Genetic modification of Clostridium kluyveri for heterologous n-butanol and n-hexanol production

The mesophilic microbe Clostridium kluyveri serves as the most commonly used model microbe to elucidate the physiology and biochemistry of ethanol-based chain elongation via reverse {beta}-oxidation. In this pathway, ethanol and acetate are converted into short- and medium-chain carboxylates. However, to date, no genetic system has been published in a peer-reviewed publication. Here, we report the development of versatile genetic tools for C. kluyveri, utilizing the pMTL Clostridia shuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system of C. kluyveri as a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern of C. kluyveri, we performed in-vivo methylation of the shuttle vector plasmid by expressing the methyltransferase in Escherichia coli, followed by DNA transfer via conjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH and NADPH-dependent alcohol dehydrogenases from Clostridium acetobutylicum. The expression of these genes was controlled by the Pthl promoter, which is commonly used in Clostridia, and the PadhE2 promoter, leading to n-butanol and n-hexanol production of the mutant strains. This genetic system for C. kluyveri will not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general. IMPORTANCEMedium-chain carboxylates are required in various everyday products, including cosmetics, pharmaceuticals, and fragrances, and show a natural antimicrobial property. Further, they represent food additives and serve as chemical building blocks for several other compounds. Traditionally, these carboxylates are produced from fossil resources, contributing to increased greenhouse gas emissions. Alternatively, they are derived from animal- or plant-based fat (e.g., coconut oil), which competes with agricultural land that is needed for food production. However, microbial chain elongation, which is a biotechnological approach relying on microbes, such as Clostridium kluyveri, is sustainable and a promising alternative to the conventional production of medium-chain carboxylates. Notably, it enables the use of industrial waste streams (e.g., off-gases, carbohydrate-rich industrial waste) as substrates, making the process more environmentally friendly. By applying our genetic system for C. kluyveri, a better understanding of microbial chain elongation can be achieved, and potentially even an extension of its product portfolio.