Deregulation of the Caldicellulosiruptor bescii Rex regulon and methods to quantify intracellular redox and energy metabolites

Branched and redundant fermentation pathways in bacterial metabolisms can make Le-Chatlier channeling of resources toward a single end-product through genetic manipulations of enzymes alone difficult.  We propose, instead, altering metabolic regulation of fermentation pathways to effect bioproductivity.  We aim to demonstrate this by disrupting the rex gene in Caldicellulosiruptor bescii, a promising consolidated bioprocessing organism.  Fermentation product profiles have been shown to be impacted by redox perturbations in closely related Caldicellulosiruptor species.  Rex is a redox-sensing global transcription factor, which responds to the intracellular state of the NADH/NAD+ redox couple.  In order to better understand Caldicellulosiruptor redox metabolism and regulation we generated two C. bescii rex knockout mutants, in different fermentative backgrounds.  We will report a bioinformatically predicted Rex binding sites, together with RNA-Seq analyses and describe binding sites confirmed through in vitro electromobility shift assays.

We have also conducted an investigation aimed at attaining the best possible extraction and quantitation of intracellular redox and energy metabolites NAD(P)+, NAD(P)H, A(T,D,M)P, which will enable us to elucidate correlations between NADH/NAD+ ratio and fermentation product formation dynamics, while simultaneously monitoring labile chemical degradation.  We have assessed our protocols on the basis of yield losses, extraction and quantification efficacy and sensitivity.  We are able to reproducibly extract and quantify these metabolites while maintaining physiological adenylate charge values.  Further, we have validated our handling, storage and detection methods to preclude degradation, recovery losses and solvent induced ion-suppression.  Preliminary intracellular redox characterizations for several bacteria with altered redox metabolisms will be reported.