Expansion of the genome editing toolbox of Clostridium autoethanogenum

Current environmental crises – from rising global temperatures to biodiversity collapse – have created an urgent need for new, circular and sustainable ways of producing and recycling materials and energy independently of fossil resources. Gas fermentation, by consuming greenhouse gases to produce fuels and chemicals, offers human society an attractive route to sustainability. This thesis describes the development of genome editing tools to facilitate the engineering of the gas fermenting Clostridium autoethanogenum. Initial studies illustrated, for the first time, the concept of genomic Bookmarks, which streamline complementation studies in any organism compatible with Cas9-mediated homology-directed mutagenesis. An array of nine of these 24 nt sequences were inserted in the genome of C. autoethanogenum in place of the pyrE gene. Each was subsequently targeted in a second round of Cas9-mediated homology-directed mutagenesis to restore the pyrE locus, generating a perfect complemented strain. Then, a mysterious drop in the efficiency of gene transfer was subjected to an in-depth analysis. The absence of mutations uncovered by whole genome sequencing suggested that epigenetic changes likely affect the conjugation efficiency of donor and/or recipient cells. By inoculating the donor strain directly from the transformation plate and significantly reducing the number of donor cells, improved conjugation efficiencies were reliably restored. Finally, Target-AID, a state-of-the-art genome editing tool that creates premature STOP codons by substituting selected C nucleotides with T, was exemplified in C. autoethanogenum. While its initial application to the pyrE locus was inconclusive, its application to knock out three genes encoding distinct alcohol dehydrogenases was successful. Up to two targeted mutations could be carried out simultaneously, although three should be possible. In the process, the targeting space of Target-AID was expanded to encompass NG and NAA protospacer adjacent motives (PAM) and showed that an array of multiple sgRNA expression cassettes was a better multiplexing strategy than a single expression cassette processed by intervening tRNAs or direct repeats (DR) from the native Streptococcus pyogenes clustered regularly interspersed repeat (CRISPR) system. The research presented in this thesis provides valuable tools and insights which will help engineering C. autoethanogenum and many other organisms. Among other biotechnological applications, this research could thus facilitate the emergence of industrial processes with a lower environmental footprint