TY - JOUR
T1 - A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis
AU - Schroeder, Wheaton L.
AU - Kuil, Teun
AU - van Maris, Antonius J.A.
AU - Olson, Daniel G.
AU - Lynd, Lee R.
AU - Maranas, Costas D.
N1 - Funding Information:
This work was supported by the Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . TK and AVM were supported by the Novo Nordisk Foundation Grant ( NNF20OC0064164 ).
Funding Information:
A preliminary analysis of the thermodynamic viability of glycogen and Ppdk-malate shunt cycles are performed using standard Gibbs free energy of reaction (ΔGrxn o) for each cycle step using the eQuilibrator tool (Beber et al., 2022), evaluated at a pH of 7, a concentration of Mg2+ ions (pMG) of 3 mM, and an ionic strength of 0.25 M. The enzyme-catalyzed steps of glycogen cycling, Ags, Glcs, and Glcp (see Fig. 1) appear thermodynamically favorable with ΔGrxn o values of −8.3±2.7, −4.1±9.4, and 2.1±9.1 kJ∙mol-1, respectively, in the direction required for the cycle to produce PPi. On the other hand, the Ppdk-malate shunt cycle involves two thermodynamic bottlenecks. When run in the PPi-generating direction, reactions PPDK, PEPCK, MDH, and ME (see Fig. 1) have ΔGrxn o values of 17.4±1, −13.1±6.6, −26.5±0.6, and 12.2±6.1 kJ∙mol-1, respectively. This suggests that the PPi-generating step of the cycle, Ppdk catalysis of the PPi-evolving reaction, is thermodynamically unfavorable. This is supported by a 13C MFA experiment, which investigated flux around the PEP-to-pyruvate node and determined that 33±2% of flux from pyruvate to PEP is accounted for by the malate shunt, with the balance accounted for by requiring Ppdk to proceed in the PPi-using direction (Olson et al., 2017). This thermodynamic and MFA data casts serious doubt on the Ppdk-malate shunt as a primary PPi source while maintaining the viability of glycogen cycling.Only two of the 34 present investigated cycles were also identified in iCTH669, namely glycogen and Ppa/ATPase cycles. By analysis of reaction thermodynamics, balances, and constituent members, we have identified the reasons why the remaining 32 cycles were not identified in iCTH669. These cycles were not identified for the following (non-exclusive) reasons: i) constituent reactions are no longer present in the model due to lack of genome support (16 cycles), ii) cycles did not account for proton transport from PPA (11 cycles), iii) acetyl-CoA synthase is no longer present in the model as (Kuil et al., 2021) found no ACS activity (8 cycles), iv) the cycle requires a reaction to proceed in a direction where ΔGrxn o>10 (5 cycles), and/or v) the cycle required the ATP maintenance reaction to have flux in the reverse (ATP-generating) direction (3 cycles) in addition to other reasons noted in Supplemental File 5. The issue of proton transport across the membrane may not have been of concern when identifying these cycles in iCBI655 as the model is stoichiometrically inconsistent with respect to protons (as identified by MEMOTE) but it is problematic in iCTH669 as energy must be expended to transport those protons again (for instance, through ATPase which is assumed to use one ATP per two protons transported). Therefore, while these cycles were identified in iCBI655, from which iCTH669 was created, the 32 unidentified present investigated cycles are deemed as no longer stoichiometrically viable.This work was supported by the Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. TK and AVM were supported by the Novo Nordisk Foundation Grant (NNF20OC0064164).
Publisher Copyright:
© 2023 The Authors
PY - 2023/5
Y1 - 2023/5
N2 - Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.
AB - Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.
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U2 - 10.1016/j.ymben.2023.04.003
DO - 10.1016/j.ymben.2023.04.003
M3 - Article
C2 - 37085141
AN - SCOPUS:85156276223
SN - 1096-7176
VL - 77
SP - 306
EP - 322
JO - Metabolic engineering
JF - Metabolic engineering
ER -
