Functional genomics of sporulation and microcompartment formation in Clostridium beijerinckii

Clostridium beijerinckii has been an organism of great interest for industrial biotechnology for about a century because it can degrade a wide range of sugars to produce acetone, butanol, ethanol, and isopropanol, chemicals used in consumer goods (paints, cosmetics, food ingredients) and as alternatives to transport fuel. Still, C. beijerinckii has other less studied physiological traits, such as the formation of intracellular compartments, spores and microcompartments. Therefore, we decided to study sporulation and microcompartment formation in C. beijerinckii using functional genomics.Sporulation is a cell differentiation process that occurs in all Firmicutes. It is triggered when the cells are exposed to an unfavorable environmental condition (presence of oxygen, high pH variation), and yields spores, metabolically inactive cells resistant to harsh conditions (UV, chemicals, heat, oxygen). In Chapter 1, the research that has been done in the last 30 years on sporulation in solvent-producing clostridia, including the latest updates on the molecular regulation of sporulation in solventogenic clostridia, is presented. Common triggers and regulation mechanisms of sporulation in several solventogenic species are identified, underlining the differences and similarities between species. Potential links in the regulation of sporulation and solvent production are pinpointed. This chapter highlights the need for more studies on signal transduction pathways, transcriptional and post-translational regulation of sporulation, and solvent production. A better understanding of the connections between both physiological phenomena would provide us with relevant targets for strain engineering of solventogenic clostridia.Metabolic engineering of Clostridium used to be tedious and hampered the studies on strain physiology. An efficient and fast engineering tool was highly desired. Chapter 2 describes the adaptation of a CRISPR-Cas9 method for use in Clostridium beijerinckii. The tool