| Clostridia species can potentially be industrial-hosts for sustainable biofuel and chemical production. However, the metabolic engineering tools for clostridia are limiting. Subsequently, we developed and applied differentiation engineering to improve butanol production in Clostridium acetobutylicum.;Previous analysis of the spo0A knockout, the master transcriptional regulator (Harris et al., 2002), and detailed transcriptional analysis of clostridial sporulation (Jones et al., 2008), suggest that a transcription regulator, downstream (temporally) of Spo0A, likely blocks sporulation and preserves solventogenesis. Subsequently, we knocked out sigE and sigG by a novel gene disruption approach. The DeltasigE strain stalled sporulation prior to stage II, resulting in only vegetative-like phenotypes. The DeltasigE cells did not synthesize granulose, but did produce solvents, albeit in a bifurcating mode (normal versus low levels) that was dependent on the physiological state of the inoculum. The Delta sigG strain stalled sporulation at stage V, exhibiting engulfment and partial cortex and spore-coat formation, and displayed enhanced solventogenesis.;As mentioned, I invented a novel gene disruption approach that is applicable to all Clostridia. Previous attempts to disrupt genes in any Clostridia were inefficient, which we argue is due to the lack of a resolvase protein. By over-expressing the Bacillus subtilis resolvase, RecU, from a replicating gene disruption plasmid, we successfully knocked out spo0A by Campbell-like double crossover integration and sigE and sigG by single crossover integration. Characteristics of the disrupting plasmid that affected the efficiency of integration were investigated. Integration was most efficient when the repL origin of replication was in the opposite coding strand of the disrupting antibiotic marker, and when the disrupting antibiotic marker was in the same coding strand as the gene targeted for disruption.;Lastly, we attempted to determine the minimal genetic loci on the pSOL1 megaplasmid necessary for sporulation by constructing a functional pSOL1 complementation library in degenerate strains of C. acetobutylicum. Assuming the loci could be determined, they could serve as additional differentiation engineering targets to abolish sporulation and maintain solventogenesis. Thus far, we have been unsuccessful, and I suggest a pSOL1 specific gene knockout library in wild-type cells to determine these loci. |
