(597c) Improving C4 to C2 Ratio for n-Butanol Production in Mixotrophic Fermentation By Engineered Clostridium Carboxidivorans

Nowadays, biobutanol has become a promising biofuel for its advantages over ethanol such as higher energy density, lower volatility, lower hygroscopicity, and excellent compatibility with vehicle engines and existing fuel infrastructures. It is considered as an important chemical in industry and has a high chance to replace gasoline in the near future. In order to commercialize bio-butanol on a large scale, its production cost must be sufficiently reduced. Since raw materials and their pretreatment cost more than half of overall operating expenses, we should maximize the conversion of raw materials into the product of interest. However, nearly one-third of the carbon in the feedstock is lost as CO₂ in the traditional acetone-butanol-ethanol (ABE) fermentation process, rendering a low mass yield. The high feedstock cost and low mass yield cause bio-butanol production uneconomical. C. carboxidivorans can naturally recapture evolved carbon dioxide using the most energy efficient carbon fixation pathway -- Wood–Ljungdahl pathway in a mixotrophic fermentation, where carbon dioxide and electrons generated from glycolysis are converted into additional acetyl-CoA, thus achieving a higher butanol yield.

In this study, metabolic engineering was successfully used to enhance bio-butanol production by C. carboxidivorans. Heterologoues thiolase (thl), hydroxybutyryl-CoA dehydrogenase (hbd), and alcohol/aldehyde dehydrogenase (adhE2) from other Clostridium species were introduced into C. carboxidivorans to increase C4/C2 ratio and butanol production in both autotrophic and mixotrophic fermentations. The effects of these mutations on flux distributions and redox balance were analyzed in a genome-scale metabolic model, and the results can be used for further metabolic engineering. This study demonstrated that C. carboxidivorans is a promising host for bio-butanol production from low-cost feedstocks and its use can reduce CO₂ emission.