(582cb) Engineering Acetogenic Clostridia for Ethanol and n-Butanol Production From CO2

Some Clostridia, including C. ljungdahlii and C. carboxidivorans, are capable of producing ethanol and butanol from CO₂ and H₂. However, the alcohol productivity, yield, and titer from these native strains are low and uneconomical for industrial applications. In contrast, several acetogenic Clostridia can convert CO₂ and H₂ to acetate with high yield, titer, and productivity comparable to those from glucose as the substrate. A high metabolic flux from CO₂ to acetyl-CoA, the immediate precursor for both acetate and ethanol, is the prerequisite for developing a highly productive ethanol and butanol producers. In this study, C. aceticum was used as a host to produce ethanol by expressing adhE2 gene under the control of the thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from C. difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and ethanol production by different mutants of C. aceticum were investigated. Ethanol production from diverse electron donors, including CO₂ and H₂, CO₂ and glycerol, cellobiose, xylose, fructose, and glucose were then investigated with the best mutant strain harboring adhE2. We also engineered C. aceticum for n-butanol production by introducing butanol biosynthesis pathway genes (thl, hbd, crt, bcd-etfB-etfA, and adhE2) using modular clostridia plasmids. We then knocked out genes (pta and ack) in acetate biosynthesis pathway to further enhance ethanol and butanol production. The fermentation kinetics of these engineered mutants and their ability to produce ethanol and butanol from diverse electron donors were studied and will be presented in this paper.