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Abstract
The growing global population and the rising middle class in developing countries drive the demand for consumer goods. However, the manufacture of these products is entirely resting on the petrochemical industry. Since fossil fuel resources are limited, there is a great need for sustainable alternatives. In the search for alternatives to petrochemicals, the interest in bioprocesses is growing. The production of the solvents acetone, butanol and ethanol (ABE) by fermentation of renewable resources is one of the bioprocesses being explored currently by industry and academics. The ABE fermentation converts a wide range of carbon sources such as organic wastes and complex sugars to solvents by natural solvent producing bacteria, called solventogenic clostridia.
Clostridia are gram-positive anaerobic bacteria that form spores. The Clostridium genus harbors around 250 species, among which 10 species can utilize sugars to produce organic solvents such as acetone, isopropanol, ethanol or butanol. These chemicals are commonly sold as paint thinners, glue additives or perfumes. Even so, the ABE fermentation process is not cost-competitive with the petrochemical processes currently used to manufacture these products. Therefore, researchers are looking for ways to improve the process and the strains used. One of the approaches investigated is strain engineering. However, the regulation and triggers of the main physiological traits (sporulation and solvent production) are not fully understood. A better knowledge of the physiology of solventogenic clostridia is necessary to generate more productive strains. This thesis describes the research on the physiology of Clostridium beijerinckii, which is the most studied solventogenic Clostridium after Clostridium acetobutylicum.Two intriguing physiological processes were tackled: firstly, the regulation of the sporulation, which is intimately coupled to solventogenesis and secondly, the metabolism of L-rhamnose, which involves the generation of particular cellular compartments called bacterial microcompartments (BMC).
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). The spores are metabolically inactive and resistant to harsh conditions (UV, chemicals, heat, oxygen). Sporulation is seen as a hindrance, in industry, for several reasons: it occurs during the fermentation, stops the solvent production, and is energy costly for the cells. The regulation of sporulation and its connection to solvent production is still not elucidated. In Chapter 1,the research done in the last 30 years on sporulation in solventogenic clostridia is reviewed, and the latest updates on the molecular regulation of sporulation in solventogenic clostridia are presented. Common triggers and regulation mechanisms of sporulation in several solventogenic species are identified, underlining the differences and similarities between the various species. Potential links in the regulation of sporulation and solvent production are pinpointed. Around fifty non-sporulating mutant strains have been reported and studied in the literature, with, for most of these strains differences in solvent productivity compared to the wild-type strain. This chapter highlights the need for more studies on signal transduction pathways, transcriptional and posttranslational 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.
Despite being studied since the 1920s, many unknowns remain in our understanding of the physiology of clostridia. These gaps in the knowledge can be linked to the lack of efficient genome engineering tools for clostridia. Recent developments in CRISPR technologies have opened new possibilities for developing and improving genome editing tools dedicated to the Clostridium genus. A two-plasmid CRISPR genome engineering system is described in Chapter 2. This tool enabled scarless modification of the genome of two reference strains of Clostridium beijerinckii, the ABE-producing strain NCIMB 8052 and the natural IBE-producing strain DSM 6423. As a proof of principle, we performed gene deletions in both strains and even cured the endogenous pNF2 plasmid of the DSM 6423 strain. In the NCIMB 8052 strain, the disruption of the spoIIE coding sequence resulted in sporulation-deficient mutants. This phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. The fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar approach was employed to engineer the genome of C. beijerinckii DSM 6423. The catB gene conferring thiamphenicol resistance was deleted with a single plasmid CRISPR tool to make this strain compatible with our dual-plasmid editing system. The dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatBto remove the endogenous pNF2 plasmid, which increased transformation efficiencies.
