Novel genetic tools for Clostridia and a synthetic biology approach to control spore formation in process organisms

Through the application of engineering principles, synthetic biology provides the means to harness the power of living things for the good of humankind. Central to this discipline is the ability to create systems through the assembly of standardised biological parts that may be incorporated into living cells. As a consequence, such synthetic chassis are endowed with biological properties of limitless, beneficial potential in applications as divergent as medicine, energy, food production and the environment. The genus Clostridium holds particular potential as a chassis for synthetic biology applications. The beneficial attributes of its members range from their ability to convert industrial, greenhouse gases into chemicals and fuels to their use as anticancer agents. Despite their tremendous diversity, clostridial species are unified by their ability to form endospores. Dormant, non-reproductive structures, spores are increasingly finding use in a wide range of applications. These seek to exploit their resilient structure and high degree of resistance to all manner of chemical and physical agents. Such properties, however, are also of concern as they could enable recombinant spores to survive for prolonged periods of time if shed into the environment. To anticipate potential risks and facilitate future applications, innovative tools that address the biological containment of spores are therefore needed. Regardless of the extraordinary diversity of clostridia, deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires ever more sophisticated approaches. Hoping to accomplish this, the body of work described in this thesis explored a wide range of inducible systems that enabled precise control of gene expression in clostridia. The utility of these systems, that included riboregulators and a novel dual mechanism that acts at the level of transcription and translation, was exemplified by the creation of a conditionally sporulating strain of Clostridium sporogenes. This clostridial species, the most promising candidate in clostridial-based cancer therapies, was able to sporulate only in the presence of specific ligands, providing a solution to the prevention of the release of recombinant spores into the environment. Additionally, the clostridial synthetic biology toolbox was expanded by developing a novel and universal CRISPR-based genome editing tool. This has provided an extremely rapid and highly effective method for Clostridium engineering in synthetic biology applications