microbial lethality. The isolation and engineering of a microbe that is capable of growth and production in the presence of scCO2 thus represents an opportunity to create a production environment that is both resist to contamination and capable of sequestering toxic products through phase separation. Using a targeted bioprospecting approach by sampling fluid from a natural, deep-subsurface scCO2 well, a strain of Bacillus megaterium was isolated that is able to germinate and grow in the presence of scCO2. Transformation is possible using a protoplast-based method, which permitted the identification of promoters capable of inducible heterologous protein expression in both aerobic and anaerobic conditions. A xylose-inducible promoter was evaluated under scCO2 and found to have similar expression under both conditions. We engineered the B. megaterium strain to produce isobutanol from 2-ketoisovalerate by introducing a two-enzyme pathway (2- ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)). Due to the strong partition of the aldehyde to the scCO2 phase, we tested five homologous Adh enzymes and found that YqhD from E. coli resulted in greater than 85% conversion when grown aerobically. Isobutanol production was also observed when our recombinant strain was cultured under scCO2. Finally, we have developed a process model for an integrated bioprocess and have found conditions that are comparable if not better than existing in situ extraction techniques such as gas stripping. Boock, J.T., A.J.E. Freedman, G.A. Tompsett, S.K. Muse, A.J. Allen, L.A. Jackson, B. Castro-Dominguez, M.T. Timko, K.L.J. Prather (co-corresponding author), J.R. Thompson. 2019. “Engineered microbial biofuel production and recovery under supercritical carbon dioxide.” Nat. Commun. 10:587. DOI: 10.1038/s41467- 019-08486-6.
Kristala Jones Prather, Jason T. Boock, Adam J. E. Freedman, Geoffrey Tompsett, Michael T. Timko, and Janelle R. Thompson, "Engineering of an environmental isolate of bacillus megaterium for biochemical production under supercritical CO2" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/71