Biology subjects

Olson, D. G.

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

8 recordsLinked to original sources

bioRxiv2026

Understanding Substrate Transport and Metabolism in Clostridium thermocellum to Improve Biofuel Production

As a cellulolytic specialist microbe, Clostridium thermocellum primarily ferments cellulose and its breakdown products. However, the full breadth of its substrate range remains poorly understood. In this work, we investigated the ability of C. thermocellum to transport and metabolize a diverse range of alternative carbon substrates. We performed tetrazolium dye assays to assess the ability of wild-type (WT) C. thermocellum (strain LL1004) to transport and metabolize 190 different substrates and identified 18 positive hits and 27 false positives. These findings guided the selection of 22 substrates for batch fermentations of WT on 10 g/L alternative substrate plus 1 g/L cellobiose. We identified eight substrates that were either fully or partially consumed (cellobiose, glucose, fructose, sorbitol, arbutin, turanose, pyruvate, glucose-6-phosphate), two that were interconverted (malate, fumarate), seven that may undergo conversion to unidentified fermentation products (xylose, sorbose, tagatose, galactose, mannose, rhamnose, fucose), and five that were not consumed (glycerol, acetate, lactate, propionate, xylitol). Sorbitol and pyruvate, which are more and less reduced than cellobiose, respectively, were selected for further analysis of their effects on redox balance and product formation. Batch fermentations on sorbitol and pyruvate demonstrated that ethanol production in C. thermocellum may be limited in part by the availability of reducing equivalents. Collectively, this work demonstrates the utility of alternative carbon substrates as a tool for perturbing metabolism, redox balance, and fermentation product distributions to further our understanding of the metabolism of anaerobic, thermophilic bacteria such as C. thermocellum for the production of biofuels and biochemicals of interest.

microbiology↗

bioRxiv2025

Characterization of new thermophilic antibiotic resistance markers

The genetic engineering of thermophilic bacteria is constrained by limited availability of thermostable antibiotic resistance markers for selection. Clostridium thermocellum, a promising candidate for consolidated bioprocessing of lignocellulosic biomass, requires reliable selection systems that function at elevated temperatures. Here, we systematically evaluated antibiotic susceptibility profiles and identified novel resistance markers for this thermophile through bioinformatic screening and experimental validation. We screened 823 thermophilic genomes against the Comprehensive Antibiotic Resistance Database, identifying 1,115 antibiotic resistance genes. From these, we selected candidates with highest homology to resistance determinants for rifampicin, tetracycline, erythromycin, thiamphenicol, and neomycin. We identified three novel antibiotic resistance systems that function in this organism: tetracycline/tet(45), erythromycin/cmeC, and rifampicin/rbpA. Of these, the rifampicin/rbpA provided the highest selection range , > 10,000-fold. Our results establish rbpA as an outstanding selectable marker for thermophilic genetic engineering and provide a validated workflow for discovering thermostable resistance determinants in high-temperature microorganisms. ImportanceThermophilic bacteria like Clostridium thermocellum hold tremendous potential for sustainable biofuel production from plant biomass, but their genetic manipulation has been severely limited by the lack of selection markers that work at high temperatures. Many existing antibiotic resistance systems do not function at thermophilic temperatures, and many approaches to genetic manipulation require multiple antibiotic resistance markers. Currently only two markers are available for C. thermocellum, and only one (cat) functions well. The newly-developed rbpA marker functions well in C. thermocellum and is likely to provide dramatic new opportunities for engineering thermophilic host organisms.

microbiology↗

bioRxiv2025

Engineering Clostridium thermocellum for production of 2,3-butanediol from cellulose

Clostridium thermocellum is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering C. thermocellum for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing C. thermocellum into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.

synthetic biology↗

bioRxiv2025

Metabolic imbalance limits fermentation in microbes engineered for high-titer ethanol production

Microbial strains engineered for high-titer ethanol production achieve lower maximum titers compared to native producers such as Zymomonas mobilis. A central unresolved question is why fermentation ceases before substrate has been exhausted by these strains. Here, we integrate metabolite profiling with thermodynamic analysis to examine this phenomenon in engineered strains of Escherichia coli and Thermoanaerobacterium saccharolyticum and compare them to Z. mobilis, a native ethanol producer. In the engineered strains, fermentation cessation coincided with marked pyruvate accumulation, due to a lack of ability to convert pyruvate to ethanol. This resulted in a local thermodynamic equilibrium at the pyruvate kinase reaction, as determined by Max-Min Driving Force (MDF) analysis. Relaxing constraints on pyruvate and related metabolites restored positive MDF values, implicating thermodynamic limitations as the underlying constraint. By contrast, Z. mobilis maintained a positive thermodynamic driving force throughout fermentation, suggesting that product titer is limited by a different mechanism in this organism. These findings establish a systems-level framework linking metabolite concentrations to pathway thermodynamics and highlight opportunities for improving microbial performance in ethanol and other bioproduction contexts.

systems biology↗

bioRxiv2025

Parameterization of cell-free systems with time-series data using KETCHUP

Kinetic models mechanistically link enzyme levels, metabolite concentrations, and allosteric regulation to metabolic reaction fluxes. This coupling allows for the quantitative elucidation of the dynamics of the evolution of metabolite concentrations and metabolic fluxes as a function of time. So far, most large-scale kinetic model parameterizations are carried out using mostly steady-state flux measurements supplemented with metabolomics and/or proteomics data when available. Even though the parameterized kinetic model can trace a temporal evolution of the system, lack of anchoring to temporal data reduces confidence in the dynamics predictions. Notably, the simulation of enzymatic cascade reactions requires the full description of the dynamics of the system as a steady-state is not applicable given that all measured metabolite concentrations vary with time. Here we describe how kinetic parameters fitted to the dynamics of single-enzyme assays remain accurate for the simulation of multi-enzyme cell-free systems. Herein, we demonstrate two extensions for the Kinetic Estimation Tool Capturing Heterogeneous datasets Using Pyomo (KETCHUP) software tool for parameterizing a kinetic model of the cell-free kinetics of formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase (BDH) through the use of time-course data across various initial conditions. An implemented extension of KETCHUP allowing for the reconciliation of measurement time-lag errors present in datasets was used to parameterize multiple datasets. By combining the kinetic parameters identified by the FDH and BDH assays, accurate simulation of the binary FDH-BDH system was achieved. KETCHUP can be accessed at https://github.com/maranasgroup/KETCHUP. Author SummaryMetabolic engineering of microorganisms offers a sustainable and renewable alternative for industrial scale production of commodity chemicals. Large-scale metabolic stoichiometric models enable means to elucidate an organisms physiological behavior towards both environment and genetic perturbations and predict optimal interventions to maximize production. Kinetic models extend stoichiometric models capabilities in characterizing metabolic phenotypes by mathematically linking enzyme catalyzed reactions in metabolic networks as functions of metabolite concentrations, enzyme levels, and allosteric regulations. Although these kinetic descriptions improve predictive accuracy and strain design, the lack of enzyme information, raw experimental data, and high computational cost preclude the construction and parameterization of detailed large-scale kinetic models. Here, we introduce an extension of KETCHUP, an open-source semi-automatic kinetic construction and parameterization tool that utilizes steady-state data as a pilot for the parameterization of single-enzyme cell-free time-course data. We show that this tool offers comparable performance to existing tools during parameterization of single enzymes and successful recapitulation of metabolite profiles for a two-enzyme system when simulating a kinetic model with parameters from the two single-enzymes. This study showcases a high throughput pipeline that can be integrated into our framework for larger scaled networks.

systems biology↗

bioRxiv2025

Rapid expression of pyruvate decarboxylase from Zymomonas mobilis in E. coli BL21 LysY/Iq

E. coli BL21 DE3 strain is commonly used to express and purify non-toxic prokaryotic proteins in high yields. Traditionally, IPTG based induction of the host strain at reduced temperatures for extended period (12-16 h) is performed to obtain high yield of functional proteins. It is desirable to explore methods which result in high yield of protein within a short period of time. We report rapid purification of pyruvate decarboxylase (PDC) enzyme from Zymomonas mobilis using E. coli BL21 pLysY/Iq as a host strain. High yield of purified PDC at 0.33 ({+/-}0.02) mg was obtained after two-hour induction by 0.6 mM IPTG at 37{degrees}C. The enzyme yield was comparable to 0.37 ({+/-}0.08) mg obtained in E. coli BL21 DE3 strain (used as control) after 16 h induction by 0.6 mM IPTG at 18{degrees}C. Similar values of the maximum specific activity of the enzyme expressed and purified at 37{degrees}C and 18{degrees}C were obtained at 78.31 ({+/-}1.13) in strain LysY/Iq and 85.73 ({+/-}4.39) {micro}mol/min/mg protein in strain DE3, respectively. In almost all IPTG treatments, the kinetic parameters of the purified enzyme - app Km, app Vmax, Kcat and Kcat/Km -also did not vary remarkably between the two temperature regimes. Based upon the data presented here, we propose that E. coli BL21 LysY/Iq strain has potential to serve as a host for efficient and rapid expression (2 h) of non-toxic proteins. Results of this study will aid in cell free system study which require rapid scale up of the complexity of metabolic pathways by utilizing multiple purified enzymes involved in bioconversion of the substrate of interest.

biochemistry↗

bioRxiv2024

Structural characterization and dynamics of AdhE ultrastructures from Clostridium thermocellum: A containment strategy for toxic intermediates?

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe, however ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 [A] structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

biophysics↗

bioRxiv2021

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 reintroduce 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 appears to function by interfering with metal ion coordination in the PHP domain responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. ImportanceCellulosic biofuels are a promising approach to decarbonize the heavy duty transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of the material to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production, however they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially-relevant organism, which can reduce the time needed for adaptive evolution experiments.

bioengineering↗