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Article ; Online: Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield.

Herring, Christopher D / Ajie, Maulana Permana / Lynd, Lee R

2023 Volume 13, Issue 1, Page(s) 2394

Abstract: Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species ... ...

Abstract	Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.
MeSH term(s)	Propylene Glycol/metabolism ; Ethanol/metabolism ; Propylene Glycols ; Adenosine Triphosphate/metabolism ; Fermentation
Chemical Substances	Propylene Glycol (6DC9Q167V3) ; Ethanol (3K9958V90M) ; 1,3-propanediol (5965N8W85T) ; Propylene Glycols ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2023-02-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-29220-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: The grand challenge of cellulosic biofuels.

Lynd, Lee R

Nature biotechnology

2017 Volume 35, Issue 10, Page(s) 912–915

Language	English
Publishing date	2017-10-10
Publishing country	United States
Document type	Journal Article
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/nbt.3976
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Article ; Online: Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation and diagnostic tests.

Kubis, Matthew R / Holwerda, Evert K / Lynd, Lee R

Biotechnology for biofuels and bioproducts

2022 Volume 15, Issue 1, Page(s) 12

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 ... ...

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 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.
Language	English
Publishing date	2022-02-05
Publishing country	England
Document type	Journal Article
ISSN	2731-3654
ISSN (online)	2731-3654
DOI	10.1186/s13068-022-02110-4
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Article ; Online: Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations.

Sharma, Bishal D / Olson, Daniel G / Giannone, Richard J / Hettich, Robert L / Lynd, Lee R

Applied and environmental microbiology

2023 Volume 89, Issue 4, Page(s) e0040623

Abstract: 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 (> ... ...

Abstract	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. 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.
MeSH term(s)	Sugars/metabolism ; Clostridium thermocellum/metabolism ; Metabolic Engineering ; Ethanol/metabolism
Chemical Substances	Sugars ; Ethanol (3K9958V90M)
Language	English
Publishing date	2023-04-11
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/aem.00406-23
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Article ; Online: Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield

Christopher D. Herring / Maulana Permana Ajie / Lee R. Lynd

Scientific Reports, Vol 13, Iss 1, Pp 1-

2023 Volume 10

Abstract: Abstract Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting ... ...

Abstract	Abstract Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2023-02-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis.

Schroeder, Wheaton L / Kuil, Teun / van Maris, Antonius J A / Olson, Daniel G / Lynd, Lee R / Maranas, Costas D

Metabolic engineering

2023 Volume 77, Page(s) 306–322

Abstract: 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 ... ...

Abstract	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
MeSH term(s)	Clostridium thermocellum/genetics ; Clostridium thermocellum/metabolism ; Diphosphates/metabolism ; Glycolysis/genetics ; Fermentation ; Adenosine Triphosphate/metabolism
Chemical Substances	diphosphoric acid (4E862E7GRQ) ; Diphosphates ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2023-04-20
Publishing country	Belgium
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2023.04.003
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Article ; Online: Conversion of phosphoenolpyruvate to pyruvate in

Cui, Jingxuan / Maloney, Marybeth I / Olson, Daniel G / Lynd, Lee R

Metabolic engineering communications

2020 Volume 10, Page(s) e00122

Abstract: Thermoanaerobacterium ... ...

Abstract	Thermoanaerobacterium saccharolyticum
Language	English
Publishing date	2020-01-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2821894-2
ISSN	2214-0301 ; 2214-0301
ISSN (online)	2214-0301
ISSN	2214-0301
DOI	10.1016/j.mec.2020.e00122
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Article ; Online: A Single Nucleotide Change in the

Lanahan, Anthony / Zakowicz, Kamila / Tian, Liang / Olson, Daniel G / Lynd, Lee R

Applied and environmental microbiology

2021 Volume 88, Issue 1, Page(s) e0153121

Abstract: 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 ... ...

Abstract	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
MeSH term(s)	Base Composition ; Clostridium thermocellum/genetics ; DNA Polymerase III ; Nucleotides ; Phenotype ; Phylogeny ; RNA, Ribosomal, 16S ; Sequence Analysis, DNA
Chemical Substances	Nucleotides ; RNA, Ribosomal, 16S ; DNA Polymerase III (EC 2.7.7.7)
Language	English
Publishing date	2021-10-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.01531-21
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Article ; Online: In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum.

Mazzoli, Roberto / Olson, Daniel G / Concu, Angela Maria / Holwerda, Evert K / Lynd, Lee R

New biotechnology

2021 Volume 67, Page(s) 12–22

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Clostridium thermocellum/genetics ; Clostridium thermocellum/metabolism ; Ethanol/metabolism ; Fermentation ; Lactic Acid ; Metabolic Engineering
Chemical Substances	Lactic Acid (33X04XA5AT) ; Ethanol (3K9958V90M)
Language	English
Publishing date	2021-12-13
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2400836-9
ISSN	1876-4347 ; 1876-4347
ISSN (online)	1876-4347
ISSN	1876-4347
DOI	10.1016/j.nbt.2021.12.003
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Cross-national analysis of food security drivers: comparing results based on the Food Insecurity Experience Scale and Global Food Security Index

Allee, Andrew / Lynd, Lee R. / Vaze, Vikrant

Food security. 2021 Oct., v. 13, no. 5

2021

Abstract: ... of variation (based on out-of-sample R²) in GFSI and FI<ₘₒd, respectively. The relative strength of HFCE ...

Abstract	The second UN Sustainable Development Goal establishes food security as a priority for governments, multilateral organizations, and NGOs. These institutions track national-level food security performance with an array of metrics and weigh intervention options considering the leverage of many possible drivers. We studied the relationships between several candidate drivers and two response variables based on prominent measures of national food security: the 2019 Global Food Security Index (GFSI) and the Food Insecurity Experience Scale’s (FIES) estimate of the percentage of a nation’s population experiencing food security or mild food insecurity (FI<ₘₒd). We compared the contributions of explanatory variables in regressions predicting both response variables, and we further tested the stability of our results to changes in explanatory variable selection and in the countries included in regression model training and testing. At the cross-national level, the quantity and quality of a nation’s agricultural land were not predictive of either food security metric. We found mixed evidence that per-capita cereal production, per-hectare cereal yield, an aggregate governance metric, logistics performance, and extent of paid employment work were predictive of national food security. Household spending as measured by per-capita final consumption expenditure (HFCE) was consistently the strongest driver among those studied, alone explaining a median of 92% and 70% of variation (based on out-of-sample R²) in GFSI and FI<ₘₒd, respectively. The relative strength of HFCE as a predictor was observed for both response variables and was independent of the countries used for model training, the transformations applied to the explanatory variables prior to model training, and the variable selection technique used to specify multivariate regressions. The results of this cross-national analysis reinforce previous research supportive of a causal mechanism where, in the absence of exceptional local factors, an increase in income drives increase in food security. However, the strength of this effect varies depending on the countries included in regression model fitting. We demonstrate that using multiple response metrics, repeated random sampling of input data, and iterative variable selection facilitates a convergence of evidence approach to analyzing food security drivers.
Keywords	agricultural land ; employment ; food security ; governance ; income ; regression analysis ; sustainable development
Language	English
Dates of publication	2021-10
Size	p. 1245-1261.
Publishing place	Springer Netherlands
Document type	Article
ZDB-ID	2486755-X
ISSN	1876-4525 ; 1876-4517
ISSN (online)	1876-4525
ISSN	1876-4517
DOI	10.1007/s12571-021-01156-w
Database	NAL-Catalogue (AGRICOLA)

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