Transcriptomic analysis of a Clostridium thermocellum strain engineered to utilize xylose: responses to xylose versus cellobiose feeding

Abstract

Clostridium (Ruminiclostridium) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335's transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log₂ fold change (FC) >|1| and 106 highly DEGs (log₂ FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD, were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes (tktAB) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation-reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.

Figure 1

A volcano plot representing DEGs in the engineered C. thermocellum strain (KJC335) capable of growing on xylose as the main carbon source. DEGs were obtained from comparisons made between cultures grown in D-xylose vs. D-cellobiose. Down-regulated genes (log₂ FC < − 1 and adjusted p value < 0.05) are shown in blue, and up-regulated genes (log₂ FC > 1 and adjusted p value < 0.05) are shown in red. RS numbers in bold correspond to the numerical suffix of locus tags in C. thermocellum DSM 1313 (e.g., RS10310 = CLO1313_RS10310). The figure was created using the R package ggplot2.

Figure 2

Main GO terms for down-regulated genes in KJC335. (A) Biological process. (B) Molecular function. (C) Cellular Component. The figure was created using the R package ggplot2.

Figure 3

Main GO terms for up-regulated genes in KJC335. (A) Biological process, (B) molecular function, (C) cellular component. The figure was created using the R package ggplot2.

Figure 4

Five ATP-binding sugar transporters in C. thermocellum and the putative operons of the transport systems. The numbers in parenthesis are log₂ fold changes. RS numbers in bold correspond to the numerical suffix of locus tags in C. thermocellum DSM 1313 (e.g., RS09235 = CLO1313_RS09235). Previous characterization of cellodextrin-binding proteins (Cbp) subunits revealed that CbpA binds only to cellotriose (G3), CbpB binds to cellodextrins ranging from cellobiose to a cellopentose (G2-G5), while CbpC and CbpD preferentially bind to cellotriose, -tetrose, and -pentose (G3–G5). Differential fold changes among the putative cbpC, cbpD, and lbp bearing operons suggest that one or more of these solute-binding proteins may bind promiscuously to xylose and facilitate xylose transport. Abbreviations: msd for membrane-spanning domain, nbd for nucleotide-binding domain, tktA for transketolase subunit A, tktB for transketolase subunit B, xdh for xylitol dehydrogenase.

Figure 5

Effect of overexpressing transketolase in KJC335 on growth when either cellobiose or xylose is provided as the primary carbon source. (A) Constructs of a control plasmid pKJC84, which does not bear transketolase genes, and a plasmid, pTC131374-5, expressing tktAB genes. The non-coding regions upstream of glyceraldehyde phosphate dehydrogenase gene, also referred to as the “gapDH promoter,” is transcriptionally fused to the genes shown in the synthetic operons. Chloramphenicol resistance gene (CAT) is used for selection of the plasmid using thiamphenicol, a thermostable analog of chloramphenicol. (B) The growth curves of strains with or without expressing the tktAB genes on 5 g/L of cellobiose. (C) Concentrations of major metabolic end-products measured at the end of cellobiose fermentation shown in (B). (D) The growth curves of strains with or without expressing the tktAB genes on 5 g/L of xylose. (E) Concentrations of major metabolic end-products measured at the end of xylose fermentation shown in (D) (n ≥ 3, data reported as average ± SD). Student’s t tests were performed to assess significance, where (*) corresponds to a p value < 0.05, (**) to a p value < 0.01, (***) to a p value < 0.001, and (****) p value < 0.0001.

Figure 6

Proposed D-xylose utilization pathway in KJC335. Previous engineered genes from T. ethanolicus are shown in blue, and genes and pathways natives to C. thermocellum are in red. The numbers in parenthesis are log₂ fold changes. RS numbers in bold correspond to the numerical suffix of locus tags in C. thermocellum DSM 1313 (e.g., RS00385 = CLO1313_RS00385).

Figure 7

A general scheme for bacterial chemotaxis. The numbers in parenthesis are log₂ FC. RS numbers in blue are the numerical suffix of locus tags in C. thermocellum DSM 1313 (e.g., RS08740 = CLO1313_RS08740). Blue boxed represents proteins that are grouped based on the KEGG map. Arrows represent activation, whereas the “T” shaped stick represents inactivation. The line with a “-” represents dissociation. The symbol “ + p” denotes phosphorylation and “-p” dephosphorylation. This general scheme is adapted from KEGG maps with modifications^,. MCP symbolizes methyl-accepting chemotaxis proteins, the Che genes are involved in chemotaxis, Mot genes are involved in motility, and FliM is a flagellar motor switch protein.