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Olson, Daniel G.

Publications and source records attributed to Olson, Daniel G..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Effects_of_heterologous_expression_of_glycolysis_enzymes_on_product_formation_in_Clostridium_thermocellum

Glycolysis is central toClostridium thermocellummetabolism; however, engineered strains for high ethanol titer exhibit a decrease in yield, suggesting the presence of glycolytic bottlenecks. We expressed heterologoustpi,fba, andgapDHgenes fromThermoanaerobacterium saccharolyticumandZymomonas mobilis, along with 26 non-phosphorylatinggapNvariants, to identify limiting reactions. We demonstrated functional expression and increased activity for several Fba and Tpi enzymes in the engineered strain LL1711. DespiteC. thermocellum'slow native FBA activity compared to other industrial strains, increasing Fba or Tpi activity via heterologous expression had no significant effect on cellobiose uptake or ethanol titers in high-substrate fermentations. Furthermore, 25 of 26 tested gapN genes proved highly toxic toC. thermocellumupon transformation. In conclusion, none of the tested glycolytic enzyme modifications improved fermentation profiles. These results suggest that the primary metabolic limitation is not at the FBA or TPI reactions, supporting a shift in future engineering efforts toward downstream fermentation pathways.

6-bisphosphate aldolase; glyceraldehyde-3-phosphat↗
DOE OSTI2026

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗
DOE OSTI2024

Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call “cell-free systems biology”, where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a threeenzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO 2 from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

09 BIOMASS FUELS↗
DOE OSTI2023

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13 C and 2 H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2 H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13 C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water ( 2 H 2 O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2 H 2 O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2 H 2 O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13 C and 2 H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2 H 2 O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2 H-labeled nutrient substrates.

09 BIOMASS FUELS↗
DOE OSTI2023

Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains

Abstract Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans—furfural and 5-hydroxymethyl furfural—and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.

5-hydroxymethyl furfural↗
DOE OSTI2023

Ethanol tolerance in engineered strains of Clostridium thermocellum

Abstract Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum . We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE . Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

09 BIOMASS FUELS↗
DOE OSTI2023

A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PP i ) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PP i sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PP i synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PP i -generating cycles. In all cases, the metabolic cost of PP i synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PP i could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PP i sources likely account for most PP i production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PP i levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations

Clostridium thermocellum , a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum , we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum . Here, we addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PP i ) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PP i -PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, in this study, we replace the PP i -PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by 13 C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PP i -PFK reaction are not the sole factor limiting product titer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional Analysis of H + -Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum

Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H + -pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PP i supply, this study adds functional annotation of four genes and availability of an updated PP i stoichiometry from biosynthesis to the scientific domain.

Clostridium thermocellum↗
DOE OSTI2022

A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. Here, the ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13 C-metabolic flux analysis ( 13 C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13 C labeling data were used with 13 C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50–85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗