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Lynd, Lee R.

Publications and source records attributed to Lynd, Lee R..

At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
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

Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation and diagnostic tests

Abstract Background For economically viable 2nd generation biofuels, processing of high solid lignocellulosic substrate concentrations is a necessity. The cellulolytic thermophilic anaerobe Clostridium thermocellum is one of the most effective biocatalysts for solubilization of carbohydrate harbored in lignocellulose. This study aims to document the solubilization performance of Clostridium thermocellum at increasing solids concentrations for two lignocellulosic feedstocks, corn stover and switchgrass, and explore potential effectors of solubilization performance. Results Monocultures of Clostridium thermocellum demonstrated high levels of carbohydrate solubilization for both unpretreated corn stover and switchgrass. However, fractional carbohydrate solubilization decreases with increasing solid loadings. Fermentation of model insoluble substrate (cellulose) in the presence of high solids lignocellulosic spent broth is temporarily affected but not model soluble substrate (cellobiose) fermentations. Mid-fermentation addition of cells ( C. thermocellum ) or model substrates did not significantly enhance overall corn stover solubilization loaded at 80 g/L, however cultures utilized the model substrates in the presence of high concentrations of corn stover. An increase in corn stover solubilization was observed when water was added, effectively diluting the solids concentration mid-fermentation. Introduction of a hemicellulose-utilizing coculture partner, Thermoanaerobacterium thermosaccharolyticum, increased the fractional carbohydrate solubilization at both high and low solid loadings. Residual solubilized carbohydrates diminished significantly in the presence of T. thermosaccharolyticum compared to monocultures of C. thermocellum, yet a small fraction of solubilized oligosaccharides of both C 5 and C 6 sugars remained unutilized. Conclusion Diminishing fractional carbohydrate solubilization with increasing substrate loading was observed for C. thermocellum -mediated solubilization and fermentation of unpretreated lignocellulose feedstocks. Results of experiments involving spent broth addition do not support a major role for inhibitors present in the liquid phase. Mid-fermentation addition experiments confirm that C. thermocellum and its enzymes remain capable of converting model substrates during the middle of high solids lignocellulose fermentation. An increase in fractional carbohydrate solubilization was made possible by (1) mid-fermentation solid loading dilutions and (2) coculturing C. thermocellum with T. thermosaccharolyticum , which ferments solubilized hemicellulose. Incomplete utilization of solubilized carbohydrates suggests that a small fraction of the carbohydrates is unaffected by the extracellular carbohydrate-active enzymes present in the culture.

09 BIOMASS FUELS↗
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↗
DOE OSTI2021

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

The pyruvate kinase (PYK) isozyme from Thermoanaerobacterium saccharolyticum (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). Our use of sugar-phosphate analogs generated a surprising result in that IMP and GMP are allosteric inhibitors (rather than activators) of TsPYK. We believe this to be the first report of any PYK isozyme being inhibited by IMP and GMP. A truncated protein that lacks the extra C-domain is also inhibited by IMP. A screen of several other bacterial PYK enzymes (include several that have the extra-C domain) indicates that the inhibition by IMP is specific to only a subset of those isozymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners— Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum —exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

09 BIOMASS FUELS↗
DOE OSTI2020

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum . Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P i → ADP + PP i .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Development of a thermophilic coculture for corn fiber conversion to ethanol

Abstract The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp . strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum . Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

09 BIOMASS FUELS↗
DOE OSTI2020

In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 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 2 H and 13 C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli . The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellummthan in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae , the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

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

Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum 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. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.

Biotechnology & Applied Microbiology↗