Atypical Glycolysis in Clostridium thermocellum

Abstract

dCofactor 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 (PP i )-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 PP i in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PP i -dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PP i 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 PP i 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.

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

“…Strains grown for transformation, biochemical characterization, and growth curves were cultured in a modified DSMZ M122 medium containing 5 g/liter cellobiose (2). For analysis of fermentation products, strains were grown in serum bottles (Fisher Scientific) on 50 ml of defined MTC medium for 72 h, as previously described (26), with the following modifications for T. saccharolyticum strains: urea was replaced with ammonium chloride, and thiamine hydrochloride was supplemented at a final concentration of 4 mg/liter. For growth measurements, strains were grown in 200 l of medium in a 96-well plate.…”

“…Fermentation products in the liquid fraction were measured using a Waters (Milford, MA) high-pressure liquid chromatograph (HPLC) with an HPX-87H column with a UV and refractive index detector as previously described (26). H 2 was measured using a model 310 SRI Instruments gas chromatograph (Torrence, CA) with a HayeSep D packed column using a thermal conductivity detector and nitrogen carrier gas.…”

“…H 2 was measured using a model 310 SRI Instruments gas chromatograph (Torrence, CA) with a HayeSep D packed column using a thermal conductivity detector and nitrogen carrier gas. Pellet carbon and nitrogen were measured with a Shimadzu TOC-V CPH elemental analyzer with TNM-1 and ASI-V modules (Shimadzu Corp., Columbia, MD) as previously described (26). Mass spectrometry analysis was performed with an HPLC pump (u3000; Dionex, Sunnvale, CA) coupled to an LTQ XL Orbitrap (Thermo Scientific, Waltham, MA) as previously described (28).…”

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

“…The concentration of pyruvate found here is relatively low (325 mg/L, see Figure 3F and Figure 4). The additional presence of the glycolysis/citric acid cycle intermediates malate (509 mg/L) and fumarate (775 mg/L) indicates metabolic imbalance ( Figure 3F) [9,69]. Desvaux et al reported that C. cellulolyticum stopped growing in a defined medium before complete utilization of substrate when cultured on concentrations higher than 6.7 g/L cellulose.…”

“…and lacks pyruvate kinase [9]. The recent development and application of genetic tools [10][11][12] has accelerated both applied and fundamental investigations of this organism.…”