Genetic modification ofClostridium kluyverifor heterologousn-butanol andn-hexanol production
Abstract
The mesophilic microbeClostridium kluyveriserves as the most commonly used model microbe to elucidate the physiology and biochemistry of ethanol-based chain elongationviareverse ß-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 forC. kluyveri, utilizing the pMTLClostridiashuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system ofC. kluyverias a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern ofC. kluyveri, we performedin-vivomethylation of the shuttle vector plasmid by expressing the methyltransferase inEscherichia coli, followed by DNA transferviaconjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH and NADPH-dependent alcohol dehydrogenases fromClostridium acetobutylicum. The expression of these genes was controlled by the Pthlpromoter, which is commonly used inClostridia,and the PadhE2promoter, leading ton-butanol andn-hexanol production of the mutant strains. This genetic system forC. kluyveriwill not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general.
IMPORTANCE
Medium-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 asClostridium 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 forC. kluyveri, a better understanding of microbial chain elongation can be achieved, and potentially even an extension of its product portfolio.
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