Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers.

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 (PP) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor.

Cell-Free Systems Biology: Characterizing Central Metabolism of 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.

Expression and characterization of monofunctional alcohol dehydrogenase enzymes in .

is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase ( genes.

The role of AdhE on ethanol tolerance and production in Clostridium thermocellum.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations.

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. C and H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to C-tracers.

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

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.

Ethanol tolerance in engineered strains of Clostridium thermocellum.

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.

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) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PP sources are key to engineering its metabolism.

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.

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in .

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) instead of ATP as the phosphate donor for the PFK reaction.

A Single Nucleotide Change in the 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 with a C669Y mutation in the 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.

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.

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

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PP) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PP was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PP.

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.

Inhibition of Pyruvate Kinase From by IMP Is Independent of the Extra-C Domain.

The pyruvate kinase (PYK) isozyme from (TsPYK) has previously been used in metabolic engineering for improved ethanol production. This isozyme belongs to a subclass of PYK isozymes that include an extra C-domain. Like other isozymes that include this extra C-domain, we found that TsPYK is activated by AMP and ribose-5-phosphate (R5P).

Laboratory Evolution and Reverse Engineering of for Growth on Glucose and Fructose.

The native ability of to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of , as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations.

Methods for Metabolic Engineering of Thermoanaerobacterium saccharolyticum.

In this work, we describe genetic tools and techniques for engineering Thermoanaerobacterium saccharolyticum. In particular, the T. saccharolyticum transformation protocol and the methods for selecting for transformants are described.

Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using C and H Tracers.

and are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with H and C metabolic flux analysis to investigate the reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of or anaerobically grown The limited thermodynamic driving force of glycolysis in could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate.

Metabolic and evolutionary responses of to genetic interventions aimed at improving ethanol production.

Background: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined.

Conversion of phosphoenolpyruvate to pyruvate in .

is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis.

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium .

The robust lignocellulose-solubilizing activity of makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer , we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C precursors (such as ribose) during growth on other (non-C) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP-PFK) instead of transaldolase.

Metabolic engineering of for -butanol production from cellulose.

Background: Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose.

Results: We tested 12 different enzyme combinations to identify an -butanol pathway with high titer and thermostability in .

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

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C.