Effect of Oxygen Availability on n-Caprylate Production from Ethanol and Acetate

The absence of efficient genetic tools has long hindered the metabolic engineering of Clostridium kluyveri, which, due to its high production rates of medium- and short-chain carboxylates, serves as a model microbe for ethanol-based chain elongation. To overcome this bottleneck, we developed a genetic system tailored to the specific requirements of C. kluyveri. It enables reliable plasmid transfer and heterologous gene expression, opening new opportunities for engineering the metabolic pathways of this organism. We designed this system to be compatible with anoxic conditions, and it accommodates regulatory elements for genetic manipulation. As a proof of concept, we applied this system to enable the conversion of carboxylates into alcohols. We constructed and tested different gene-promoter combinations, integrating both NADH- and NADPH- dependent alcohol dehydrogenases from Clostridium acetobutylicum in combination with different constitutive promoter to control the expression levels. While all tested plasmid constructs resulted in detectable alcohol production, the efficiency varied considerably between constructs, with some combinations substantially improving conversion rates. Overall, our findings demonstrate the effectiveness of the developed toolkit and its promising potential for expanding the metabolic capabilities of C. kluyveri. In conclusion, this work establishes a robust genetic tool for C. kluyveri and demonstrates its applicability for future synthetic biology and biotechnological applications in this model microbe.