Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii

Abstract

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

Keywords: Caldicellulosiruptor; Caldicellulosiruptor bescii; carbohydrate metabolism; carbohydrate utilization; comparative genomics; lignocellulose degradation; metabolic reconstruction; plant biomass degradation; regulon; transcriptional regulation.

FIG 1

Reconstruction of metabolic pathways and regulons involved in plant polysaccharide degradation and carbohydrate utilization in C. bescii. Polysaccharide substrates are in red bent rectangles. Extracellular enzymes, transporters, and cytoplasmic enzymes are indicated by rectangles with red, blue, and black text, respectively. CAZymes and other classes of enzymes are indicated by large and small arrows, respectively. Novel carbohydrate-specific ABC transporters from the CUT1 and CUT2 families are in boxes with red and green outlines, respectively. The PTS transporter for fructose is in a blue outline box. The genes regulated by the same TF are indicated by matching colored symbols described in the lower inset. The names of differentially expressed (DE) genes on five carbohydrate substrates measured in RNA-Seq experiments are highlighted by light blue (cellulose), green (cellobiose), yellow (glucose), pink (xylan), and pastel blue (xylose). The central carbohydrate metabolism, fermentation, and hydrogen production pathways are indicated by a light yellow background. Detailed information on each gene from the reconstructed CU pathways, including gene IDs, functional roles, etc., is provided in Data Set S1A.

FIG 2

DNA-binding motifs for reconstructed carbohydrate utilization regulons. TFBS sequence logos were built by WebLogo using all candidate TFBSs identified by comparative genomics techniques in C. bescii and related Caldicellulosiruptor genomes. The identified TFBSs have either inverted-repeat (palindromes, represented as a single DNA element) or direct-repeat (tandems, represented by two identical DNA elements with a nonconserved linker of specified length) structures. Predicted repression or activation mechanism of regulation for each TF is specified by circled “R” and “A” letters, respectively.

FIG 3

Genomic context of reconstructed regulons for catabolic and transporter genes involved in glucan, cellulose, mannan and pectin degradation in C. bescii. Carbohydrate utilization genes are indicated by arrows with colors according to the functional gene classification and numbers corresponding to their locus tag with Athe_ prefix. Candidate TF binding sites are indicated by colored symbols and connected to a cognate TF. Putative DNA-binding sites of as-yet-unknown TFs are indicated by circled numbers. Predicted effectors of TFs are given in square brackets. Transcription start sites (TSSs) identified by in RNA-Seq experiments are indicated by standing arrows. Genes upregulated on a particular substrate according to RNA-Seq and their corresponding log₂-fold change are shown in Fig. S3.

FIG 4

Genomic context of reconstructed regulons for catabolic and transporter genes involved in xylan degradation in C. bescii. For abbreviations, see the Fig. 3 legend.

FIG 5

Comparison of growth for the ΔmsmK strain and the parent strain on various carbon substrates. The ΔmsmK strain and the parent strain were grown in biological triplicate (n = 3) at 75°C on a variety of soluble and insoluble carbohydrate substrates. Growth was compared on glucose (A), xylose (B), xylan (C), cellobiose (D), cellulose (E), and pectin (F). Insoluble xylan particles with similar scale to C. bescii cells interfered with direct quantification of growth (counting cells). Therefore, for growth on xylan, the growth was reported as the endpoint (28 h) protein concentration. For all other substrates, growth was reported as the cell density (cells/ml) over the course of growth (24 to 28 h).