Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Abstract

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK) as well as a malate shunt for the conversion of PEP to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present whereas the activities of a malate shunt enzymes were low in T. saccharolyticum. In C. thermocellum glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi —> 2 pyruvate + 5 NTP + Pi. Metabolic flux analysis of chemostat data with the wild-type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi —> ADP + PPi. Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation as large amounts of pyruvate and amino acids, mainly valine, were excreted: up to 50% of the nitrogen consumed was excreted again as amino acids. Importance. This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of a reversible pyrophosphate-dependent glycolysis, as occurring in Clostridium thermocellum, could not be properly evaluated as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.

Pyrophosphate and Irreversibility in Evolution, or why PPi Is Not an Energy Currency and why Nature Chose Triphosphates

“…Guanosine triphosphate (GTP) hydrolyzing reactions are not uncommon in LUCA's biosynthetic core (Figure 1), in line with its ancient role in metabolism (Martin and Russell, 2007) and the observation that in some organisms where it has been investigated, GTP is readily used as a substrate in reactions that are typically regarded as ATP dependent (Holwerda et al, 2020). About 26% of a cell's energy budget is consumed in the GTP-dependent steps of translation.…”

“…Soluble pyrophosphatases (sPPases) are ubiquitous in distribution ( Lahti, 1983 ). Ion-pumping mPPases are found in various microbes and plants ( Serrano et al, 2007 ), and PP i -dependent glycolytic enzymes occur as alternatives of ATP-dependent forms, at the phosphofructokinase (PFK) and pyruvate kinase (PYK) steps ( Heinonen, 2001 ; Siebers and Schönheit, 2005 ; Bräsen et al, 2014 ; Holwerda et al, 2020 ). Though PP i- dependent glycolysis is often interpreted as an adaptation that reduces ATP expense ( Heinonen, 2001 ) or that salvages energy from PP i produced from translation ( Reeves, 1968 ), PP i -utilizing glycolytic enzymes have a conspicuous tendency to occur among microbes that have specialized to sugar-rich environments.…”

“…Though PP i- dependent glycolysis is often interpreted as an adaptation that reduces ATP expense ( Heinonen, 2001 ) or that salvages energy from PP i produced from translation ( Reeves, 1968 ), PP i -utilizing glycolytic enzymes have a conspicuous tendency to occur among microbes that have specialized to sugar-rich environments. Such specialists include human parasites, such as Entamoeba ( Reeves, 1984 ), Giardia ( Müller et al, 2012 ), and trypanosomes ( Michels et al, 2006 ), as well as non-parasitic cellulose-, saccharose-, and sugar-degrading bacteria ( Bielen et al, 2010 ; Holwerda et al, 2020 ) and archaea ( Bräsen et al, 2014 ). In addition, PP i -dependent enzymes are particularly common in the strictly sugar-based carbon metabolism of plants ( Serrano et al, 2007 ).…”

“…In the well-studied example of trypanosomes, loss of regulation allows flux through the pathway to be governed by sugar concentrations in the medium (blood sugar), an ecological adaptation of growth rates to substrate availability, not energetic efficiency, especially as trypanosomes excrete the energy rich compound pyruvate as a metabolic end product ( Michels et al, 2006 ). Even in the well-studied glucose fermenting bacterium Clostridium thermocellum , which also excretes pyruvate, a clear energetic advantage of its PP i -dependent glycolysis is not evident ( Holwerda et al, 2020 ). Moreover, deletion of C. thermocellum ’s mPPase has no impact on growth ( Holwerda et al, 2020 ), a finding that is hard to reconcile with a central role for energy conservation via PP i in energy metabolism of the bacterium, although sPPase activity was not reported in the mPPase mutant.…”

“…Even in the well-studied glucose fermenting bacterium Clostridium thermocellum , which also excretes pyruvate, a clear energetic advantage of its PP i -dependent glycolysis is not evident ( Holwerda et al, 2020 ). Moreover, deletion of C. thermocellum ’s mPPase has no impact on growth ( Holwerda et al, 2020 ), a finding that is hard to reconcile with a central role for energy conservation via PP i in energy metabolism of the bacterium, although sPPase activity was not reported in the mPPase mutant. By contrast, deletion mutants of sPPases are lethal in Escherichia coli ( Chen et al, 1990 ) and in yeast ( Pérez-Castiñeira et al, 2002 ).…”