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Atypical glycolysis in Clostridium thermocellum.

ABSTRACT: Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PPi in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.

SUBMITTER: Zhou J

PROVIDER: S-EPMC3623140 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Atypical glycolysis in Clostridium thermocellum.

Zhou Jilai J Olson Daniel G DG Argyros D Aaron DA Deng Yu Y van Gulik Walter M WM van Dijken Johannes P JP Lynd Lee R LR

Applied and environmental microbiology 20130222 9

Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome ...[more]

PMID: 23435896

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Project description:Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ?-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ?glyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ?glyR2 strain, and ?-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ?glyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Dataset Information

Atypical glycolysis in Clostridium thermocellum.

Publications

Atypical glycolysis in Clostridium thermocellum.

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