(773a) Engineering an Environmentally-Isolated Bacterium for Continuous Biofuel Production and Recovery Under Supercritical CO2

Model organisms such as E. coli and S. cerevisiae are the workhorses of biotechnological product generation due to their well understood and easily manipulated genetics, fast doubling times, and low cost fermentation media. However, bioprocesses utilizing these hosts are prone to contamination and are negatively affected by product toxicity. Environmental strain isolation provides an opportunity to discover alternative organisms with unique growth characteristics and physiological traits to overcome these long-standing bioprocess challenges. Using a targeted bioprospecting approach by sampling fluid from a deep subsurface supercritical carbon dioxide (scCO₂) well, a bacterium was isolated that is rarely able to demonstrate consistent, robust growth in the presence of scCO₂. Due to the broad microbial lethality and solvent chemistry of scCO₂, we hypothesize that a dual-phase reactor of growth media and scCO₂ will simultaneously provide a sterile growth environment and the capacity to continuously strip off strain-produced biofuels (short chain alcohols) to alleviate product toxicity. Additionally, scCO₂ is a sustainable, labile solvent that can be separated from desired products through depressurization, leaving these products at high concentrations and providing advantages over other co-solvent extraction systems. We have begun characterizing this unique host through genome sequencing and by determining optimal growth conditions in various environments. Transformation is possible using a protoplast-based method, which has permitted the identification of promoters capable of inducible heterologous protein expression in both aerobic and anaerobic conditions. We engineered the scCO₂ tolerant strain to produce isobutanol by introducing a two-enzyme (2-ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)) pathway. The two-step conversion occurs at approximately 70-80% from 2-ketoisovalerate when grown aerobically; however, the intermediate aldehyde was found to accumulate at short culture times. Due to the high partition coefficient for the aldehyde to the scCO₂ phase, five alternative alcohol dehydrogenases were tested. A variant was identified that lowered the build-up of isobutyraldehyde and resulted in conversion of 2-ketoisovalerate to isobutanol above 85% in aerobic cultures. Efforts to scale up this process and to develop tools to integrate the biofuel pathway into the genome are currently underway.