Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum.

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum's membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrations under physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrations due to cofactor modification and reduction in ATP generation. The combination of ATP linked phosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) with NADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP+ oxidoreductase (NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balances MDF improvements with ATP generation, and appears to functionally reproduce the pathway employed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list of measured intracellular metabolites and improving the quantification accuracy of measurements was found to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This study demonstrates even before addressing an organism's enzyme kinetics and allosteric regulations, pathway thermodynamics can flag pathway bottlenecks and identify testable strategies for enhancing pathway thermodynamic feasibility and function.

[1]  Uldis Kalnenieks,et al.  The Low Energy-Coupling Respiration in Zymomonas mobilis Accelerates Flux in the Entner-Doudoroff Pathway , 2016, PloS one.

[2]  Peyman Ezzati,et al.  Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression , 2012, BMC Microbiology.

[3]  L. Lynd,et al.  Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol , 2017, Biotechnology for Biofuels.

[4]  L. Lynd,et al.  Cellulosic ethanol: status and innovation. , 2017, Current opinion in biotechnology.

[5]  Manuel Hörl,et al.  Glycolysis without pyruvate kinase in Clostridium thermocellum. , 2017, Metabolic engineering.

[6]  L. Lynd,et al.  Development of a core Clostridium thermocellum kinetic metabolic model consistent with multiple genetic perturbations , 2017, Biotechnology for Biofuels.

[7]  Cong T. Trinh,et al.  Elucidating central metabolic redox obstacles hindering ethanol production in Clostridium thermocellum. , 2015, Metabolic engineering.

[8]  Ronan M. T. Fleming,et al.  Consistent Estimation of Gibbs Energy Using Component Contributions , 2013, PLoS Comput. Biol..

[9]  E. Papoutsakis,et al.  Increased levels of ATP and NADH are associated with increased solvent production in continuous cultures of Clostridium acetobutylicum , 1989, Applied Microbiology and Biotechnology.

[10]  R. Carlson,et al.  The fractional contributions of elementary modes to the metabolism of Escherichia coli and their estimation from reaction entropies. , 2006, Metabolic engineering.

[11]  Sean F. Covalla,et al.  Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood , 2016, Biotechnology for Biofuels.

[12]  M. Ataman,et al.  Heading in the right direction: thermodynamics-based network analysis and pathway engineering. , 2015, Current opinion in biotechnology.

[13]  A. Redaelli,et al.  Review: Engineering of thermostable enzymes for industrial applications , 2018, APL bioengineering.

[14]  S. Schuster,et al.  How important is thermodynamics for identifying elementary flux modes? , 2017, PloS one.

[15]  R. Wolfenden,et al.  A proficient enzyme. , 1995, Science.

[16]  Jonathan R. Mielenz,et al.  Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum , 2011, Applied Microbiology and Biotechnology.

[17]  Wolfram Liebermeister,et al.  Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism , 2014, PLoS Comput. Biol..

[18]  Griffin M. Weber,et al.  BioNumbers—the database of key numbers in molecular and cell biology , 2009, Nucleic Acids Res..

[19]  Angel Rubio,et al.  Computing the shortest elementary flux modes in genome-scale metabolic networks , 2009, Bioinform..

[20]  W. Shinoda.  Permeability across lipid membranes. , 2016, Biochimica et biophysica acta.

[21]  Masato Ikeda,et al.  l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene. , 2016, Metabolic engineering.

[22]  L. Lynd,et al.  The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum. , 2017, Metabolic engineering.

[23]  Liang Zhang,et al.  Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast , 2011, Journal of Industrial Microbiology & Biotechnology.

[24]  Lee R Lynd,et al.  Recent progress in consolidated bioprocessing. , 2012, Current opinion in biotechnology.

[25]  Soha Hassoun,et al.  Establishing synthesis pathway‐host compatibility via enzyme solubility , 2019, Biotechnology and bioengineering.

[26]  Daniel Amador-Noguez,et al.  The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading , 2014, Biotechnology for Biofuels.

[27]  A. Demain,et al.  Cellulase, Clostridia, and Ethanol , 2005, Microbiology and Molecular Biology Reviews.

[28]  Cong T Trinh,et al.  Overflow metabolism and growth cessation in Clostridium thermocellum DSM1313 during high cellulose loading fermentations , 2017, Biotechnology and bioengineering.

[29]  Jerry M. Parks,et al.  Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum , 2011, Proceedings of the National Academy of Sciences.

[30]  L. Lynd,et al.  Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase , 2017, Microbial Cell Factories.

[31]  Lee R. Lynd,et al.  Atypical Glycolysis in Clostridium thermocellum , 2013, Applied and Environmental Microbiology.

[32]  G. Bennett,et al.  Engineering E. coli Central Metabolism for Enhanced Primary Metabolite Production , 2009 .

[33]  J. Rabinowitz,et al.  Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. , 2007, Analytical chemistry.

[34]  L. Lynd,et al.  Ethanol production by engineered thermophiles. , 2015, Current opinion in biotechnology.

[35]  Steffen Klamt,et al.  OptMDFpathway: Identification of metabolic pathways with maximal thermodynamic driving force and its application for analyzing the endogenous CO2 fixation potential of Escherichia coli , 2018, PLoS Comput. Biol..

[36]  I. S. Pretorius,et al.  Microbial Cellulose Utilization: Fundamentals and Biotechnology , 2002, Microbiology and Molecular Biology Reviews.

[37]  L. Lynd,et al.  Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum , 2015, Journal of bacteriology.

[38]  L. Lynd.  The grand challenge of cellulosic biofuels , 2017, Nature Biotechnology.

[39]  Matthias Heinemann,et al.  Condition-Dependent Cell Volume and Concentration of Escherichia coli to Facilitate Data Conversion for Systems Biology Modeling , 2011, PloS one.

[40]  Costas D Maranas,et al.  OptStrain: a computational framework for redesign of microbial production systems. , 2004, Genome research.

[41]  L. Lynd,et al.  Nicotinamide cofactor ratios in engineered strains of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. , 2016, FEMS microbiology letters.

[42]  Shihui Yang,et al.  Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress , 2012, BMC Genomics.

[43]  F. Srienc,et al.  Elementary mode analysis: a useful metabolic pathway analysis tool for characterizing cellular metabolism , 2009, Applied Microbiology and Biotechnology.