Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic <i>Caldicellulosiruptorales</i>

Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The order <i>Caldicellulosiruptorales</i> has drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction of <i>Anaerocellum (f. Caldicellulosiruptor) bescii</i> identified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair within <i>A. bescii</i> is conserved in all sequenced species of <i>Caldicellulosiruptorales</i>, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes for <i>A. bescii</i>. These results show that two separate regulatory schemes (dual repression) are coordinated by <i>A. bescii</i> to effectively regulate the hemicellulose inventory and xylan catabolism.IMPORTANCETo take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned. <i>Anaerocellum bescii</i>, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.