Investigating the Central Metabolism of Clostridium thermosuccinogenes

Abstract

is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C with the C/C metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes. Running industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production. seems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PP cofactor utilization.

Identification of a Novel Fumarate Reductase Potentially Involved in Electron Bifurcation

“…Pseudoclostridium thermosuccinogenes is the only thermophile known to produce succinic acid as one of its main products of fermentation, together with acetic acid, formic acid and several minor products (1)(2)(3).…”

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

“…A third PFK has been annotated in the genome of C. thermosuccinogenes for which no activity had been detected, and which is absent in C. thermocellum. From assays with cell-free extract, it was already clear that PP i -dependent PFK is the dominant isoform (1,25). We repeated the aforementioned assays with C. thermosuccinogenes cell-free extract in the presence of NH 4 , which we had serendipitously found to activate ATP/ GTP-PFK, but were still unable to detect ATP-dependent activ-ity (data not shown), confirming that PP i -dependent PFK is the dominant isoform in this organism as well.…”

“…C. thermosuccinogenes was grown anaerobically in adapted CP medium (35), as described previously (1), which contained per liter 0.408 g of KH 2 PO 4 , 0.534 g of Na 2 HPO 4 ⅐2H 2 O, 0.3 g of NH 4 Cl, 0.3 g of NaCl, 0.1 g of MgCl 2 ⅐6H 2 O, 0.11 g of CaCl 2 ⅐2H 2 O, 4.0 g of NaHCO 3 , 0.1 g of Na 2 SO 4 , 1.0 g of L-cysteine, 1.0 g of yeast extract (BD Pharmingen, BD Bacto), 1 ml of vitamin solution, 1 ml of trace elements solution I, and 1 ml of trace elements solution II. No resazurin was added to eliminate the possibility of it interfering with the metabolomics, as it appeared to adsorb to the nylon filter used for making the metabolome extracts.…”

“…In C. thermosuccinogenes only the genes in the PPP responsible for the conversions of C 5 sugars to xylulose 5-phosphate (i.e. xylose transporters, xylose isomerase, and xylulokinase) were up-regulated during growth on xylose versus glucose (1). In Clostridium termitidis, a transketolase was found to be up-regulated as well, during growth on xylose or xylan versus cellobiose (2).…”

“…Several cellulolytic clostridia 4 have been reported to lack an annotated transaldolase gene, whereas at least a few of those are able to grow very efficiently on pentose sugars, including Clostridium thermosuccinogenes and Clostridium cellobioparum subsp. termitidis, and Clostridium stercorarium (1)(2)(3). This implies that an alternative route to link C 5 to the rest glycolysis must be present in those organisms.…”