CRISPR/Cas9n knock-out plasmid construction and transformation into Clostridium autoethanogenum for deletion of a formate dehydrogenase gene

Abstract

Gas fermentation using acetogen bacteria is an attractive technology for tackling the problem of the accumulation of solid waste and greenhouse gases while contributing towards a circular economy through sustainable production of chemicals and fuels. Acetogens are the preferred biocatalysts for gas fermentation as they can use gases such as carbon monoxide (CO) and carbon dioxide (CO2) as their sole carbon source. They fix carbon oxides via the Wood-Ljungdahl pathway (WLP), but specific functionalities of several key enzymes in the WLP are unclear. This thesis constructed a CRISPR/Cas9 nickase (Cas9n) plasmid for targeted deletion of the formate dehydrogenase gene fdhA in the model-acetogen Clostridium autoethanogenum that can catalyse the reduction of CO2 to formate in the WLP. The fdhA knock-out plasmid was constructed using PCR-based cloning methods and InFusion cloning. The latter was also used to insert homology arms flanking the fdhA gene into the knock-out plasmid that are needed to facilitate gene deletion via homologous recombination in C. autoethanogenum. The plasmid was successfully transformed into C. autoethanogenum but fdhA deletion-bearing cells were not identified. Future work includes sub-culturing and plating the transformed cells to isolate fdhA knock-out cells, plasmid curing, and growth characterisation of the fdhA knock-out strain. This thesis provides both an intermediate strain for further study and valuable information for optimisation of future genetic engineering efforts.