Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers

Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers Bishal Dev Sharma, Shuen Hon, Eashant Thusoo, David M. Stevenson, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5027329/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 10 You are reading this latest preprint version Abstract Background Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild type organism, this reaction uses pyrophosphate (PPi) as an energy cofactor, which is thermodynamically less favorable compared reactions that use ATP as a cofactor. Previously we showed that replacing the PPi-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer. Results In this study, we substituted PPi- pfk with ATP- pfk , deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase ( ppdk ), and expressed a soluble pyrophosphatase ( PPase ) and pyruvate kinase ( pyk ) genes to engineer PPi-free glycolysis in C. thermocellum . We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 g/L to 21.0 g/L) by using PPi-free glycolysis. Conclusions By engineering PPi-free glycolysis in C. thermocellum , we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers. Acetivibrio thermocellus biofuels Clostridium thermocellum ethanol production metabolic bottleneck metabolic engineering phosphofructokinase thermodynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. BACKGROUND Technologies are needed to decarbonize the transportation sector to help reduce the need for petroleum usage that is driving catastrophic climate change. While electrification may help replace the current usage of gasoline for light duty transportation, sustainable liquid fuel solutions are critically needed for aviation, rail, and marine sectors [ 1 ]. Conversion of lignocellulose into ethanol can be part of the solution because ethanol can be catalytically oligomerized to hydrocarbons that can be used for sustainable aviation fuel (SAF). However, better conversion technologies are needed to make SAF a reality. Clostridium thermocellum (also known as ​​ Acetivibrio thermocellus , Hungateiclostridium thermocellum , and Ruminiclostridium thermocellum ) is a thermophilic anaerobe that has been extensively studied and engineered for consolidated bioprocessing of lignocellulose to biofuels such as ethanol [ 2 , 3 ], due to its strong native ability to solubilize and ferment lignocellulose [ 4 , 5 ]. One of the unique properties of this organism is that it consumes crystalline cellulose (e.g. Avicel) at the same rate or faster than soluble substrates, such as cellobiose [ 6 ]. Previous metabolic engineering efforts aimed at transforming C. thermocellum into a commercial platform for lignocellulosic biofuel production have primarily concentrated on enhancing fuel yield. Notably, high ethanol yields exceeding 80% of the theoretical maximum have been achieved [ 7 , 8 ]. Current research is now directed towards further engineering the bacterium to achieve higher ethanol titers. Existing engineered strains produce titers in the range of 25–30 g/L [ 7 – 9 ]. Concentrations in the 40–50 g/L range are thought to be necessary for commercial applications [ 1 , 10 ]. The atypical Embden-Meyerhof-Parnas (EMP) glycolytic pathway found in wild type C. thermocellum [ 11 ] was previously observed to function closer to thermodynamic equilibrium compared to organisms such as Thermoanaerobacterium saccharolyticum or Escherichia coli that utilize a canonical glycolytic pathway [ 12 , 13 ], and may limit final product titer in C. thermocellum . For our purposes, we define a canonical glycolytic pathway as one that uses an ATP-consuming reaction to generate fructose 1,6-bisphosphate and an ATP-producing reaction to convert phosphoenolpyruvate to pyruvate, and both of these steps have a high thermodynamic driving force that allows for allosteric regulation. Differences in cofactor usage for key glycolytic reactions were proposed as the underlying reason for the distinct pathway thermodynamics [ 12 ]. Specifically, the pyrophosphate (PPi)-dependent phosphofructokinase (PPi-PFK, ∆G PPi−pfk = -1.45 kJ/mol) reaction is thought to significantly contribute to the lower thermodynamic driving force observed in C. thermocellum . In microbes like T. saccharolyticum , which utilize an ATP-dependent phosphofructokinase (ATP-PFK, ∆G ATP−pfk = -22.57 kJ/mol), this reaction exhibits the largest drop in Gibbs free energy change of the entire pathway [ 12 ]. Moreover, research findings have suggested that the ATP/ADP ratio tends to be significantly higher than the PPi to inorganic phosphate (PPi/Pi) ratio in many organisms [ 14 ], and thus the actual further widening the difference in thermodynamic driving force between the two reactions. Additionally, it has been previously noted that the two native pathways employed by C. thermocellum for the conversion of phosphoenolpyruvate (PEP) to pyruvate, namely the pyruvate:phosphate dikinase (PPDK) that uses PPi to convert PEP and AMP to pyruvate and ATP [ 15 ] and the malate shunt that converts PEP to oxaloacetate to malate to pyruvate [ 15 ], also represent thermodynamic bottlenecks [ 16 ]. We recently engineered C. thermocellum to use ATP as the main cofactor for the PFK reaction [ 17 ]. Although this increased the thermodynamic driving force of the PFK reaction, the effects on intracellular metabolites were limited to a small region adjacent to this reaction, and there was no effect on ethanol titer. Furthermore, resequencing analyses found that the resulting strain (LL1660) had a partial genome duplication, complicating subsequent genetic engineering. In this work, we make further progress toward engineering a PPi-free glycolysis in C. thermocellum glycolysis by eliminating the PPi-consuming reactions PFK and PPDK and providing an alternative pathway for PEP to pyruvate conversion via the pyruvate kinase (PYK) reaction. We define “PPi-free glycolysis” strain as one that does not require pyrophosphate (PPi) for any of the reactions in glycolysis. A key enabling step of engineering this into C. thermocellum was the expression of a soluble pyrophosphatase (PPase). Finally, we show that PPi-free glycolysis increases the levels of downstream glycolysis metabolites, increasing ethanol titers. This demonstrates the potential of engineering pathway thermodynamics as a general strategy for improving product titers. 2. MATERIAL AND METHODS 2.1 Strains used in this work Table 1 Strains used in this work are described below. Tsc and Taq refers to genes introduced from T. saccharolyticum and T. aquaticus , respectively. Organism Strain ID Description Accession Number References C. thermocellum LL1592 DSM 1313 ∆hpt, ∆ldh, ΔreIII , P2638:: adhA(Tsc), nfnAB(Tsc), adhEG544D(Tsc ), Clo1313_2637::Ptsc0046-pforA(Tsc)-ferredoxin(Tsc), ∆pfor1, ∆pfor2, ∆pfor3, ∆pfor4, ∆pfor5 SRX5290154 [17] C. thermocellum LL1590 LL1592 ∆adhE SRX5290158 [18] C. thermocellum LL1649 LL1592 tal(Tsc) , ATP- pfk(Tsc) , ∆ags, pPfk(Tsc) SRX6875980 [17] C. thermocellum LL1689 LL1649 pyk(Tsc) , PPase(Taq) SRX7724531 This study C. thermocellum LL1710 LL1689 ∆ppdk, adhE E784* SRX9409012 This study C. thermocellum LL1711 LL1710 ∆ PPi- pfk SRX9409011 This study 2.2 Plasmid and strain construction Plasmids were constructed via isothermal assembly [ 19 ], using commercially available kits (New England Biolabs NEBuilder® HiFi DNA Assembly Master Mix, catalog number E2621). Plasmids were maintained and propagated from BL21 derivative E. coli strains (New England Biolabs T7 Express E. coli , catalog number C2566) to ensure proper methylation of the plasmid DNA [ 20 ]. Plasmid DNA was purified using commercial kits available from either New England Biolabs or Zymo Research. Transformation of C. thermocellum was performed as previously described [ 21 ]. Table 2 Plasmids used in this study Plasmid Description Accession no. References pDGO143 C. thermocellum expression vector KX259110 [22] pDGO145 Deletion/Integration vector backbone KY852359 [23] pMU2051 Plasmid for deletion of ppdk gene KC146550 [11] pLL1392 Deletion vector; deletes C. thermocellum PPi-pfk only in strain LL1647 and its derivatives ON809513 [17] pLL1498 pDGO143 with C. thermocellum Clo 1313_2638 promoter driving T. saccharolyticum ATP- pfk and T. aquaticus PPase PP855251 This work pLL1499 pDGO143 with C. thermocellum Clo 1313_2638 promoter driving T. aquaticus PPase PP855252 This work pLL1500 pDGO143 with C. thermocellum Clo 1313_2638 promoter driving Geobacillus stearothermophilus PPase PP855253 This work pLL1501 pDGO143 with T. saccharolyticum enolase promoter driving Geobacillus stearothermophilus PPase PP855254 This work pLL1502 pDGO143 with T. saccharolyticum enolase promoter driving Geobacillus thermoglucosidasius PPase PP855255 This work pLL1503 pDGO143 with T. saccharolyticum enolase promoter driving T. acidophilum PPase PP855256 This work pLL1504 pDGO143 with T. saccharolyticum enolase promoter driving T. aquaticus PPase PP855257 This work pLL1505 pDGO143 with T. saccharolyticum enolase promoter driving T. thermophilus PPase PP855258 This work pLL1506 pDGO145 with T. saccharolyticum pyk and G. thermoglucosidasius cytosolic PPase driven by T. saccharolyticum enolase promoter PP855259 This work pLL1507 pDGO145 with T. saccharolyticum pyk and T. aquaticus cytosolic PPase driven by T. saccharolyticum enolase promoter PP855260 This work 2.3 Media and growth conditions All reagents used in this study were of molecular grade and obtained from Sigma Aldrich or Fisher Scientific, unless otherwise noted. C. thermocellum strains were grown at 55°C under anaerobic conditions, either in anaerobic chambers (Coy Laboratory Products, Grass Lakes, MI), with previously described environmental settings [ 23 ], or in sealed serum bottles that were prepared as previously described [ 23 ]. Complex medium CTFUD was prepared as previously described [ 21 ] and used for culturing cells for use in transformations, or for preparing genomic DNA for strain resequencing. Defined MTC-5 medium was used for all other purposes [ 23 ]. 2.4 Enzyme assays All chemicals were ordered from MilliporeSigma unless otherwise specified. All enzyme assays were performed anaerobically unless otherwise mentioned. For enzyme assays, cells were grown to mid-exponential phase (OD 600 between 0.6 to 1.0), harvested and lysed to obtain cell extract as described earlier [ 23 ]. Protein concentrations were determined using Bradford assay (Coomassie Plus Reagent, Thermo Scientific), with bovine serum albumin used as a protein standard. All the enzyme assays were performed at 55°C except for the phosphofructokinase assay, which was performed at 40°C due to precipitation issues associated with the aldolase coupling enzyme. 2.4.1 Phosphofructokinase (PFK) assay. PFK activity was assayed by measuring the formation of fructose 1,6-bisphosphate coupled with consumption of NADH as previously described [ 11 , 17 ]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5mM MgCl 2 , 0.15 mM NADH, 1 mM fructose-6-phosphate, 4 U/mL fructose bisphosphate aldolase, 4 U/mL triosephosphate isomerase, 4 U/mL a-glycerophosphate dehydrogenase, cell extract, and 2 mM either PPi or ATP. The assay reaction was started by the addition of the phosphate donor (PPi or ATP). 2.4.2 Pyruvate phosphate dikinase (PPDK) assay. PPDK activity was assayed by measuring the consumption of NADH by coupled assay with lactate dehydrogenase (LDH) as previously described with slight modification [ 11 ]. The assay mixture contained 100 mM Tris-HCl (pH 7.0), 5 mM MgCl 2 , 2 mM AMP, 0.15 mM NADH, 20 mM NH 4 Cl, 2 mM PEP, 1 mM fructose-1,6-bisphosphate, 4 U/ml lactate dehydrogenase, cell extract, and 2 mM PPi. The assay reaction was started by the addition of PPi. 2.4.3 Pyruvate kinase (PYK) assay. PYK activity was measured by coupled assay with lactate dehydrogenase (LDH) as described earlier with slight modification [ 11 , 15 , 24 ]. The assay buffer contained 100 mM Tris-HCl, pH 7.5 (at 55°C), 5 mM dithiothreitol (DTT), 10 mM KCl, 12 mM MgCl 2 , 10 mM ADP, 0.3 mM NADH, 0.1 mM 3-phosphoglyceric acid (3PG), 5 mM PEP, 12 U LDH enzyme, and cell extract. This reaction could be started with either ADP or PEP. For this experiment, the assay reaction was started by the addition of ADP. 2.4.4 Soluble pyrophosphatase (PPase) assay. PPase activity was measured aerobically based on hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi) using malachite green assay as defined earlier [ 25 ] with some modifications. The assay mixture contained 100 mM Tris-HCl at pH 8.0, 1 mM MgCl 2 , 1 mM sodium pyrophosphate and 2, 4 or 8 µl cell extract. This assay mixture was incubated at 55°C for 2 minutes and the reaction was stopped by transferring the samples to ice. 2.5 ul of this sample was added to 40 µl of malachite green reagent (Malachite green:Ammonium molybdate at 3:1) in 96 wells plate. The wells with PPase activity turn green in color. 7.5 µl of 34% sodium citrate was added, mixed and incubated at room temperature for 30 minutes to allow full color development [ 26 ]. Phosphate concentration was calculated by measuring absorbance at 660 nm using a calibration curve with phosphate concentration ranging from (0–5) mM. 2.4.5 Aldehyde dehydrogenase (ALDH) assay. ALDH activity was measured anaerobically as described earlier [ 27 ]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5 µM FeSO 4 , 0.25 mM NADH or NADPH, 1.25 mM acetyl-CoA, 1 mM DTT, and cell extract. The assay reaction was started by the addition of acetyl-CoA. 2.4.6 Alcohol dehydrogenase (ADH) assay. ADH activity was measured anaerobically as described earlier [ 27 ]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5 µM FeSO 4 , 0.25 mM NADH or NADPH, 18 mM acetaldehyde, 1 mM DTT, and cell extract. The assay reaction was started by the addition of acetaldehyde. 2.5 13 C labeling conditions For 13 C labeling experiments, C. thermocellum cells were grown anaerobically on MTC-5 medium consisting of 3 mM naturally labeled cellobiose (Sigma C7252) and 3 mM uniformly labeled 13 C-cellobiose (Omicron Biomedicals, CEL-002) as mentioned earlier [ 17 ]. 10 mL of this media was inoculated with 10 µl freezer stock of LL1590, LL1592 and LL1711 and incubated at 55°C. The freezer stocks were prepared by growing the cells to mid-exponential phase (OD 600 ~ 0.6) on MTC-5 with 5 g/L naturally labeled cellobiose. Metabolite samples were extracted at three different phases during growth i.e. early log phase (OD 600 ~ 0.4), late log phase (OD 600 ~ 0.8) and stationary phase (12 hours after maximum OD has reached). The growth was monitored continuously using a custom-built absorbance reader. Intracellular metabolites were collected using vacuum filtration as previously described [ 12 , 28 – 30 ]. For each sample, 2.5 ml of culture medium was filtered through 3.0 µm hydrophilic nylon filter (SF14529, Tisch Scientific) to separate cells from medium components. The filter with cells was placed in 1.6 mL of cold extraction solvent (40% acetonitrile, 40% methanol, and 20% water) with the cell side facing down and kept on aluminum block from − 80°C to quench metabolism and extract metabolites. Cells were washed off the filter using extraction solvent and then centrifuged for 5 minutes to remove cell debris. The supernatant with metabolites was collected, dried using a sample concentrator (catalog no. EW-36620-40; Cole-Parmer) to remove the metabolite extraction buffer and resuspended in molecular-grade water. This metabolite extract was analyzed using LC-MS. Metabolites of interest were detected based on retention time and mass-to-charge ratio, compared with pure standards, using El-MAVEN software [ 31 ]. 2.6 Quantification of PPi in cells To quantify the concentration of PPi in cells, samples were resuspended in molecular-grade water as mentioned earlier (section 3.5 ). Inorganic pyrophosphate (PPi) was quantified using a combination of two different kits; a PPiLight inorganic pyrophosphate assay kit (catalog no. LT07-610; Lonza) which measures the sum of PPi and ATP and an ADP/ATP ratio assay kit (catalog no. MAK135; Sigma-Aldrich) which measures only ATP without cross-reacting with PPi. Then, PPi in cells was calculated using the first kit, and corrected for the effects of ATP using the second kit using the formula below: PPi (mM) per cell = (PPi (mM) in metabolite extract × Volume of metabolite extraction buffer )/Intracellular volume (ml) where, PPi (mM) in metabolite extract was calculated by using the kits above as PPi (mM) = ((ATP + PPi) LT07−610 - ATP MAK135 )/1000, and Intracellular volume = cell volume x cell number [ 30 ]. 2.7 pH-controlled bioreactor fermentation conditions and metabolites extraction C. thermocellum cultures were grown anaerobically at 55°C in a Coy (Ann Arbor, MI) anaerobic chamber with a gas phase of 85% N 2 , 10% CO 2 , and 5% H 2 . Fermentation was carried out at 300 mL working volume in MTC-7 (medium for thermophilic clostridia) with 100 g/L cellobiose as substrate as mentioned earlier [ 30 ]. MTC-7 medium contained 100 g/L cellobiose, 9.3 g/L MOPS (morpholinepropanesulfonic acid) sodium salt, 2 g/L potassium citrate monohydrate, 1.3 g/L citric acid monohydrate, 1 g/L Na 2 SO 4 , 1 g/L KH 2 PO 4 , 2.5 g/L NaHCO 3 , 2 g/L urea, 1 g/L MgCl 2 ⋅6H 2 O, 0.2 g/L CaCl 2 ⋅2H 2 O, 0.1 g/L FeCl 2 ⋅4H 2 O, 1 g/L L-cysteine HCl monohydrate, 0.02 g/L pyridoxamine HCl, 0.004 g/L p-aminobenzoic acid, 0.002 g/L d-biotin, 0.002 g/L vitamin B12, 0.004 g/L thiamine, 0.0005 g/L MnCl 2 ⋅4H2O, 0.0005 g/L CoCl 2 ⋅6H 2 O, 0.0002 g/L ZnCl 2 , 0.0001 g/L CuCl 2 ⋅2H2O, 0.0001 g/L H 3 BO 3 , 0.0001 g/L Na 2 MoO 4 ⋅2H 2 O, and 0.0001 g/L NiCl 2 ⋅6H 2 O. MTC-7 is MTC-5 with the addition of 0.004 g/L thiamine. The pH was controlled at 7.0 using Mettler-Toledo (Columbus, OH, USA) pH probe with the addition of KOH. The extracellular metabolites/fermentation products (ethanol, lactate, formate, acetate, pyruvate) at different time points during the fermentation were quantified by HPLC (Waters, Milford, MA or LC-2030, Shimadzu) with refractive index (RI) and UV detection using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) with a 5 mM sulfuric acid solution eluent [ 32 ]. To quantify intracellular/glycolysis metabolites, samples were taken at the same time points as HPLC. Samples were prepared using a protocol we previously developed for metabolite quantification from high-substrate fermentations [ 30 ]. Briefly, samples were extracted using vacuum filtration followed by quenching. For each sample, (0.5–8) ml of culture medium was filtered through 3.0 µm hydrophilic nylon filter based on OD 600 of cells. The samples were then quenched, dried, resuspended in molecular grade water and analyzed using LC-MS. 2.8 Liquid chromatography-mass spectrometry (LC-MS) analysis A Vanquish ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific) coupled to a hybrid quadrupole-Orbitrap™ mass spectrometer (Q Exactive™; Thermo Scientific) equipped with electrospray ionization operating in negative-ion mode as defined earlier [ 12 ] was used for LCMS analysis. The chromatography was performed at 25°C using a 2.1 × 100 mm reverse-phase C18 column with a 1.7 µm particle size (Water™; Acquity UHPLC BEH). Two chromatography gradients were used. The first used Solvent A (97:3 H2O: methanol + 10 mM tributylamine) and Solvent B (100% methanol) as follows: 0–2.5 min, 5% B; 2.5–17 min, linear gradient from 5% B to 95% B; 17–19.5 min, 95% B; 19.5–20 min, linear gradient from 95% B to 5% B; 20–25 min, 5% B. The second also used Solvent A and Solvent B (100% methanol) and was as follows: 0–2.5 min, 5% B; 2.5–7.5 min, linear gradient from 5% B to 20% B; 7.5–13 min, 20% B to 55% B; 13–18.5min, 55% B to 95% B; 18.5–19 min linear gradient from 95% B to 5% B; 19–25 min, 5% B. The flow rate was held constant at 0.2 mL/min for both chromatograph methods. Metabolites of interest were identified by retention times (based on pure standards) and monoisotopic mass using MAVEN [ 33 ] and El-MAVEN [ 31 ] software. 2.9 Sequencing methods Routine Sanger sequencing was outsourced to Azenta Life Sciences. Whole genome resequencing was performed by the Department of Energy Joint Genome Institute using the Illumina MiSeq sequencing platform, with a minimum of 100-fold coverage. Strains were analyzed with the software CLC Genomics Workbench (Qiagen) using strain DSM1313 as the reference genome (Genbank accession NC_017304.1); reads were filtered against strain LL1649 (accession number SRP222669) [ 17 ] to exclude inherited mutations. A summary of the identified mutations is provided in Additional File 4. 3. RESULTS 3.1 Strain construction to eliminate and replace PPi-dependent glycolytic reactions In our previous work, we created strain LL1660 (Fig. 1 ), where the PPi- pfk gene was replaced with an ATP- pfk gene [ 17 ], eliminating a major sink for PPi. We also observed the spontaneous occurrence of a large partial genome duplication. This partial genome duplication region encodes proteins that may serve as alternative sinks for PPi, including a membrane-bound pyrophosphatase gene ( HppA - Clo1313_0823 ) and pyruvate:phosphate dikinase gene ( PpdK - Clo1313_0949 ). We therefore hypothesized that the partial genome duplication event might have occurred to address an imbalance in the supply and demand of PPi created by the mutations in strain LL1660. To address this potential problem, we set out to construct a new strain where PPi-consuming reactions in glycolysis were eliminated, and where the excess PPi generated by metabolism was hydrolyzed using a soluble PPase [ 25 ]. Initially, we attempted to express soluble PPase in wild-type C. thermocellum , but we did not get any colonies in at least two transformations. This was not surprising, given our prior work showing the importance of PPi as a cofactor for the PFK reaction in glycolysis [ 11 , 15 ]. Next, we tried to express the soluble PPase in a strain with ATP-linked PFK activity (strain LL1649), which we hypothesized would be more compatible with the metabolic effects of the PPase. In this strain, we occasionally obtained a small number of colonies with our PPase expression plasmid. However, in those colonies, the PPase gene was invariably inactivated either by a transposon insertion or inactivating nonsense mutation. In this phase, we tested PPases from Geobacillus stearothermophilus , Geobacillus thermoglucosidasius , Thermoplasma acidophilum , Thermus aquaticus , and Thermus thermophilus (plasmids pLL1501 to pLL1505, Table 2 ). Out of these, only PPase from Geobacillus thermoglucosidasius and Thermus aquaticus showed PPase activity (data not shown). We further hypothesized that in addition to eliminating the need for PPi in the PFK reaction, we also needed to eliminate the need for PPi in the PPDK reaction (which converts PEP to pyruvate) [ 15 , 34 ]. To do this, we designed an integration construct to simultaneously introduce pyruvate kinase and a soluble PPase onto the chromosome, in this case using the PPases from G. thermoglucosidasius and T. aquaticus since we had in some instances observed PPase activity from cell extracts of C thermocellum that expressed those PPases [ 25 ]. Successful strain construction was obtained only with T. aquaticus PPase containing construct, pLL1507 (Table 2 ), which was validated by the gain of both pyruvate kinase and cytosolic PPase activity (Fig. 2 ). Our ability to observe functional PPase expression only in strains with both ATP-PFK and PYK activity provides further evidence of progress toward eliminating PPi as a key energy carrying cofactor in C. thermocellum . Strain LL1592, which uses the native C. thermocellum glycolytic pathway, with T. saccharolyticum pyruvate-to-ethanol pathway [ 17 ] was selected as reference strain for engineering. Strain LL1649 was derived from strain LL1592, by introduction of the T. saccharolyticum transaldolase, followed by simultaneous deletion of the ADP-glucose synthase operon and expression of T. saccharolyticum ATP- pfk . Strain LL1689 was derived from strain LL1649 by the simultaneous integration of the T. saccharolyticum pyruvate kinase and the cytosolic pyrophosphatase from Thermus aquaticus. The purpose of introducing a heterologous pyruvate kinase was to allow subsequent deletion of the ppdk gene. The purpose of introducing a soluble PPase was to serve as an alternative sink for PPi, once both the PPi- pfk and ppdk genes were deleted. With these modification in place, we were then able to successfully delete glycolytic sinks for PPi, including the ppdk gene (resulting in strain LL1710), and then, subsequently, the PPi -pfk gene (resulting in strain LL1711) (Fig. 1 ). Biochemical assays were performed to confirm the effect of the genetic modifications of the strains used in this study. As expected, strain LL1592 and strain LL1711 exclusively have PPi-PFK and ATP-PFK activity, respectively. Strains LL1649, LL1689, and LL1710 have both ATP- and PPi-linked PFK activity, as expected (PFK reaction, Fig. 2 ). Functional expression of the pyk gene was confirmed by the presence of PYK activity in strains LL1689, LL1710 and LL1711 (PYK reaction, Fig. 2 ). The effect of the ppdk gene deletion was confirmed in strains LL1710 and LL1711 based on elimination of PPDK activity (PPDK reaction, Fig. 2 ). Low levels of PPase activity were still detected in strains LL1592 and LL1649 (Paired t-test, p > 0.5); this could be attributed to the native membrane bound PPase enzyme being present in the cell extracts. Previous studies have reported the presence of membrane-bound pyrophosphatase in C. thermocellum cells [ 25 ]. Nonetheless, about 5- to 13-fold higher PPase activities (Paired t-test, p < 0.005) were observed in strains LL1689, LL1710 and LL1711, which we attribute to expression of a soluble PPase. As such, it was determined that strain LL1711 had been engineered to not require PPi as a cofactor in glycolysis, and that it now possessed a more canonical glycolytic pathway [ 12 ]. 3.2 Use of ATP-dependent pathway alters thermodynamics of glycolysis To determine the effect the genetic modifications had on the reversibility of the PFK reaction, we performed 13 C labeling on C. thermocellum strains LL1590, LL1592 and LL1711. Initially, we were planning to compare only the parent strain (LL1592) and the PPi-free glycolysis strain (LL1711); however, whole genome sequencing of strain LL1711 revealed a frameshift mutation in the wild type C. thermocellum adhE gene corresponding to amino acid position E784 that eliminated its function (Figure S1 ). We therefore included a control strain (LL1590) derived from the parent strain with a targeted deletion of the wild type C. thermocellum adhE gene [ 18 ], to better understand the effects of our changes to the glycolytic pathway without the confounding effects of the modification to ethanol production pathway. Note that all three strains (LL1590, LL1592, and LL1711) still produce ethanol via the heterologous T. saccharolyticum adhE G544D (Fig. 1 ). C. thermocellum does not readily consume glucose [ 35 ], and thus cellobiose is the most commonly-used soluble sugar for laboratory growth experiments. Strains were cultured on a 1:1 ratio of naturally labeled to uniformly 13 C-labeled cellobiose. We confirmed that 13 C cellobiose and naturally labeled cellobiose are taken up equally by measuring the ratio of M + 0 to M + 6 isotopomers for glucose 6-phosphate, which ranged from 0.8–1.2 (Additional File 2). Samples were collected at three different phases (early-log, late-log and stationary) during growth (Figure S2 ). As previously described [ 12 ], the forward glycolysis reaction should generate a 1:1 mixture of fully labeled (M + 6) and fully unlabeled (M + 0) glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). The forward flux of fructose-bisphosphate aldolase reaction (FBA) will generate 50% unlabeled (M + 0) and 50% fully labeled (M + 3) dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). However, the reverse FBA reaction flux generates a 1:2:1 mixture of M + 0, M + 3 and M + 6 FBP. The reverse flux from phosphofructokinase (PFK) and phosphoglycerate isomerase (PGI) reactions transfer these M + 3 species into the F6P and G6P pools. Appearance of the M + 3 isotopomer in the F6P and G6P pools allows us to uniquely identify reverse flux through the PFK reaction. If the PFK reaction were completely irreversible, we would expect to see no M + 3 species in either the F6P or G6P pools. As expected, based on our prior work [ 17 ], we observed that the M + 3 pool in the glycolytic intermediates G6P and F6P decreased substantially in the PPi-free glycolysis strain (LL1711) vs. the parent and control strains (LL1590 and LL1592), indicating that the genetic modifications introduced into LL1711 reduced the reversibility of the PFK reaction (G6P, F6P; Fig. 3 ). We saw an unexpected decrease in the M + 3 fraction of FBP in mid-log phase in the PPi-free glycolysis strain (LL1711) (Fig. 3 , Figure S3 ). This suggests reduced reversibility of Fba reaction, although the reason for this change is not known. In addition to comparing the isotope ratios, we also compared the ratio of the FBP and F6P metabolite pool sizes. This gives us an additional indication of the thermodynamic driving force of the reaction. A high thermodynamic driving force at the PFK reaction would result in a high FBP/F6P ratio. Comparisons of FBP/F6P ratio showed a lower ratio for the parent and control strains that use the PPi-PFK reaction (LL1590 and LL1592) and a much higher ratio for the PPi-free glycolysis strain (LL1711), which uses the ATP-PFK reaction (Fig. 3 ). This ratio is 5- to 17-fold and 74- to 120-fold higher in ATP- pfk strain compared to PPi- pfk strains during log phases and stationary phases, respectively. The higher FBP/F6P ratio is similar to what is observed in organisms with canonical glycolytic pathways, such as E. coli and T. saccharolyticum [ 12 , 36 ]. Taken together, these two lines of evidence (M + 3 abundance changes and FBP/F6P ratio changes) support the conclusion that we have increased the thermodynamic driving force of the PFK reaction in our PPi-free glycolysis strain. 3.3 Expression of PPase results in lower PPi levels To determine the effect of PPase expression in PPi levels of C. thermocellum strains with wild type glycolysis (strains LL1590 and LL1592) and PPi-free glycolysis (strain LL1711), we quantified the concentration of PPi in cells. We observed a significant decrease in PPi concentration in the PPi-free glycolysis strain (strain LL1711) (Fig. 4 ) during the early-log and late-log growth phases. During the stationary phase, the difference between strain LL1592 and strain LL1711 isn’t as significant, which might be due to the depletion of the PPi pool during this phase. We see consistently higher levels of PPi during the early-log and late-log phases in strains LL1590 and LL1592, which then drop during the stationary phase in both strains. The constant low level of PPi in strain LL1711 during all the growth phases might be the basal level of PPi in cells, even with the expression of PPase. 3.4 Increased driving force leads to higher level of fermentation products in ATP- pfk strain Having demonstrated increased thermodynamic driving force in glycolysis in the PPi-free glycolysis strain, we explored the impact of fermentation behavior and ethanol production using batch fermentations of all three strains with high substrate concentrations (100 g/L cellobiose). The fermentations had three distinct phases: 1. growth coupled fermentation, where both the cell growth and ethanol production are at the highest rate, 2. growth uncoupled fermentation, where cell growth stops and the rate of ethanol production decreases, and 3. cessation of both growth and ethanol production. During this final phase, cellobiose is hydrolyzed to glucose at a slow rate, but metabolism appears to be inactive. The final ethanol titer was 329 ± 8 mM (15.1 ± 0.4 g/L) for strain LL1590, 403 ± 11 mM (18.6 ± 0.5 g/L) for strain LL1592, and 455 ± 12 mM (21.0 ± 0.6 g/L) for strain LL1711. Titer of another fermentation product, acetate, was 65 ± 0 mM (3.8 ± 0 g/L) for strain LL1590, 51 ± 9 mM (3.0 ± 0.5 g/L) for strain LL1592 and 88 ± 7 (5.2 ± 0.4 g/L) for strain LL1711. (Fig. 5 , Figure S4 , Additional File 3). Comparison of the fermentation products between LL1592 and LL1590 suggests that deletion of C. thermocellum adhE impairs the production of ethanol. Taking the effect of the native adhE inactivation into account (i.e. comparing LL1711 to LL1590), allows us to observe the effect of PPi-free glycolysis on ethanol production without the confounding effects of changes in the ethanol production pathway. In this comparison (LL1711 vs LL1590), we observe a 38% increase in ethanol titer, and a 35.4% increase in acetate titer. Together, these suggest that our PPi-free glycolysis strain exhibits increased glycolytic flux, resulting in an increased abundance of acetyl-CoA, which is then converted to more ethanol and acetate. 3.5 Changes in glycolysis metabolite levels at elevated substrate concentrations in pH-controlled fermentations To better understand the changes in central metabolism that allowed for increased ethanol titer in the PPi-free glycolysis strain, we measured intracellular glycolytic metabolites over the course of the fermentation using the same fermentation conditions from Fig. 5 (100 g/L cellobiose, pH-controlled batch fermentations). Since preliminary data showed similar pattern of glycolytic metabolites (Figure S5 ) and 13 C labeling patterns were similar for both the PPi- pfk strains (Fig. 3 ), and strain LL1590 produced lower levels of ethanol than strain LL1592 (Fig. 5 ), we compared glycolytic metabolite levels in strains LL1592 and LL1711 . Three major changes were observed in the glycolytic metabolite profiles between these two strains. First, in the parent strain (LL1592, with wild type glycolysis) we see gradual accumulation of hexose phosphates (G6P, F6P) as the fermentation progresses. In the PPi-free glycolysis strain (LL1711), hexose phosphates were still observed to accumulate, but to a lesser extent as the fermentation progressed. Second, during the growth-uncoupled fermentation phase, we observed a dramatic increase in the levels of lower glycolysis metabolites (DHAP, 3PG, and PEP) in the PPi-free glycolysis strain. Finally, in the phase where fermentation and ethanol production stop, the parent strain shows a large accumulation of upper glycolysis metabolites (glucose, G6P, and F6P), while the PPi-free glycolysis strain shows low levels of almost all metabolites (Fig. 6 , Figure S6). These intracellular metabolites data over the course of fermentation also underscores the importance of conducting metabolomics studies throughout the fermentation process. Most previous metabolomics studies in C. thermocellum and other microbes have focused on metabolite levels at a single-time point, typically during the mid-exponential growth phase [ 12 , 36 , 37 ]. However, analyzing metabolites at multiple time points during fermentation reveals important dynamic changes in the metabolite pools. 4. DISCUSSION We have successfully engineered a strain of C. thermocellum where PPi no longer plays a role in central metabolism, which increased the thermodynamic driving force in glycolysis. In our previous work, where we replaced the PPi-PFK reaction with an ATP-PFK reaction without engineering other aspects of PPi metabolism [ 17 ], we observed an increased thermodynamic driving force at the PFK reaction, and an increased FBP/F6P ratio, but this metabolic perturbation appeared to be confined to a localized region around the PFK reaction. For example, the FBP level increased, but the downstream metabolites (DHAP, 3PG, or PEP) did not. In this work, by contrast, the levels of these downstream metabolites do increase. There are several possible explanations for this difference. One is that changes in PPi levels resulting from expression of a soluble PPase affect glycolytic flux. It is known that PPi concentrations allosterically regulate enzymes in the malate shunt [ 38 ], and it is possible that PPi concentrations regulate other enzymes in central metabolism as well. Another possible explanation is that we previously measured intracellular metabolite concentrations in bottle fermentations where pH was only controlled by MOPS buffer, whereas in this work, the pH was actively controlled. Changes in pH may have limited the effects of the ATP-PFK reaction in those experiments. Finally, a third possible explanation is that the previously observed localization of the effect of ATP-PFK was a temporal effect, since our prior work only measured intracellular metabolites at a single time point in mid-log phase. Among these three possibilities, we have no evidence to distinguish between the first two. We can, however, tentatively reject the third one. In the PPi-free glycolysis strain (LL1711), the increased abundance of downstream metabolites (DHAP, 3PG, and PEP) is observed in both mid-log and early stationary phases (approximately 22–78 hours). Comparing any of the points in this range with our prior results shows a clear difference with the much lower levels of these metabolites observed in mid-log phase with our previously-developed ATP-PFK strain (LL1660) [ 17 ]. In our PPi-free glycolysis strain (strain LL1711), whole genome sequencing revealed a frameshift mutation (E784) in C. thermocellum adhE that eliminated its function. We have routinely observed mutations in adhE in strains that either have higher ethanol production or tolerance [ 18 , 39 , 40 ]. Previous studies have shown that inactivating mutations in adhE that eliminated NADH-linked alcohol dehydrogenase (ADH) activity increase ethanol tolerance in C. thermocellum [ 39 , 41 ]. We believe that this is an adaptive response by the strain to avoid a thermodynamic limitation that can result from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ADH reactions using the same cofactor (i.e., NADH) [ 18 , 40 , 41 ]. Although the PPi-free glycolysis strain produced 455 mM (21.0 g/L) ethanol, it is likely that titers of 40–50 g/L are needed for commercial application [ 1 ]. Nonetheless this work represents another step along the path toward converting the atypical glycolysis of C. thermocellum to the canonical glycolysis [ 11 , 12 ]. In our previous work, we replaced the PPi-linked PFK reaction with an ATP-linked PFK reaction [ 17 ]. In this work, we needed to recreate this modification due to the presence of a spontaneous partial genome duplication event in our prior strain, and additionally introduce a deletion of the ppdk gene and heterologous expression of a soluble PPase gene. The expression of a soluble PPase appears to be the enabling factor for engineering PPi-free glycolysis [ 25 ]. Subsequent steps along this path may include eliminating the malate shunt [ 15 ], and understanding the role of GTP vs. ATP in the glucokinase and phosphoglycerate kinase reactions [ 11 ]. CONCLUSIONS In the field of Metabolic Engineering, strategies for improving product yield are relatively well developed compared to strategies for improving product titer. Engineering the thermodynamic landscape of a metabolic pathway provides an interesting approach to address product titer limitations. Although there is extensive prior literature on analysis of the thermodynamic landscape of metabolic pathways [ 12 , 14 , 16 , 42 ], there have been far fewer attempts to apply this knowledge for targeted improvement of these pathways. Several prior studies have hypothesized that the low thermodynamic driving force of the atypical glycolysis of C. thermocellum , and/or a limited PPi supply for the PPi-consuming reactions might limit product titer [ 11 , 25 , 43 ]. Here we show that the improved thermodynamic driving force of our PPi-free glycolysis allowed a 38% increase in ethanol titer (Fig. 5 ), providing evidence of the utility of this approach. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of Data and Materials The datasets supporting the conclusions of this article are included within the article and supplementary materials. Competing Interests Lee R. Lynd is the co-founder and CTO of Terragia Biofuels, Inc (https://terragiabiofuel.com/). Shuen Hon is an employee of Terragia Biofuels. Terragia has a financial interest in commercialization of Clostridium thermocellum . There are no other competing interests. Funding This work was supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886. Authors’ contributions BDS: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Visualization; Writing - original draft. SH: Conceptualization; Investigation; Strain development; Formal analysis; Visualization; Writing - Review and Editing. ET: Data curation; Investigation; Writing - Review and Editing. DMS: Data curation; Investigation. DAN: Writing - Review and Editing. AMG: Conceptualization, Writing - Review and Editing. LL: Funding Acquisition, Project Administration, Writing - Review and Editing. DGO: Conceptualization, Writing - Review and Editing, Supervision, Project Administration, Funding Acquisition. Acknowledgements Not applicable References Lynd LR, Beckham GT, Guss AM, et al (2022) Toward low-cost biological and hybrid biological/catalytic conversion of cellulosic biomass to fuels. Energy Environ Sci 15:938–990 Lynd LR, Guss AM, Himmel ME, et al (2017) Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum . In: Wittmann C, Liao JC (eds) Ind. Biotechnol., 1st ed. Wiley, pp 365–394 Mazzoli R, Olson DG (2020) Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose. In: Adv. Appl. Microbiol. Elsevier, pp 111–161 Holwerda EK, Worthen RS, Kothari N, et al (2019) Multiple levers for overcoming the recalcitrance of lignocellulosic biomass. Biotechnol Biofuels 12:15 Lynd LR, Liang X, Biddy MJ, Allee A, Cai H, Foust T, Himmel ME, Laser MS, Wang M, Wyman CE (2017) Cellulosic ethanol: status and innovation. Curr Opin Biotechnol 45:202–211 Holwerda EK, Ellis LD, Lynd LR (2013) Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms. Biotechnol Bioeng 110:2380–2388 Hon S, Holwerda EK, Worthen RS, Maloney MI, Tian L, Cui J, Lin PP, Lynd LR, Olson DG (2018) Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production. Biotechnol Biofuels. https://doi.org/10.1186/s13068-018-1245-2 Tian L, Papanek B, Olson DG, et al (2016) Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum. Biotechnol Biofuels 9:1–11 Holwerda EK, Olson DG, Ruppertsberger NM, Stevenson DM, Murphy SJL, Maloney MI, Lanahan AA, Amador-Noguez D, Lynd LR (2020) Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. Biotechnol Biofuels 13:40 Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258–266 Zhou J, Olson DG, Argyros DA, Deng Y, van Gulik WM, van Dijken JP, Lynd LR (2013) Atypical Glycolysis in Clostridium thermocellum. Appl Environ Microbiol 79:3000–3008 Jacobson TB, Korosh TK, Stevenson DM, Foster C, Maranas C, Olson DG, Lynd LR, Amador-Noguez D (2020) In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers. mSystems 5:e00736-19 Park JO, Rubin SA, Xu Y-F, Amador-Noguez D, Fan J, Shlomi T, Rabinowitz JD (2016) Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat Chem Biol 12:482–489 Noor E, Bar-Even A, Flamholz A, Reznik E, Liebermeister W, Milo R (2014) Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism. PLoS Comput Biol 10:e1003483 Olson DG, Hörl M, Fuhrer T, Cui J, Zhou J, Maloney MI, Amador-Noguez D, Tian L, Sauer U, Lynd LR (2017) Glycolysis without pyruvate kinase in Clostridium thermocellum. Metab Eng 39:169–180 Dash S, Olson DG, Hung S, Chan J, Amador-noguez D, Lynd LR, Maranas CD (2019) Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum. Metab Eng 55:161–169 Hon S, Jacobson T, Stevenson DM, Maloney MI, Giannone RJ, Hettich RL, Amador-Noguez D, Olson DG, Lynd LR (2022) Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum . Appl Environ Microbiol 88:e01258-22 Olson DG, Maloney MI, Lanahan AA, et al (2023) Ethanol tolerance in engineered strains of Clostridium thermocellum. Biotechnol Biofuels Bioprod 16:137 Gibson DG (2011) Enzymatic Assembly of Overlapping DNA Fragments. In: Methods Enzymol. Elsevier, pp 349–361 Guss AM, Olson DG, Caiazza NC, Lynd LR (2012) Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum. Biotechnol Biofuels 5:30 Olson DG, Lynd LR (2012) Transformation of clostridium thermocellum by electroporation. Methods Enzymol. https://doi.org/10.1016/B978-0-12-415931-0.00017-3 Hon S, Lanahan AA, Tian L, Giannone RJ, Hettich RL, Olson DG, Lynd LR (2016) Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs). Metab Eng Commun 3:120–129 Hon S, Olson DG, Holwerda EK, Lanahan AA, Murphy SJL, Maloney MI, Zheng T, Papanek B, Guss AM, Lynd LR (2017) The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum. Metab Eng 42:175–184 Yayo J, Rydzak T, Kuil T, Karlsson A, Harding DJ, Guss AM, Van Maris AJA (2023) The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum. Appl Environ Microbiol 89:e01753-22 Kuil T, Hon S, Yayo J, Foster C, Ravagnan G, Maranas CD, Lynd LR, Olson DG, van Maris AJA (2022) Functional Analysis of H + -Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum. Appl Environ Microbiol 88:e01857-21 Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95–97 Lo J, Zheng T, Hon S, Olson DG, Lynd LR (2015) The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. J Bacteriol 197:1386–1393 Bennett BD, Yuan J, Kimball EH, Rabinowitz JD (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3:1299–1311 Callaghan MM, Thusoo E, Sharma BD, Getahun F, Stevenson DM, Maranas C, Olson DG, Lynd LR, Amador-Noguez D (2023) Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism. Metab Eng 80:254–266 Sharma BD, Olson DG, Giannone RJ, Hettich RL, Lynd LR (2023) Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations. Appl Environ Microbiol 89:e00406-23 Agrawal S, Kumar S, Sehgal R, George S, Gupta R, Poddar S, Jha A, Pathak S (2019) El-MAVEN: A Fast, Robust, and User-Friendly Mass Spectrometry Data Processing Engine for Metabolomics. In: D’Alessandro A (ed) High-Throughput Metabolomics. Springer New York, New York, NY, pp 301–321 Holwerda EK, Thorne PG, Olson DG, Amador-Noguez D, Engle NL, Tschaplinski TJ, Van Dijken JP, Lynd LR (2014) The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading. Biotechnol Biofuels 7:155 Clasquin MF, Melamud E, Rabinowitz JD (2012) LC‐MS Data Processing with MAVEN: A Metabolomic Analysis and Visualization Engine. Curr Protoc Bioinforma. https://doi.org/10.1002/0471250953.bi1411s37 Deng Y, Olson DG, Zhou J, Herring CD, Joe Shaw A, Lynd LR (2013) Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum. Metab Eng. https://doi.org/10.1016/j.ymben.2012.11.006 Yayo J, Kuil T, Olson DG, Lynd LR, Holwerda EK, van Maris AJA (2021) Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose. Appl Environ Microbiol 87:1–18 Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5:593–599 Røst LM, Brekke Thorfinnsdottir L, Kumar K, Fuchino K, Eide Langørgen I, Bartosova Z, Kristiansen KA, Bruheim P (2020) Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems. Metabolites 10:74 Taillefer M, Rydzak T, Levin DB, Oresnik IJ, Sparling R (2015) Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase. Appl Environ Microbiol 81:2423–2432 Biswas R, Prabhu S, Lynd LR, Guss AM (2014) Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum. PLoS ONE 9:e86389 Pech-Canul A, Hammer SK, Ziegler SJ, Richardson ID, Sharma BD, Maloney MI, Bomble YJ, Lynd LR, Olson DG (2024) The Role of AdhE on Ethanol Tolerance and Production in Clostridium thermocellum. J Biol Chem 107559 Tian L, Cervenka ND, Low AM, Olson DG, Lynd LR (2019) A mutation in the AdhE alcohol dehydrogenase of Clostridium thermocellum increases tolerance to several primary alcohols, including isobutanol, n-butanol and ethanol. Sci Rep 9:1736 Khana DB, Callaghan MM, Amador-Noguez D (2022) Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks. Curr Opin Microbiol 66:21–31 Schroeder WL, Kuil T, Van Maris AJA, Olson DG, Lynd LR, Maranas CD (2023) A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis. Metab Eng 77:306–322 Additional Declarations Competing interest reported. Lee R. Lynd is the co-founder and CTO of Terragia Biofuels, Inc ( https://terragiabiofuel.com/ ). Shuen Hon is an employee of Terragia Biofuels. Terragia has a financial interest in commercialization of Clostridium thermocellum. There are no other competing interests. Supplementary Files AdditionalFile1Enzymeassayrawdata.xlsx AdditionalFile2isotopepeakvalues.xlsx AdditionalFile3Completefermentationdata.xlsx AdditionalFile4Resequencing.xlsx SupplementaryfilesBBB.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2024 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted Editorial decision: Revision requested 21 Oct, 2024 Reviews received at journal 19 Oct, 2024 Reviews received at journal 13 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviewers agreed at journal 01 Oct, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers invited by journal 16 Sep, 2024 Editor assigned by journal 04 Sep, 2024 Submission checks completed at journal 04 Sep, 2024 First submitted to journal 03 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5027329","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":361037609,"identity":"60fb2415-43a4-4245-8ef6-ee618fc0bb86","order_by":0,"name":"Bishal Dev Sharma","email":"","orcid":"","institution":"Thayer School of Engineering, Dartmouth College","correspondingAuthor":false,"prefix":"","firstName":"Bishal","middleName":"Dev","lastName":"Sharma","suffix":""},{"id":361037610,"identity":"b49836db-004b-4dce-9600-9e98b32dae6b","order_by":1,"name":"Shuen Hon","email":"","orcid":"","institution":"Thayer School of Engineering, Dartmouth College","correspondingAuthor":false,"prefix":"","firstName":"Shuen","middleName":"","lastName":"Hon","suffix":""},{"id":361037612,"identity":"bdb75afe-b380-433c-9f5c-da1b488467cc","order_by":2,"name":"Eashant Thusoo","email":"","orcid":"","institution":"Department of Bacteriology, University of Wisconsin–Madison","correspondingAuthor":false,"prefix":"","firstName":"Eashant","middleName":"","lastName":"Thusoo","suffix":""},{"id":361037615,"identity":"242e33bb-1b1f-4d72-a2e5-ed06a0f52abb","order_by":3,"name":"David M. Stevenson","email":"","orcid":"","institution":"Department of Bacteriology, University of Wisconsin–Madison","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"M.","lastName":"Stevenson","suffix":""},{"id":361037616,"identity":"ceddf9c2-6ecb-4390-b5cd-76d1e53dddd3","order_by":4,"name":"Daniel Amador-Noguez","email":"","orcid":"","institution":"Department of Bacteriology, University of Wisconsin–Madison","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Amador-Noguez","suffix":""},{"id":361037617,"identity":"f38c5ecc-8ae9-4d89-8891-6e586fd4e587","order_by":5,"name":"Adam M. Guss","email":"","orcid":"","institution":"Biosciences Division, Oak Ridge National Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"M.","lastName":"Guss","suffix":""},{"id":361037618,"identity":"5928c7a2-545a-4722-a8ad-e30f9934158b","order_by":6,"name":"Lee R. Lynd","email":"","orcid":"","institution":"Thayer School of Engineering, Dartmouth College","correspondingAuthor":false,"prefix":"","firstName":"Lee","middleName":"R.","lastName":"Lynd","suffix":""},{"id":361037619,"identity":"7e9be35b-e37d-4ce4-9321-35b1ea083609","order_by":7,"name":"Daniel G. Olson","email":"data:image/png;base64,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","orcid":"","institution":"Thayer School of Engineering, Dartmouth College","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"G.","lastName":"Olson","suffix":""}],"badges":[],"createdAt":"2024-09-03 21:07:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5027329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5027329/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-024-02591-5","type":"published","date":"2024-12-18T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65936223,"identity":"f5c177c2-c63f-4b4e-8e93-ac4508885715","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":479590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrain lineage diagram showing the sequence of genetic modifications for strains presented in this work and relevant prior work.\u003c/strong\u003e Strains shown in blue, orange and pink colors correspond to previous metabolic engineering work in our group. The green color represents the strains that were created in this study. Genetic modification between the strains are mentioned below specific arrows. Multiple arrows indicate multiple genetic modifications between different strains. The complete genotype of strains used in this work are presented in Table 1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/3d912edac7f180d6e27f5300.png"},{"id":65937166,"identity":"8e34ef69-8760-43b2-b201-3e530e45f9df","added_by":"auto","created_at":"2024-10-04 15:12:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":197889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzyme activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. thermocellum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains for reactions related to PPi-linked reactions. \u003c/strong\u003eThese reactions include phosphofructokinase (PFK, EC 2.7.1.11 or 2.7.1.90), pyruvate phosphate dikinase (PPDK, EC 2.7.9.1), pyruvate kinase (PYK, EC 2.7.1.40) and thermostable soluble PPase (EC 3.6.1.1). In the PFK reaction panel, red bars and blue bars represent activity with either ATP or PPi as the cofactor. In the table at the bottom of the figure, the ‘+’ and ‘-’ signs indicate the presence or absence of heterologous genes at a given locus. The ‘WT’ and ‘Δ’ signs indicate the presence or absence (i.e. by deletion) of native genes. All the reactions were carried out using cell free extract. Missing bars indicate the absence of activity (i.e. below the \u0026nbsp;limit of detection, \u0026lt;0.02 μmol/min/mg). Error bars represent 1 standard \u0026nbsp;deviation (n ≥ 3 biological replicates). Raw enzyme assay data is available as Additional File 1.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/ddca200b5c3167bd5b642ac5.png"},{"id":65936214,"identity":"07322579-0bc0-437f-9cd3-1df252a58e4b","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC-labeling patterns of key glycolysis metabolites near the PFK reaction.\u003c/strong\u003e The metabolites glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (FBP) and dihydroxyacetone phosphate (DHAP) were measured at different stages of growth for cells grown with a 50:50 mixture of uniformly labeled and naturally labeled cellobiose. ‘M+’ notation indicates the number of \u003csup\u003e13\u003c/sup\u003eC-labeled carbon atoms. One set of representative data is shown for each strain (n = 2 biological replicates). Strain LL1592 is the parent strain, strain LL1590 is a control for loss of wild type \u003cem\u003eC. thermocellum\u003c/em\u003e AdhE activity, strain LL1711 is the PPi-free glycolysis strain. The ratio represents the ratio of absolute abundance of FBP to F6P. Figure S3 shows a comparison between biological duplicates of this experiment. Raw data used for the analysis is available as Additional File 2.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/1fb0178f606de00e46eaaf0f.png"},{"id":65937167,"identity":"294843df-c6df-43fa-b7ba-f7dfd4d94be3","added_by":"auto","created_at":"2024-10-04 15:12:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPi concentration in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. thermocellum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains without PPase expression (strain LL1590 and strain LL1592) and with PPase expression (strain LL1711).\u003c/strong\u003e The bar plot represents average values. The colored circles represent individual data points. PPi levels were measured in cell extracts and the intracellular concentration was calculated as described in the materials and methods section. P-values were calculated using one-sided paired t-test statistics. (n =6; 3 biological replicates, 2 technical replicates)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/cd2fc6ff497bb71191af06ba.png"},{"id":65937168,"identity":"8ae92d96-5482-46eb-af14-07c8cb654e72","added_by":"auto","created_at":"2024-10-04 15:12:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":343107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBatch fermentations of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. thermocellum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains with wild type glycolysis (strains LL1590 and LL1592) and PPi-free glycolysis (strain LL1711).\u003c/strong\u003e Strain LL1592 is the parent strain. Strain LL1590 is a control for loss of wild type \u003cem\u003eC. thermocellum\u003c/em\u003e AdhE activity (note that strain LL1590 is descended from strain LL1592 despite what might be assumed from the strain ID, see Figure 1). Strain LL1711 is the PPi-freeglycolysis strain. Strain LL1590 (LL1592 with a targeted deletion of \u003cem\u003eC. thermocellum adhE\u003c/em\u003e) is included to control for the effects of inactivation of the native \u003cem\u003eadhE \u003c/em\u003egene, to allow the effects of the changes to glycolysis to be seen more clearly. In strains LL1590 and LL1711, the only functional \u003cem\u003eadhE \u003c/em\u003egene is the one heterologously expressed from \u003cem\u003eT. saccharolyticum.\u003c/em\u003e Fermentations were performed with 100 g/L cellobiose in MTC-7 chemically defined medium with pH maintained at 7.0 +\\- 0.05 by addition of 4 M potassium hydroxide. The shaded background represents different phases of fermentation: dark gray, growth coupled fermentation; medium gray, growth uncoupled fermentation; and light gray, no ethanol production phases. One representative fermentation profile is shown for each strain (n≥2). Figure S4 shows a biological duplicate of this experiment. Additional File 3 (LL1590-1, LL1592-1, LL1711-1) shows additional fermentation product data for this experiment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/5ecff45a6d900e65794df82a.png"},{"id":65937672,"identity":"fb28cbee-e89f-4968-81bb-8838c0fcc87d","added_by":"auto","created_at":"2024-10-04 15:20:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbundance of glycolytic metabolites in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. thermocellum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains LL1592 and LL1711 during the course of fermentation.\u003c/strong\u003e The dotted lines differentiate different growth phases (from left to right, growth coupled fermentation, growth uncoupled fermentation and no ethanol production respectively). Absolute metabolite concentrations were determined by using external standards. Metabolite concentrations were normalized (per metabolite) from 0 to 1, with 0 being the least and 1 being the highest concentration measured. The green box shows the location of the PFK reaction in glycolysis. Figure S6shows a biological duplicate of this experiment. Key: cellobiose (Cel); glucose (Glu); glucose 6-phosphate (G6P); fructose 6-phosphate (F6P); fructose 1,6-bisphosphate (FBP); dihydroxyacetone phosphate (DHAP); 3-phosphoglycerate (3PG); phosphoenolpyruvate (PEP); pyruvate (Pyr)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/ed1f5bf00342ae18df526a91.png"},{"id":72201867,"identity":"503d51ec-8faa-4bbc-a025-9bdd95b036d2","added_by":"auto","created_at":"2024-12-23 16:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2283606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/2d32ca98-0e87-41aa-bd89-4525070c81b9.pdf"},{"id":65936215,"identity":"6284e830-b6fc-47b9-9462-dad2cca5b190","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23945,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1Enzymeassayrawdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/a7615683ddb21f9c59be900c.xlsx"},{"id":65936222,"identity":"cf5f198e-7d77-49f4-9531-d9bf050aa857","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16190,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile2isotopepeakvalues.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/ce2c62687e2a6c7e2e3dade1.xlsx"},{"id":65936218,"identity":"2ccd2b2d-cdfa-4a39-9c2e-655c1c4cafcd","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":35505,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile3Completefermentationdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/87ff3f98907b554db8d4c2c9.xlsx"},{"id":65936221,"identity":"6bf1bcdc-d11c-4553-888b-ed72ef9c0263","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":27801,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile4Resequencing.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/0d55ad212decbaf971bee359.xlsx"},{"id":65936224,"identity":"6abb209a-481e-4db0-a877-9c37f8648690","added_by":"auto","created_at":"2024-10-04 15:04:54","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1095907,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfilesBBB.docx","url":"https://assets-eu.researchsquare.com/files/rs-5027329/v1/0cb9e27bebf0d47ff2d954a9.docx"}],"financialInterests":"Competing interest reported. Lee R. Lynd is the co-founder and CTO of Terragia Biofuels, Inc (https://terragiabiofuel.com/). Shuen Hon is an employee of Terragia Biofuels. Terragia has a financial interest in commercialization of Clostridium thermocellum. There are no other competing interests.","formattedTitle":"Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers","fulltext":[{"header":"1. BACKGROUND","content":"\u003cp\u003eTechnologies are needed to decarbonize the transportation sector to help reduce the need for petroleum usage that is driving catastrophic climate change. While electrification may help replace the current usage of gasoline for light duty transportation, sustainable liquid fuel solutions are critically needed for aviation, rail, and marine sectors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Conversion of lignocellulose into ethanol can be part of the solution because ethanol can be catalytically oligomerized to hydrocarbons that can be used for sustainable aviation fuel (SAF). However, better conversion technologies are needed to make SAF a reality.\u003c/p\u003e \u003cp\u003e \u003cem\u003eClostridium thermocellum\u003c/em\u003e (also known as ​​\u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e, \u003cem\u003eHungateiclostridium thermocellum\u003c/em\u003e, and \u003cem\u003eRuminiclostridium thermocellum\u003c/em\u003e) is a thermophilic anaerobe that has been extensively studied and engineered for consolidated bioprocessing of lignocellulose to biofuels such as ethanol [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], due to its strong native ability to solubilize and ferment lignocellulose [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One of the unique properties of this organism is that it consumes crystalline cellulose (e.g. Avicel) at the same rate or faster than soluble substrates, such as cellobiose [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous metabolic engineering efforts aimed at transforming \u003cem\u003eC. thermocellum\u003c/em\u003e into a commercial platform for lignocellulosic biofuel production have primarily concentrated on enhancing fuel yield. Notably, high ethanol yields exceeding 80% of the theoretical maximum have been achieved [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Current research is now directed towards further engineering the bacterium to achieve higher ethanol titers. Existing engineered strains produce titers in the range of 25\u0026ndash;30 g/L [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Concentrations in the 40\u0026ndash;50 g/L range are thought to be necessary for commercial applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe atypical Embden-Meyerhof-Parnas (EMP) glycolytic pathway found in wild type \u003cem\u003eC. thermocellum\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] was previously observed to function closer to thermodynamic equilibrium compared to organisms such as \u003cem\u003eThermoanaerobacterium saccharolyticum\u003c/em\u003e or \u003cem\u003eEscherichia coli\u003c/em\u003e that utilize a canonical glycolytic pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and may limit final product titer in \u003cem\u003eC. thermocellum\u003c/em\u003e. For our purposes, we define a canonical glycolytic pathway as one that uses an ATP-consuming reaction to generate fructose 1,6-bisphosphate and an ATP-producing reaction to convert phosphoenolpyruvate to pyruvate, and both of these steps have a high thermodynamic driving force that allows for allosteric regulation.\u003c/p\u003e \u003cp\u003eDifferences in cofactor usage for key glycolytic reactions were proposed as the underlying reason for the distinct pathway thermodynamics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Specifically, the pyrophosphate (PPi)-dependent phosphofructokinase (PPi-PFK, ∆G\u003csub\u003ePPi\u0026minus;pfk\u003c/sub\u003e = -1.45 kJ/mol) reaction is thought to significantly contribute to the lower thermodynamic driving force observed in \u003cem\u003eC. thermocellum\u003c/em\u003e. In microbes like \u003cem\u003eT. saccharolyticum\u003c/em\u003e, which utilize an ATP-dependent phosphofructokinase (ATP-PFK, ∆G\u003csub\u003eATP\u0026minus;pfk\u003c/sub\u003e = -22.57 kJ/mol), this reaction exhibits the largest drop in Gibbs free energy change of the entire pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, research findings have suggested that the ATP/ADP ratio tends to be significantly higher than the PPi to inorganic phosphate (PPi/Pi) ratio in many organisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and thus the actual further widening the difference in thermodynamic driving force between the two reactions. Additionally, it has been previously noted that the two native pathways employed by \u003cem\u003eC. thermocellum\u003c/em\u003e for the conversion of phosphoenolpyruvate (PEP) to pyruvate, namely the pyruvate:phosphate dikinase (PPDK) that uses PPi to convert PEP and AMP to pyruvate and ATP [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and the malate shunt that converts PEP to oxaloacetate to malate to pyruvate [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], also represent thermodynamic bottlenecks [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe recently engineered \u003cem\u003eC. thermocellum\u003c/em\u003e to use ATP as the main cofactor for the PFK reaction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although this increased the thermodynamic driving force of the PFK reaction, the effects on intracellular metabolites were limited to a small region adjacent to this reaction, and there was no effect on ethanol titer. Furthermore, resequencing analyses found that the resulting strain (LL1660) had a partial genome duplication, complicating subsequent genetic engineering.\u003c/p\u003e \u003cp\u003eIn this work, we make further progress toward engineering a PPi-free glycolysis in \u003cem\u003eC. thermocellum\u003c/em\u003e glycolysis by eliminating the PPi-consuming reactions PFK and PPDK and providing an alternative pathway for PEP to pyruvate conversion via the pyruvate kinase (PYK) reaction. We define \u0026ldquo;PPi-free glycolysis\u0026rdquo; strain as one that does not require pyrophosphate (PPi) for any of the reactions in glycolysis. A key enabling step of engineering this into \u003cem\u003eC. thermocellum\u003c/em\u003e was the expression of a soluble pyrophosphatase (PPase). Finally, we show that PPi-free glycolysis increases the levels of downstream glycolysis metabolites, increasing ethanol titers. This demonstrates the potential of engineering pathway thermodynamics as a general strategy for improving product titers.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Strains used in this work\u003c/h2\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStrains used in this work are described below. Tsc and Taq refers to genes introduced from \u003cem\u003eT. saccharolyticum\u003c/em\u003e and \u003cem\u003eT. aquaticus\u003c/em\u003e, respectively.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrganism\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAccession Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReferences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1592\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDSM 1313 \u003cem\u003e∆hpt, ∆ldh, \u0026Delta;reIII\u003c/em\u003e, P2638::\u003cem\u003eadhA(Tsc), nfnAB(Tsc), adhEG544D(Tsc\u003c/em\u003e), \u003cem\u003eClo1313_2637::Ptsc0046-pforA(Tsc)-ferredoxin(Tsc), ∆pfor1, ∆pfor2, ∆pfor3, ∆pfor4, ∆pfor5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX5290154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[17]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1592 \u003cem\u003e∆adhE\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX5290158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[18]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1592 \u003cem\u003etal(Tsc)\u003c/em\u003e, ATP-\u003cem\u003epfk(Tsc)\u003c/em\u003e, \u003cem\u003e∆ags, pPfk(Tsc)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX6875980\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[17]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1649 \u003cem\u003epyk(Tsc)\u003c/em\u003e, \u003cem\u003ePPase(Taq)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX7724531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1689 \u003cem\u003e∆ppdk, adhE\u003c/em\u003e E784*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX9409012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLL1710 \u003cem\u003e∆\u003c/em\u003ePPi-\u003cem\u003epfk\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSRX9409011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Plasmid and strain construction\u003c/h2\u003e\n \u003cp\u003ePlasmids were constructed via isothermal assembly [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], using commercially available kits (New England Biolabs NEBuilder\u0026reg; HiFi DNA Assembly Master Mix, catalog number E2621). Plasmids were maintained and propagated from BL21 derivative \u003cem\u003eE. coli\u003c/em\u003e strains (New England Biolabs T7 Express \u003cem\u003eE. coli\u003c/em\u003e, catalog number C2566) to ensure proper methylation of the plasmid DNA [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Plasmid DNA was purified using commercial kits available from either New England Biolabs or Zymo Research.\u003c/p\u003e\n \u003cp\u003eTransformation of \u003cem\u003eC. thermocellum\u003c/em\u003e was performed as previously described [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePlasmids used in this study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlasmid\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAccession no.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReferences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e expression vector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKX259110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[22]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDeletion/Integration vector backbone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKY852359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[23]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epMU2051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlasmid for deletion of \u003cem\u003eppdk\u003c/em\u003e gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKC146550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[11]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDeletion vector; deletes \u003cem\u003eC. thermocellum PPi-pfk\u003c/em\u003e only in strain LL1647 and its derivatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eON809513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[17]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eC. thermocellum\u003c/em\u003e Clo 1313_2638 promoter driving \u003cem\u003eT. saccharolyticum\u003c/em\u003e ATP-\u003cem\u003epfk\u003c/em\u003e and \u003cem\u003eT. aquaticus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eC. thermocellum\u003c/em\u003e Clo 1313_2638 promoter driving \u003cem\u003eT. aquaticus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eC. thermocellum\u003c/em\u003e Clo 1313_2638 promoter driving \u003cem\u003eGeobacillus stearothermophilus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter driving \u003cem\u003eGeobacillus stearothermophilus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter driving \u003cem\u003eGeobacillus thermoglucosidasius PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter driving \u003cem\u003eT. acidophilum PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter driving \u003cem\u003eT. aquaticus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO143 with \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter driving \u003cem\u003eT. thermophilus PPase\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO145 with \u003cem\u003eT. saccharolyticum pyk\u003c/em\u003e and \u003cem\u003eG. thermoglucosidasius\u003c/em\u003e cytosolic \u003cem\u003ePPase\u003c/em\u003e driven by \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epLL1507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epDGO145 with \u003cem\u003eT. saccharolyticum pyk\u003c/em\u003e and \u003cem\u003eT. aquaticus\u003c/em\u003e cytosolic \u003cem\u003ePPase\u003c/em\u003e driven by \u003cem\u003eT. saccharolyticum\u003c/em\u003e enolase promoter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP855260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Media and growth conditions\u003c/h2\u003e\n \u003cp\u003eAll reagents used in this study were of molecular grade and obtained from Sigma Aldrich or Fisher Scientific, unless otherwise noted.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e strains were grown at 55\u0026deg;C under anaerobic conditions, either in anaerobic chambers (Coy Laboratory Products, Grass Lakes, MI), with previously described environmental settings [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e], or in sealed serum bottles that were prepared as previously described [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eComplex medium CTFUD was prepared as previously described [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] and used for culturing cells for use in transformations, or for preparing genomic DNA for strain resequencing. Defined MTC-5 medium was used for all other purposes [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Enzyme assays\u003c/h2\u003e\n \u003cp\u003eAll chemicals were ordered from MilliporeSigma unless otherwise specified. All enzyme assays were performed anaerobically unless otherwise mentioned. For enzyme assays, cells were grown to mid-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e between 0.6 to 1.0), harvested and lysed to obtain cell extract as described earlier [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Protein concentrations were determined using Bradford assay (Coomassie Plus Reagent, Thermo Scientific), with bovine serum albumin used as a protein standard. All the enzyme assays were performed at 55\u0026deg;C except for the phosphofructokinase assay, which was performed at 40\u0026deg;C due to precipitation issues associated with the aldolase coupling enzyme.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e2.4.1 Phosphofructokinase (PFK) assay. PFK activity was assayed by measuring the formation of fructose 1,6-bisphosphate coupled with consumption of NADH as previously described [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.15 mM NADH, 1 mM fructose-6-phosphate, 4 U/mL fructose bisphosphate aldolase, 4 U/mL triosephosphate isomerase, 4 U/mL a-glycerophosphate dehydrogenase, cell extract, and 2 mM either PPi or ATP. The assay reaction was started by the addition of the phosphate donor (PPi or ATP).\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.4.2 Pyruvate phosphate dikinase (PPDK) assay. PPDK activity was assayed by measuring the consumption of NADH by coupled assay with lactate dehydrogenase (LDH) as previously described with slight modification [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. The assay mixture contained 100 mM Tris-HCl (pH 7.0), 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM AMP, 0.15 mM NADH, 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 2 mM PEP, 1 mM fructose-1,6-bisphosphate, 4 U/ml lactate dehydrogenase, cell extract, and 2 mM PPi. The assay reaction was started by the addition of PPi.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.4.3 Pyruvate kinase (PYK) assay. PYK activity was measured by coupled assay with lactate dehydrogenase (LDH) as described earlier with slight modification [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The assay buffer contained 100 mM Tris-HCl, pH 7.5 (at 55\u0026deg;C), 5 mM dithiothreitol (DTT), 10 mM KCl, 12 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM ADP, 0.3 mM NADH, 0.1 mM 3-phosphoglyceric acid (3PG), 5 mM PEP, 12 U LDH enzyme, and cell extract. This reaction could be started with either ADP or PEP. For this experiment, the assay reaction was started by the addition of ADP.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.4.4 Soluble pyrophosphatase (PPase) assay. PPase activity was measured aerobically based on hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi) using malachite green assay as defined earlier [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] with some modifications. The assay mixture contained 100 mM Tris-HCl at pH 8.0, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM sodium pyrophosphate and 2, 4 or 8 \u0026micro;l cell extract. This assay mixture was incubated at 55\u0026deg;C for 2 minutes and the reaction was stopped by transferring the samples to ice. 2.5 ul of this sample was added to 40 \u0026micro;l of malachite green reagent (Malachite green:Ammonium molybdate at 3:1) in 96 wells plate. The wells with PPase activity turn green in color. 7.5 \u0026micro;l of 34% sodium citrate was added, mixed and incubated at room temperature for 30 minutes to allow full color development [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Phosphate concentration was calculated by measuring absorbance at 660 nm using a calibration curve with phosphate concentration ranging from (0\u0026ndash;5) mM.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.4.5 Aldehyde dehydrogenase (ALDH) assay. ALDH activity was measured anaerobically as described earlier [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5 \u0026micro;M FeSO\u003csub\u003e4\u003c/sub\u003e, 0.25 mM NADH or NADPH, 1.25 mM acetyl-CoA, 1 mM DTT, and cell extract. The assay reaction was started by the addition of acetyl-CoA.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.4.6 Alcohol dehydrogenase (ADH) assay. ADH activity was measured anaerobically as described earlier [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The assay reaction contained 100 mM Tris-HCl (pH 7.0), 5 \u0026micro;M FeSO\u003csub\u003e4\u003c/sub\u003e, 0.25 mM NADH or NADPH, 18 mM acetaldehyde, 1 mM DTT, and cell extract. The assay reaction was started by the addition of acetaldehyde.\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 \u003csup\u003e13\u003c/sup\u003eC labeling conditions\u003c/h2\u003e\n \u003cp\u003eFor \u003csup\u003e13\u003c/sup\u003eC labeling experiments, \u003cem\u003eC. thermocellum\u003c/em\u003e cells were grown anaerobically on MTC-5 medium consisting of 3 mM naturally labeled cellobiose (Sigma C7252) and 3 mM uniformly labeled \u003csup\u003e13\u003c/sup\u003eC-cellobiose (Omicron Biomedicals, CEL-002) as mentioned earlier [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. 10 mL of this media was inoculated with 10 \u0026micro;l freezer stock of LL1590, LL1592 and LL1711 and incubated at 55\u0026deg;C. The freezer stocks were prepared by growing the cells to mid-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.6) on MTC-5 with 5 g/L naturally labeled cellobiose.\u003c/p\u003e\n \u003cp\u003eMetabolite samples were extracted at three different phases during growth i.e. early log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.4), late log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.8) and stationary phase (12 hours after maximum OD has reached). The growth was monitored continuously using a custom-built absorbance reader. Intracellular metabolites were collected using vacuum filtration as previously described [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. For each sample, 2.5 ml of culture medium was filtered through 3.0 \u0026micro;m hydrophilic nylon filter (SF14529, Tisch Scientific) to separate cells from medium components. The filter with cells was placed in 1.6 mL of cold extraction solvent (40% acetonitrile, 40% methanol, and 20% water) with the cell side facing down and kept on aluminum block from \u0026minus;\u0026thinsp;80\u0026deg;C to quench metabolism and extract metabolites. Cells were washed off the filter using extraction solvent and then centrifuged for 5 minutes to remove cell debris. The supernatant with metabolites was collected, dried using a sample concentrator (catalog no. EW-36620-40; Cole-Parmer) to remove the metabolite extraction buffer and resuspended in molecular-grade water. This metabolite extract was analyzed using LC-MS. Metabolites of interest were detected based on retention time and mass-to-charge ratio, compared with pure standards, using El-MAVEN software [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Quantification of PPi in cells\u003c/h2\u003e\n \u003cp\u003eTo quantify the concentration of PPi in cells, samples were resuspended in molecular-grade water as mentioned earlier (section \u003cspan class=\"InternalRef\"\u003e3.5\u003c/span\u003e). Inorganic pyrophosphate (PPi) was quantified using a combination of two different kits; a PPiLight inorganic pyrophosphate assay kit (catalog no. LT07-610; Lonza) which measures the sum of PPi and ATP and an ADP/ATP ratio assay kit (catalog no. MAK135; Sigma-Aldrich) which measures only ATP without cross-reacting with PPi. Then, PPi in cells was calculated using the first kit, and corrected for the effects of ATP using the second kit using the formula below:\u003c/p\u003e\n \u003cp\u003ePPi (mM) per cell = (PPi (mM) in metabolite extract \u0026times; Volume of metabolite extraction buffer )/Intracellular volume (ml) where, PPi (mM) in metabolite extract was calculated by using the kits above as PPi (mM) = ((ATP\u0026thinsp;+\u0026thinsp;PPi)\u003csub\u003eLT07\u0026minus;610\u003c/sub\u003e - ATP\u003csub\u003eMAK135\u003c/sub\u003e)/1000, and Intracellular volume\u0026thinsp;=\u0026thinsp;cell volume x cell number [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 pH-controlled bioreactor fermentation conditions and metabolites extraction\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eC. thermocellum\u003c/em\u003e cultures were grown anaerobically at 55\u0026deg;C in a Coy (Ann Arbor, MI) anaerobic chamber with a gas phase of 85% N\u003csub\u003e2\u003c/sub\u003e, 10% CO\u003csub\u003e2\u003c/sub\u003e, and 5% H\u003csub\u003e2\u003c/sub\u003e. Fermentation was carried out at 300 mL working volume in MTC-7 (medium for thermophilic clostridia) with 100 g/L cellobiose as substrate as mentioned earlier [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. MTC-7 medium contained 100 g/L cellobiose, 9.3 g/L MOPS (morpholinepropanesulfonic acid) sodium salt, 2 g/L potassium citrate monohydrate, 1.3 g/L citric acid monohydrate, 1 g/L Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.5 g/L NaHCO\u003csub\u003e3\u003c/sub\u003e, 2 g/L urea, 1 g/L MgCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;6H\u003csub\u003e2\u003c/sub\u003eO, 0.2 g/L CaCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.1 g/L FeCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;4H\u003csub\u003e2\u003c/sub\u003eO, 1 g/L L-cysteine HCl monohydrate, 0.02 g/L pyridoxamine HCl, 0.004 g/L p-aminobenzoic acid, 0.002 g/L d-biotin, 0.002 g/L vitamin B12, 0.004 g/L thiamine, 0.0005 g/L MnCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;4H2O, 0.0005 g/L CoCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;6H\u003csub\u003e2\u003c/sub\u003eO, 0.0002 g/L ZnCl\u003csub\u003e2\u003c/sub\u003e, 0.0001 g/L CuCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;2H2O, 0.0001 g/L H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.0001 g/L Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026sdot;2H\u003csub\u003e2\u003c/sub\u003eO, and 0.0001 g/L NiCl\u003csub\u003e2\u003c/sub\u003e\u0026sdot;6H\u003csub\u003e2\u003c/sub\u003eO. MTC-7 is MTC-5 with the addition of 0.004 g/L thiamine. The pH was controlled at 7.0 using Mettler-Toledo (Columbus, OH, USA) pH probe with the addition of KOH.\u003c/p\u003e\n \u003cp\u003eThe extracellular metabolites/fermentation products (ethanol, lactate, formate, acetate, pyruvate) at different time points during the fermentation were quantified by HPLC (Waters, Milford, MA or LC-2030, Shimadzu) with refractive index (RI) and UV detection using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) with a 5 mM sulfuric acid solution eluent [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eTo quantify intracellular/glycolysis metabolites, samples were taken at the same time points as HPLC. Samples were prepared using a protocol we previously developed for metabolite quantification from high-substrate fermentations [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, samples were extracted using vacuum filtration followed by quenching. For each sample, (0.5\u0026ndash;8) ml of culture medium was filtered through 3.0 \u0026micro;m hydrophilic nylon filter based on OD\u003csub\u003e600\u003c/sub\u003e of cells. The samples were then quenched, dried, resuspended in molecular grade water and analyzed using LC-MS.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Liquid chromatography-mass spectrometry (LC-MS) analysis\u003c/h2\u003e\n \u003cp\u003eA Vanquish ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific) coupled to a hybrid quadrupole-Orbitrap\u0026trade; mass spectrometer (Q Exactive\u0026trade;; Thermo Scientific) equipped with electrospray ionization operating in negative-ion mode as defined earlier [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e] was used for LCMS analysis. The chromatography was performed at 25\u0026deg;C using a 2.1 \u0026times; 100 mm reverse-phase C18 column with a 1.7 \u0026micro;m particle size (Water\u0026trade;; Acquity UHPLC BEH). Two chromatography gradients were used. The first used Solvent A (97:3 H2O: methanol\u0026thinsp;+\u0026thinsp;10 mM tributylamine) and Solvent B (100% methanol) as follows: 0\u0026ndash;2.5 min, 5% B; 2.5\u0026ndash;17 min, linear gradient from 5% B to 95% B; 17\u0026ndash;19.5 min, 95% B; 19.5\u0026ndash;20 min, linear gradient from 95% B to 5% B; 20\u0026ndash;25 min, 5% B. The second also used Solvent A and Solvent B (100% methanol) and was as follows: 0\u0026ndash;2.5 min, 5% B; 2.5\u0026ndash;7.5 min, linear gradient from 5% B to 20% B; 7.5\u0026ndash;13 min, 20% B to 55% B; 13\u0026ndash;18.5min, 55% B to 95% B; 18.5\u0026ndash;19 min linear gradient from 95% B to 5% B; 19\u0026ndash;25 min, 5% B. The flow rate was held constant at 0.2 mL/min for both chromatograph methods. Metabolites of interest were identified by retention times (based on pure standards) and monoisotopic mass using MAVEN [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] and El-MAVEN [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Sequencing methods\u003c/h2\u003e\n \u003cp\u003eRoutine Sanger sequencing was outsourced to Azenta Life Sciences. Whole genome resequencing was performed by the Department of Energy Joint Genome Institute using the Illumina MiSeq sequencing platform, with a minimum of 100-fold coverage. Strains were analyzed with the software CLC Genomics Workbench (Qiagen) using strain DSM1313 as the reference genome (Genbank accession NC_017304.1); reads were filtered against strain LL1649 (accession number SRP222669) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e] to exclude inherited mutations. A summary of the identified mutations is provided in Additional File 4.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Strain construction to eliminate and replace PPi-dependent glycolytic reactions\u003c/h2\u003e \u003cp\u003eIn our previous work, we created strain LL1660 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where the PPi-\u003cem\u003epfk\u003c/em\u003e gene was replaced with an ATP-\u003cem\u003epfk\u003c/em\u003e gene [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], eliminating a major sink for PPi. We also observed the spontaneous occurrence of a large partial genome duplication. This partial genome duplication region encodes proteins that may serve as alternative sinks for PPi, including a membrane-bound \u003cem\u003epyrophosphatase\u003c/em\u003e gene (\u003cem\u003eHppA\u003c/em\u003e - \u003cem\u003eClo1313_0823\u003c/em\u003e) and \u003cem\u003epyruvate:phosphate dikinase\u003c/em\u003e gene (\u003cem\u003ePpdK\u003c/em\u003e - \u003cem\u003eClo1313_0949\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eWe therefore hypothesized that the partial genome duplication event might have occurred to address an imbalance in the supply and demand of PPi created by the mutations in strain LL1660. To address this potential problem, we set out to construct a new strain where PPi-consuming reactions in glycolysis were eliminated, and where the excess PPi generated by metabolism was hydrolyzed using a soluble PPase [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Initially, we attempted to express soluble PPase in wild-type \u003cem\u003eC. thermocellum\u003c/em\u003e, but we did not get any colonies in at least two transformations. This was not surprising, given our prior work showing the importance of PPi as a cofactor for the PFK reaction in glycolysis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNext, we tried to express the soluble PPase in a strain with ATP-linked PFK activity (strain LL1649), which we hypothesized would be more compatible with the metabolic effects of the PPase. In this strain, we occasionally obtained a small number of colonies with our PPase expression plasmid. However, in those colonies, the PPase gene was invariably inactivated either by a transposon insertion or inactivating nonsense mutation. In this phase, we tested PPases from \u003cem\u003eGeobacillus stearothermophilus\u003c/em\u003e, \u003cem\u003eGeobacillus thermoglucosidasius\u003c/em\u003e, \u003cem\u003eThermoplasma acidophilum\u003c/em\u003e, \u003cem\u003eThermus aquaticus\u003c/em\u003e, and \u003cem\u003eThermus thermophilus\u003c/em\u003e (plasmids pLL1501 to pLL1505, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Out of these, only PPase from \u003cem\u003eGeobacillus thermoglucosidasius\u003c/em\u003e and \u003cem\u003eThermus aquaticus\u003c/em\u003e showed PPase activity (data not shown).\u003c/p\u003e \u003cp\u003eWe further hypothesized that in addition to eliminating the need for PPi in the PFK reaction, we also needed to eliminate the need for PPi in the PPDK reaction (which converts PEP to pyruvate) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To do this, we designed an integration construct to simultaneously introduce pyruvate kinase and a soluble PPase onto the chromosome, in this case using the PPases from \u003cem\u003eG. thermoglucosidasius\u003c/em\u003e and \u003cem\u003eT. aquaticus\u003c/em\u003e since we had in some instances observed PPase activity from cell extracts of \u003cem\u003eC thermocellum\u003c/em\u003e that expressed those PPases [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Successful strain construction was obtained only with \u003cem\u003eT. aquaticus\u003c/em\u003e PPase containing construct, pLL1507 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which was validated by the gain of both pyruvate kinase and cytosolic PPase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our ability to observe functional PPase expression only in strains with both ATP-PFK and PYK activity provides further evidence of progress toward eliminating PPi as a key energy carrying cofactor in \u003cem\u003eC. thermocellum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStrain LL1592, which uses the native \u003cem\u003eC. thermocellum\u003c/em\u003e glycolytic pathway, with \u003cem\u003eT. saccharolyticum\u003c/em\u003e pyruvate-to-ethanol pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] was selected as reference strain for engineering. Strain LL1649 was derived from strain LL1592, by introduction of the \u003cem\u003eT. saccharolyticum\u003c/em\u003e transaldolase, followed by simultaneous deletion of the ADP-glucose synthase operon and expression of \u003cem\u003eT. saccharolyticum\u003c/em\u003e ATP-\u003cem\u003epfk\u003c/em\u003e. Strain LL1689 was derived from strain LL1649 by the simultaneous integration of the \u003cem\u003eT. saccharolyticum\u003c/em\u003e pyruvate kinase and the cytosolic pyrophosphatase from \u003cem\u003eThermus aquaticus.\u003c/em\u003e The purpose of introducing a heterologous pyruvate kinase was to allow subsequent deletion of the \u003cem\u003eppdk\u003c/em\u003e gene. The purpose of introducing a soluble PPase was to serve as an alternative sink for PPi, once both the PPi-\u003cem\u003epfk\u003c/em\u003e and \u003cem\u003eppdk\u003c/em\u003e genes were deleted. With these modification in place, we were then able to successfully delete glycolytic sinks for PPi, including the \u003cem\u003eppdk\u003c/em\u003e gene (resulting in strain LL1710), and then, subsequently, the PPi\u003cem\u003e-pfk\u003c/em\u003e gene (resulting in strain LL1711) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiochemical assays were performed to confirm the effect of the genetic modifications of the strains used in this study. As expected, strain LL1592 and strain LL1711 exclusively have PPi-PFK and ATP-PFK activity, respectively. Strains LL1649, LL1689, and LL1710 have both ATP- and PPi-linked PFK activity, as expected (PFK reaction, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Functional expression of the \u003cem\u003epyk\u003c/em\u003e gene was confirmed by the presence of PYK activity in strains LL1689, LL1710 and LL1711 (PYK reaction, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The effect of the \u003cem\u003eppdk\u003c/em\u003e gene deletion was confirmed in strains LL1710 and LL1711 based on elimination of PPDK activity (PPDK reaction, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Low levels of PPase activity were still detected in strains LL1592 and LL1649 (Paired t-test, p \u0026gt; 0.5); this could be attributed to the native membrane bound PPase enzyme being present in the cell extracts. Previous studies have reported the presence of membrane-bound pyrophosphatase in \u003cem\u003eC. thermocellum\u003c/em\u003e cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Nonetheless, about 5- to 13-fold higher PPase activities (Paired t-test, p \u0026lt; 0.005) were observed in strains LL1689, LL1710 and LL1711, which we attribute to expression of a soluble PPase. As such, it was determined that strain LL1711 had been engineered to not require PPi as a cofactor in glycolysis, and that it now possessed a more canonical glycolytic pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Use of ATP-dependent pathway alters thermodynamics of glycolysis\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the effect the genetic modifications had on the reversibility of the PFK reaction, we performed \u003csup\u003e13\u003c/sup\u003eC labeling on \u003cem\u003eC. thermocellum\u003c/em\u003e strains LL1590, LL1592 and LL1711. Initially, we were planning to compare only the parent strain (LL1592) and the PPi-free glycolysis strain (LL1711); however, whole genome sequencing of strain LL1711 revealed a frameshift mutation in the wild type \u003cem\u003eC. thermocellum adhE\u003c/em\u003e gene corresponding to amino acid position E784 that eliminated its function (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We therefore included a control strain (LL1590) derived from the parent strain with a targeted deletion of the wild type \u003cem\u003eC. thermocellum adhE\u003c/em\u003e gene [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], to better understand the effects of our changes to the glycolytic pathway without the confounding effects of the modification to ethanol production pathway. Note that all three strains (LL1590, LL1592, and LL1711) still produce ethanol via the heterologous \u003cem\u003eT. saccharolyticum adhE\u003c/em\u003e\u003csup\u003e\u003cem\u003eG544D\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. thermocellum\u003c/em\u003e does not readily consume glucose [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and thus cellobiose is the most commonly-used soluble sugar for laboratory growth experiments. Strains were cultured on a 1:1 ratio of naturally labeled to uniformly \u003csup\u003e13\u003c/sup\u003eC-labeled cellobiose. We confirmed that \u003csup\u003e13\u003c/sup\u003eC cellobiose and naturally labeled cellobiose are taken up equally by measuring the ratio of M + 0 to M + 6 isotopomers for glucose 6-phosphate, which ranged from 0.8–1.2 (Additional File 2). Samples were collected at three different phases (early-log, late-log and stationary) during growth (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the forward glycolysis reaction should generate a 1:1 mixture of fully labeled (M + 6) and fully unlabeled (M + 0) glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). The forward flux of fructose-bisphosphate aldolase reaction (FBA) will generate 50% unlabeled (M + 0) and 50% fully labeled (M + 3) dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). However, the reverse FBA reaction flux generates a 1:2:1 mixture of M + 0, M + 3 and M + 6 FBP. The reverse flux from phosphofructokinase (PFK) and phosphoglycerate isomerase (PGI) reactions transfer these M + 3 species into the F6P and G6P pools. Appearance of the M + 3 isotopomer in the F6P and G6P pools allows us to uniquely identify reverse flux through the PFK reaction. If the PFK reaction were completely irreversible, we would expect to see no M + 3 species in either the F6P or G6P pools.\u003c/p\u003e \u003cp\u003eAs expected, based on our prior work [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we observed that the M + 3 pool in the glycolytic intermediates G6P and F6P decreased substantially in the PPi-free glycolysis strain (LL1711) vs. the parent and control strains (LL1590 and LL1592), indicating that the genetic modifications introduced into LL1711 reduced the reversibility of the PFK reaction (G6P, F6P; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We saw an unexpected decrease in the M + 3 fraction of FBP in mid-log phase in the PPi-free glycolysis strain (LL1711) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This suggests reduced reversibility of Fba reaction, although the reason for this change is not known.\u003c/p\u003e \u003cp\u003eIn addition to comparing the isotope ratios, we also compared the ratio of the FBP and F6P metabolite pool sizes. This gives us an additional indication of the thermodynamic driving force of the reaction. A high thermodynamic driving force at the PFK reaction would result in a high FBP/F6P ratio. Comparisons of FBP/F6P ratio showed a lower ratio for the parent and control strains that use the PPi-PFK reaction (LL1590 and LL1592) and a much higher ratio for the PPi-free glycolysis strain (LL1711), which uses the ATP-PFK reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This ratio is 5- to 17-fold and 74- to 120-fold higher in ATP-\u003cem\u003epfk\u003c/em\u003e strain compared to PPi-\u003cem\u003epfk\u003c/em\u003e strains during log phases and stationary phases, respectively. The higher FBP/F6P ratio is similar to what is observed in organisms with canonical glycolytic pathways, such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eT. saccharolyticum\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Taken together, these two lines of evidence (M + 3 abundance changes and FBP/F6P ratio changes) support the conclusion that we have increased the thermodynamic driving force of the PFK reaction in our PPi-free glycolysis strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Expression of PPase results in lower PPi levels\u003c/h2\u003e \u003cp\u003eTo determine the effect of PPase expression in PPi levels of \u003cem\u003eC. thermocellum\u003c/em\u003e strains with wild type glycolysis (strains LL1590 and LL1592) and PPi-free glycolysis (strain LL1711), we quantified the concentration of PPi in cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed a significant decrease in PPi concentration in the PPi-free glycolysis strain (strain LL1711) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) during the early-log and late-log growth phases. During the stationary phase, the difference between strain LL1592 and strain LL1711 isn’t as significant, which might be due to the depletion of the PPi pool during this phase. We see consistently higher levels of PPi during the early-log and late-log phases in strains LL1590 and LL1592, which then drop during the stationary phase in both strains. The constant low level of PPi in strain LL1711 during all the growth phases might be the basal level of PPi in cells, even with the expression of PPase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Increased driving force leads to higher level of fermentation products in ATP-\u003cem\u003epfk\u003c/em\u003e strain\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaving demonstrated increased thermodynamic driving force in glycolysis in the PPi-free glycolysis strain, we explored the impact of fermentation behavior and ethanol production using batch fermentations of all three strains with high substrate concentrations (100 g/L cellobiose). The fermentations had three distinct phases: 1. growth coupled fermentation, where both the cell growth and ethanol production are at the highest rate, 2. growth uncoupled fermentation, where cell growth stops and the rate of ethanol production decreases, and 3. cessation of both growth and ethanol production. During this final phase, cellobiose is hydrolyzed to glucose at a slow rate, but metabolism appears to be inactive.\u003c/p\u003e \u003cp\u003eThe final ethanol titer was 329 ± 8 mM (15.1 ± 0.4 g/L) for strain LL1590, 403 ± 11 mM (18.6 ± 0.5 g/L) for strain LL1592, and 455 ± 12 mM (21.0 ± 0.6 g/L) for strain LL1711. Titer of another fermentation product, acetate, was 65 ± 0 mM (3.8 ± 0 g/L) for strain LL1590, 51 ± 9 mM (3.0 ± 0.5 g/L) for strain LL1592 and 88 ± 7 (5.2 ± 0.4 g/L) for strain LL1711. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, Additional File 3). Comparison of the fermentation products between LL1592 and LL1590 suggests that deletion of \u003cem\u003eC. thermocellum adhE\u003c/em\u003e impairs the production of ethanol. Taking the effect of the native \u003cem\u003eadhE\u003c/em\u003e inactivation into account (i.e. comparing LL1711 to LL1590), allows us to observe the effect of PPi-free glycolysis on ethanol production without the confounding effects of changes in the ethanol production pathway. In this comparison (LL1711 vs LL1590), we observe a 38% increase in ethanol titer, and a 35.4% increase in acetate titer. Together, these suggest that our PPi-free glycolysis strain exhibits increased glycolytic flux, resulting in an increased abundance of acetyl-CoA, which is then converted to more ethanol and acetate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Changes in glycolysis metabolite levels at elevated substrate concentrations in pH-controlled fermentations\u003c/h2\u003e \u003cp\u003eTo better understand the changes in central metabolism that allowed for increased ethanol titer in the PPi-free glycolysis strain, we measured intracellular glycolytic metabolites over the course of the fermentation using the same fermentation conditions from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (100 g/L cellobiose, pH-controlled batch fermentations). Since preliminary data showed similar pattern of glycolytic metabolites (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e) and \u003csup\u003e13\u003c/sup\u003eC labeling patterns were similar for both the PPi-\u003cem\u003epfk\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and strain LL1590 produced lower levels of ethanol than strain LL1592 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), we compared glycolytic metabolite levels in strains LL1592 and LL1711 .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThree major changes were observed in the glycolytic metabolite profiles between these two strains. First, in the parent strain (LL1592, with wild type glycolysis) we see gradual accumulation of hexose phosphates (G6P, F6P) as the fermentation progresses. In the PPi-free glycolysis strain (LL1711), hexose phosphates were still observed to accumulate, but to a lesser extent as the fermentation progressed. Second, during the growth-uncoupled fermentation phase, we observed a dramatic increase in the levels of lower glycolysis metabolites (DHAP, 3PG, and PEP) in the PPi-free glycolysis strain. Finally, in the phase where fermentation and ethanol production stop, the parent strain shows a large accumulation of upper glycolysis metabolites (glucose, G6P, and F6P), while the PPi-free glycolysis strain shows low levels of almost all metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Figure S6).\u003c/p\u003e \u003cp\u003eThese intracellular metabolites data over the course of fermentation also underscores the importance of conducting metabolomics studies throughout the fermentation process. Most previous metabolomics studies in \u003cem\u003eC. thermocellum\u003c/em\u003e and other microbes have focused on metabolite levels at a single-time point, typically during the mid-exponential growth phase [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, analyzing metabolites at multiple time points during fermentation reveals important dynamic changes in the metabolite pools.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eWe have successfully engineered a strain of \u003cem\u003eC. thermocellum\u003c/em\u003e where PPi no longer plays a role in central metabolism, which increased the thermodynamic driving force in glycolysis. In our previous work, where we replaced the PPi-PFK reaction with an ATP-PFK reaction without engineering other aspects of PPi metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we observed an increased thermodynamic driving force at the PFK reaction, and an increased FBP/F6P ratio, but this metabolic perturbation appeared to be confined to a localized region around the PFK reaction. For example, the FBP level increased, but the downstream metabolites (DHAP, 3PG, or PEP) did not. In this work, by contrast, the levels of these downstream metabolites do increase. There are several possible explanations for this difference. One is that changes in PPi levels resulting from expression of a soluble PPase affect glycolytic flux. It is known that PPi concentrations allosterically regulate enzymes in the malate shunt [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and it is possible that PPi concentrations regulate other enzymes in central metabolism as well. Another possible explanation is that we previously measured intracellular metabolite concentrations in bottle fermentations where pH was only controlled by MOPS buffer, whereas in this work, the pH was actively controlled. Changes in pH may have limited the effects of the ATP-PFK reaction in those experiments. Finally, a third possible explanation is that the previously observed localization of the effect of ATP-PFK was a temporal effect, since our prior work only measured intracellular metabolites at a single time point in mid-log phase. Among these three possibilities, we have no evidence to distinguish between the first two. We can, however, tentatively reject the third one. In the PPi-free glycolysis strain (LL1711), the increased abundance of downstream metabolites (DHAP, 3PG, and PEP) is observed in both mid-log and early stationary phases (approximately 22–78 hours). Comparing any of the points in this range with our prior results shows a clear difference with the much lower levels of these metabolites observed in mid-log phase with our previously-developed ATP-PFK strain (LL1660) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our PPi-free glycolysis strain (strain LL1711), whole genome sequencing revealed a frameshift mutation (E784) in \u003cem\u003eC. thermocellum adhE\u003c/em\u003e that eliminated its function. We have routinely observed mutations in \u003cem\u003eadhE\u003c/em\u003e in strains that either have higher ethanol production or tolerance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies have shown that inactivating mutations in \u003cem\u003eadhE\u003c/em\u003e that eliminated NADH-linked alcohol dehydrogenase (ADH) activity increase ethanol tolerance in \u003cem\u003eC. thermocellum\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We believe that this is an adaptive response by the strain to avoid a thermodynamic limitation that can result from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ADH reactions using the same cofactor (i.e., NADH) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough the PPi-free glycolysis strain produced 455 mM (21.0 g/L) ethanol, it is likely that titers of 40–50 g/L are needed for commercial application [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nonetheless this work represents another step along the path toward converting the atypical glycolysis of \u003cem\u003eC. thermocellum\u003c/em\u003e to the canonical glycolysis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our previous work, we replaced the PPi-linked PFK reaction with an ATP-linked PFK reaction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this work, we needed to recreate this modification due to the presence of a spontaneous partial genome duplication event in our prior strain, and additionally introduce a deletion of the \u003cem\u003eppdk\u003c/em\u003e gene and heterologous expression of a soluble \u003cem\u003ePPase\u003c/em\u003e gene. The expression of a soluble PPase appears to be the enabling factor for engineering PPi-free glycolysis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Subsequent steps along this path may include eliminating the malate shunt [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and understanding the role of GTP vs. ATP in the glucokinase and phosphoglycerate kinase reactions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn the field of Metabolic Engineering, strategies for improving product yield are relatively well developed compared to strategies for improving product titer. Engineering the thermodynamic landscape of a metabolic pathway provides an interesting approach to address product titer limitations. Although there is extensive prior literature on analysis of the thermodynamic landscape of metabolic pathways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], there have been far fewer attempts to apply this knowledge for targeted improvement of these pathways. Several prior studies have hypothesized that the low thermodynamic driving force of the atypical glycolysis of \u003cem\u003eC. thermocellum\u003c/em\u003e, and/or a limited PPi supply for the PPi-consuming reactions might limit product titer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Here we show that the improved thermodynamic driving force of our PPi-free glycolysis allowed a 38% increase in ethanol titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), providing evidence of the utility of this approach.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and supplementary materials.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLee R. Lynd is the co-founder and CTO of Terragia Biofuels, Inc (https://terragiabiofuel.com/). Shuen Hon is an employee of Terragia Biofuels. Terragia has a financial interest in commercialization of \u003cem\u003eClostridium thermocellum\u003c/em\u003e. There are no other competing interests.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBDS: \u0026nbsp;Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Validation; Visualization; Writing - original draft. SH: Conceptualization; Investigation; Strain development; Formal analysis; Visualization; Writing - Review and Editing. ET: Data curation; Investigation; Writing - Review and Editing. DMS: Data curation; Investigation. DAN: Writing - Review and Editing. AMG: Conceptualization, Writing - Review and Editing. LL: Funding Acquisition, Project Administration, Writing - Review and Editing. DGO: Conceptualization, Writing - Review and Editing, Supervision, Project Administration, Funding Acquisition.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLynd LR, Beckham GT, Guss AM, et al (2022) Toward low-cost biological and hybrid biological/catalytic conversion of cellulosic biomass to fuels. Energy Environ Sci 15:938\u0026ndash;990\u003c/li\u003e\n\u003cli\u003eLynd LR, Guss AM, Himmel ME, et al (2017) Advances in Consolidated Bioprocessing Using \u003cem\u003eClostridium thermocellum\u003c/em\u003e and \u003cem\u003eThermoanaerobacter saccharolyticum\u003c/em\u003e. In: Wittmann C, Liao JC (eds) Ind. Biotechnol., 1st ed. Wiley, pp 365\u0026ndash;394\u003c/li\u003e\n\u003cli\u003eMazzoli R, Olson DG (2020) Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose. In: Adv. Appl. Microbiol. Elsevier, pp 111\u0026ndash;161\u003c/li\u003e\n\u003cli\u003eHolwerda EK, Worthen RS, Kothari N, et al (2019) Multiple levers for overcoming the recalcitrance of lignocellulosic biomass. Biotechnol Biofuels 12:15\u003c/li\u003e\n\u003cli\u003eLynd LR, Liang X, Biddy MJ, Allee A, Cai H, Foust T, Himmel ME, Laser MS, Wang M, Wyman CE (2017) Cellulosic ethanol: status and innovation. Curr Opin Biotechnol 45:202\u0026ndash;211\u003c/li\u003e\n\u003cli\u003eHolwerda EK, Ellis LD, Lynd LR (2013) Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms. Biotechnol Bioeng 110:2380\u0026ndash;2388\u003c/li\u003e\n\u003cli\u003eHon S, Holwerda EK, Worthen RS, Maloney MI, Tian L, Cui J, Lin PP, Lynd LR, Olson DG (2018) Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production. Biotechnol Biofuels. https://doi.org/10.1186/s13068-018-1245-2\u003c/li\u003e\n\u003cli\u003eTian L, Papanek B, Olson DG, et al (2016) Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum. Biotechnol Biofuels 9:1\u0026ndash;11\u003c/li\u003e\n\u003cli\u003eHolwerda EK, Olson DG, Ruppertsberger NM, Stevenson DM, Murphy SJL, Maloney MI, Lanahan AA, Amador-Noguez D, Lynd LR (2020) Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. Biotechnol Biofuels 13:40\u003c/li\u003e\n\u003cli\u003eDien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258\u0026ndash;266\u003c/li\u003e\n\u003cli\u003eZhou J, Olson DG, Argyros DA, Deng Y, van Gulik WM, van Dijken JP, Lynd LR (2013) Atypical Glycolysis in Clostridium thermocellum. Appl Environ Microbiol 79:3000\u0026ndash;3008\u003c/li\u003e\n\u003cli\u003eJacobson TB, Korosh TK, Stevenson DM, Foster C, Maranas C, Olson DG, Lynd LR, Amador-Noguez D (2020) \u003cem\u003eIn Vivo\u003c/em\u003e Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using \u003csup\u003e13\u003c/sup\u003e C and \u003csup\u003e2\u003c/sup\u003e H Tracers. mSystems 5:e00736-19\u003c/li\u003e\n\u003cli\u003ePark JO, Rubin SA, Xu Y-F, Amador-Noguez D, Fan J, Shlomi T, Rabinowitz JD (2016) Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat Chem Biol 12:482\u0026ndash;489\u003c/li\u003e\n\u003cli\u003eNoor E, Bar-Even A, Flamholz A, Reznik E, Liebermeister W, Milo R (2014) Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism. PLoS Comput Biol 10:e1003483\u003c/li\u003e\n\u003cli\u003eOlson DG, H\u0026ouml;rl M, Fuhrer T, Cui J, Zhou J, Maloney MI, Amador-Noguez D, Tian L, Sauer U, Lynd LR (2017) Glycolysis without pyruvate kinase in Clostridium thermocellum. Metab Eng 39:169\u0026ndash;180\u003c/li\u003e\n\u003cli\u003eDash S, Olson DG, Hung S, Chan J, Amador-noguez D, Lynd LR, Maranas CD (2019) Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum. Metab Eng 55:161\u0026ndash;169\u003c/li\u003e\n\u003cli\u003eHon S, Jacobson T, Stevenson DM, Maloney MI, Giannone RJ, Hettich RL, Amador-Noguez D, Olson DG, Lynd LR (2022) Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in \u003cem\u003eClostridium thermocellum\u003c/em\u003e. Appl Environ Microbiol 88:e01258-22\u003c/li\u003e\n\u003cli\u003eOlson DG, Maloney MI, Lanahan AA, et al (2023) Ethanol tolerance in engineered strains of Clostridium thermocellum. Biotechnol Biofuels Bioprod 16:137\u003c/li\u003e\n\u003cli\u003eGibson DG (2011) Enzymatic Assembly of Overlapping DNA Fragments. In: Methods Enzymol. Elsevier, pp 349\u0026ndash;361\u003c/li\u003e\n\u003cli\u003eGuss AM, Olson DG, Caiazza NC, Lynd LR (2012) Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum. Biotechnol Biofuels 5:30\u003c/li\u003e\n\u003cli\u003eOlson DG, Lynd LR (2012) Transformation of clostridium thermocellum by electroporation. Methods Enzymol. https://doi.org/10.1016/B978-0-12-415931-0.00017-3\u003c/li\u003e\n\u003cli\u003eHon S, Lanahan AA, Tian L, Giannone RJ, Hettich RL, Olson DG, Lynd LR (2016) Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs). Metab Eng Commun 3:120\u0026ndash;129\u003c/li\u003e\n\u003cli\u003eHon S, Olson DG, Holwerda EK, Lanahan AA, Murphy SJL, Maloney MI, Zheng T, Papanek B, Guss AM, Lynd LR (2017) The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum. Metab Eng 42:175\u0026ndash;184\u003c/li\u003e\n\u003cli\u003eYayo J, Rydzak T, Kuil T, Karlsson A, Harding DJ, Guss AM, Van Maris AJA (2023) The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum. Appl Environ Microbiol 89:e01753-22\u003c/li\u003e\n\u003cli\u003eKuil T, Hon S, Yayo J, Foster C, Ravagnan G, Maranas CD, Lynd LR, Olson DG, van Maris AJA (2022) Functional Analysis of H \u003csup\u003e+\u003c/sup\u003e -Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum. Appl Environ Microbiol 88:e01857-21\u003c/li\u003e\n\u003cli\u003eLanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95\u0026ndash;97\u003c/li\u003e\n\u003cli\u003eLo J, Zheng T, Hon S, Olson DG, Lynd LR (2015) The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, \u003cem\u003eadhE\u003c/em\u003e , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. J Bacteriol 197:1386\u0026ndash;1393\u003c/li\u003e\n\u003cli\u003eBennett BD, Yuan J, Kimball EH, Rabinowitz JD (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3:1299\u0026ndash;1311\u003c/li\u003e\n\u003cli\u003eCallaghan MM, Thusoo E, Sharma BD, Getahun F, Stevenson DM, Maranas C, Olson DG, Lynd LR, Amador-Noguez D (2023) Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism. Metab Eng 80:254\u0026ndash;266\u003c/li\u003e\n\u003cli\u003eSharma BD, Olson DG, Giannone RJ, Hettich RL, Lynd LR (2023) Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations. Appl Environ Microbiol 89:e00406-23\u003c/li\u003e\n\u003cli\u003eAgrawal S, Kumar S, Sehgal R, George S, Gupta R, Poddar S, Jha A, Pathak S (2019) El-MAVEN: A Fast, Robust, and User-Friendly Mass Spectrometry Data Processing Engine for Metabolomics. In: D\u0026rsquo;Alessandro A (ed) High-Throughput Metabolomics. Springer New York, New York, NY, pp 301\u0026ndash;321\u003c/li\u003e\n\u003cli\u003eHolwerda EK, Thorne PG, Olson DG, Amador-Noguez D, Engle NL, Tschaplinski TJ, Van Dijken JP, Lynd LR (2014) The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading. Biotechnol Biofuels 7:155\u003c/li\u003e\n\u003cli\u003eClasquin MF, Melamud E, Rabinowitz JD (2012) LC‐MS Data Processing with MAVEN: A Metabolomic Analysis and Visualization Engine. Curr Protoc Bioinforma. https://doi.org/10.1002/0471250953.bi1411s37\u003c/li\u003e\n\u003cli\u003eDeng Y, Olson DG, Zhou J, Herring CD, Joe Shaw A, Lynd LR (2013) Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum. Metab Eng. https://doi.org/10.1016/j.ymben.2012.11.006\u003c/li\u003e\n\u003cli\u003eYayo J, Kuil T, Olson DG, Lynd LR, Holwerda EK, van Maris AJA (2021) Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose. Appl Environ Microbiol 87:1\u0026ndash;18\u003c/li\u003e\n\u003cli\u003eBennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5:593\u0026ndash;599\u003c/li\u003e\n\u003cli\u003eR\u0026oslash;st LM, Brekke Thorfinnsdottir L, Kumar K, Fuchino K, Eide Lang\u0026oslash;rgen I, Bartosova Z, Kristiansen KA, Bruheim P (2020) Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems. Metabolites 10:74\u003c/li\u003e\n\u003cli\u003eTaillefer M, Rydzak T, Levin DB, Oresnik IJ, Sparling R (2015) Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase. Appl Environ Microbiol 81:2423\u0026ndash;2432\u003c/li\u003e\n\u003cli\u003eBiswas R, Prabhu S, Lynd LR, Guss AM (2014) Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum. PLoS ONE 9:e86389\u003c/li\u003e\n\u003cli\u003ePech-Canul A, Hammer SK, Ziegler SJ, Richardson ID, Sharma BD, Maloney MI, Bomble YJ, Lynd LR, Olson DG (2024) The Role of AdhE on Ethanol Tolerance and Production in Clostridium thermocellum. J Biol Chem 107559\u003c/li\u003e\n\u003cli\u003eTian L, Cervenka ND, Low AM, Olson DG, Lynd LR (2019) A mutation in the AdhE alcohol dehydrogenase of Clostridium thermocellum increases tolerance to several primary alcohols, including isobutanol, n-butanol and ethanol. Sci Rep 9:1736\u003c/li\u003e\n\u003cli\u003eKhana DB, Callaghan MM, Amador-Noguez D (2022) Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks. Curr Opin Microbiol 66:21\u0026ndash;31\u003c/li\u003e\n\u003cli\u003eSchroeder WL, Kuil T, Van Maris AJA, Olson DG, Lynd LR, Maranas CD (2023) A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis. Metab Eng 77:306\u0026ndash;322\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acetivibrio thermocellus, biofuels, Clostridium thermocellum, ethanol production, metabolic bottleneck, metabolic engineering, phosphofructokinase, thermodynamics","lastPublishedDoi":"10.21203/rs.3.rs-5027329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5027329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClostridium thermocellum\u003c/em\u003e is a promising candidate for production of cellulosic biofuels, however its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild type organism, this reaction uses pyrophosphate (PPi) as an energy cofactor, which is thermodynamically less favorable compared reactions that use ATP as a cofactor. Previously we showed that replacing the PPi-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we substituted PPi-\u003cem\u003epfk\u003c/em\u003e with ATP-\u003cem\u003epfk\u003c/em\u003e, deleted the other PPi-requiring glycolytic gene \u003cem\u003epyruvate:phosphate dikinase\u003c/em\u003e (\u003cem\u003eppdk\u003c/em\u003e), and expressed a soluble \u003cem\u003epyrophosphatase \u003c/em\u003e(\u003cem\u003ePPase\u003c/em\u003e)\u003cem\u003e \u003c/em\u003eand \u003cem\u003epyruvate kinase\u003c/em\u003e (\u003cem\u003epyk\u003c/em\u003e) genes to engineer PPi-free glycolysis in \u003cem\u003eC. thermocellum\u003c/em\u003e. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 g/L to 21.0 g/L) by using PPi-free glycolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy engineering PPi-free glycolysis in \u003cem\u003eC. thermocellum\u003c/em\u003e, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in \u003cem\u003eC. thermocellum\u003c/em\u003e to increase ethanol titers.\u003c/p\u003e","manuscriptTitle":"Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 15:04:49","doi":"10.21203/rs.3.rs-5027329/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-21T06:45:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-19T13:53:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-14T02:31:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252984161585233228526579346828287195695","date":"2024-10-05T02:02:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38333860670811116425437377276718343759","date":"2024-10-01T09:25:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147986105610005080934464264636602049110","date":"2024-09-19T04:38:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-16T09:50:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-04T06:51:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-04T06:47:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2024-09-03T21:06:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"614622bf-69ef-42f8-a077-66d77345d5da","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:03:56+00:00","versionOfRecord":{"articleIdentity":"rs-5027329","link":"https://doi.org/10.1186/s13068-024-02591-5","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2024-12-18 15:57:55","publishedOnDateReadable":"December 18th, 2024"},"versionCreatedAt":"2024-10-04 15:04:49","video":"","vorDoi":"10.1186/s13068-024-02591-5","vorDoiUrl":"https://doi.org/10.1186/s13068-024-02591-5","workflowStages":[]},"version":"v1","identity":"rs-5027329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5027329","identity":"rs-5027329","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}