Characterization and Metabolic Engineering of Transcription Factors and Redox Dynamics in Candidate Consolidated Bioprocessing Biocatalysts

This thesis studies the metabolic engineering of candidate consolidated bioprocessing biocatalyst microorganisms through targeting regulatory genes, with an emphasis on redox metabolism. Consolidated bioprocessing is the single-step hydrolysis and conversion of lignocellulosic material to biofuels. The biocatalysts considered are Clostridium thermocellum and Caldicellulosiruptor bescii, and the primary product of interest is ethanol. Both organisms are thermophilic anaerobic bacteria which encode and express genes that facilitate the deconstruction and solubilization of lignocellulose into fermentable carbohydrates. Furthermore, these organisms ferment these carbohydrates into ethanol, organic acids, as well as other fermentation products. We seek to improve redox metabolism and osmotolerance in these organsisms toward a biorefining objective goal of engineering a biocatalyst capable of facilitating economically viable consolidated bioprocessing.Expression profiling, transcription factor regulon mapping, genetic engineering, and analytical fermentation were approaches employed to assay and understand which specific traits can be beneficially altered. The traits sought to be altered are characteristically complex, co-opting many cellular sub-processes to enable a molecular mechanism resulting in an observable trait. Such traits are notoriously difficult not only to understand, but to alter through classical metabolic engineering. Instead, the possibility of making system-wide changes through a minimal number of genetic alterations to methodically selected and/or screened regulatory genes was investigated.Active redox-dependent systems were characterized in both bacteria, many of which are controlled by the global redox-state sensing transcription factor Rex. Eliminating Rex control over gene expression in C. bescii resulted in a more reduced intracellular redox state, and ultimately drives increased ethanol synthesis. A method for quantifying important redox metabolites intracellularly is also adopted and validated for use with C. thermocellum. This approach was extended to less characterized gene targets and, arguably, even more complex traits. Screening of single-gene deletion mutants identified two strains of C. bescii showing phenotypic growth differences in elevated osmolarity conditions. One strain housed a deletion of the fapR gene, while the other a deletion of the fruR/cra gene. Characterizing these transcription factors and their regulons elucidates mechanisms which this organism uses to facilitate survival at elevated osmolarities. We are also able to construct genetic variants in C. bescii which are substantially more osmotolerant than native strains, highlighting the usefulness of these genes as targets and the applicability, and important considerations, of our metabolic engineering approach.