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 was initially developed for Clostridium acetobutylicum, but several modifications of the tool were necessary for use in C. beijerinckii. This method allows the deletion and insertion of genes in C. beijerinckii NCIMB 8052 and C. beijerinckii NRRL B593, an isopropanol-producing strain.

The method developed in Chapter 2 was used to generate a SpoIIE deficient mutant in C. beijerinckii NCIMB 8052. The mutant phenotype is described in Chapter 3. Morphology, fermentation profile, and transcriptome of the mutant were investigated and compared to the wild-type. Thanks to this mutant strain, we could study the impact of the interruption of the sporulation cycle before forespore formation on other cellular events.

The mutant investigated in Chapter 3 was not the only SpoIIE deficient isolate we obtained. Five SpoIIE deficient strains were isolated, and each of them had a slightly different genotype, but all of them were asporogenic. Four of the five strains showed a solventogenic phenotype, and one was deficient in solvent production. The characterization of a non-solventogenic SpoIIE deficient strain is reported in Chapter 4. Morphology, fermentation and transcriptome were compared to the solvent producing ΔspoIIE strain described in Chapter 3.

Apart from spores, C. beijerinckii can form other intracellular compartments called bacterial microcompartments (BMCs). Like spores, BMCs are formed due to changes in the environment and, more precisely, when C. beijerinckii metabolizes L-rhamnose. In Chapter 5, the metabolism of C. beijerinckii NRRL B593 on rhamnose was investigated with respect to its fermentation profile, product formation, transcriptome.

Chapter 6 summarizes the results described in this thesis and discusses their contribution to Clostridium research in general. Moreover, an outlook on further studies on the physiology of industrially relevant clostridia is given. Based on the work described in this thesis, three interesting future research lines are presented.