42 results on '"Gregg T. Beckham"'
Search Results
2. Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440
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Kevin J. McNaught, Eugene Kuatsjah, Michael Zahn, Érica T. Prates, Huiling Shao, Gayle J. Bentley, Andrew R. Pickford, Josephine N. Gruber, Kelley V. Hestmark, Daniel A. Jacobson, Brenton C. Poirier, Chen Ling, Myrsini San Marchi, William E. Michener, Carrie D. Nicora, Jacob N. Sanders, Caralyn J. Szostkiewicz, Dušan Veličković, Mowei Zhou, Nathalie Munoz, Young-Mo Kim, Jon K. Magnuson, Kristin E. Burnum-Johnson, K.N. Houk, John E. McGeehan, Christopher W. Johnson, and Gregg T. Beckham
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Bioengineering ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2023
3. Continuous hydrodeoxygenation of lignin to jet-range aromatic hydrocarbons
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Michael L. Stone, Matthew S. Webber, William P. Mounfield, David C. Bell, Earl Christensen, Ana R.C. Morais, Yanding Li, Eric M. Anderson, Joshua S. Heyne, Gregg T. Beckham, and Yuriy Román-Leshkov
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General Energy - Published
- 2022
4. The role of binding modules in enzymatic poly(ethylene terephthalate) hydrolysis at high-solids loadings
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Rosie Graham, Erika Erickson, Richard K. Brizendine, Davinia Salvachúa, William E. Michener, Yaohao Li, Zhongping Tan, Gregg T. Beckham, John E. McGeehan, and Andrew R. Pickford
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Chemistry (miscellaneous) ,Organic Chemistry ,Physical and Theoretical Chemistry - Published
- 2022
5. Multi-pass flow-through reductive catalytic fractionation
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Jun Hee Jang, David G. Brandner, Reagan J. Dreiling, Arik J. Ringsby, Jeremy R. Bussard, Lisa M. Stanley, Renee M. Happs, Anjaneya S. Kovvali, Joshua I. Cutler, Tom Renders, James R. Bielenberg, Yuriy Román-Leshkov, and Gregg T. Beckham
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General Energy - Published
- 2022
6. Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation
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Alissa Bleem, Eugene Kuatsjah, Gerald N. Presley, Daniel J. Hinchen, Michael Zahn, David C. Garcia, William E. Michener, Gerhard König, Konstantinos Tornesakis, Marco N. Allemann, Richard J. Giannone, John E. McGeehan, Gregg T. Beckham, and Joshua K. Michener
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Chemistry (miscellaneous) ,Organic Chemistry ,Physical and Theoretical Chemistry - Published
- 2022
7. Design principles for intrinsically circular polymers with tunable properties
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Gregg T. Beckham, Changxia Shi, V. Sai Phani Kumar, Liam T. Reilly, Scott R. Nicholson, Linda J. Broadbelt, Eugene Y.-X. Chen, and Matthew W. Coile
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chemistry.chemical_classification ,Deconstruction (building) ,chemistry ,Computer science ,General Chemical Engineering ,Biochemistry (medical) ,Materials Chemistry ,Environmental Chemistry ,Design elements and principles ,Nanotechnology ,General Chemistry ,Polymer ,Biochemistry - Abstract
Summary This perspective discusses a set of design principles for next-generation kinetically trapped, intrinsically circular polymers (iCPs) that are inherently, selectively, and expediently depolymerizable to their monomer state once their kinetic barriers of deconstruction are overcome, thereby enabling not only the ideal shortest chemical circularity but also tunable performance properties. After describing four elements of the design principles—thermodynamics and kinetics, strategies to overcome trade-offs and unify conflicting properties, predictive modeling, and supply-chain life-cycle assessment and techno-economic analysis, which are illustrated with state-of-the-art examples—it concludes with presenting key challenges and opportunities for sustainable development of iCPs.
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- 2021
8. Manufacturing energy and greenhouse gas emissions associated with plastics consumption
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Gregg T. Beckham, Alberta Carpenter, Scott R. Nicholson, and Nicholas A. Rorrer
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Consumption (economics) ,Waste management ,Circular economy ,Supply chain ,Commodity ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Energy requirement ,0104 chemical sciences ,General Energy ,Greenhouse gas ,Sustainability ,Environmental science ,Production (economics) ,0210 nano-technology - Abstract
Summary Reducing the carbon intensity of plastics production by sourcing sustainable feedstocks while simultaneously enabling effective polymer recycling represents a potential transformation of 21st century manufacturing. To evaluate technologies that could enable such changes, it is imperative to compare the sustainability of bio-based and/or circular plastic flows to those of incumbent manufacturing paradigms. To that end, we estimate the supply chain energy requirements and greenhouse gas (GHG) emissions associated with US-based plastics consumption. Major commodity polymers, each of which has a global consumption of at least 1 MMT per year, account for an estimated annual 3.2 quadrillion Btutus (quads) of energy and 104 MMTCO2e of GHG emissions in the US alone. This study serves as a foundation for comparing the supply chain energy requirements and GHG emissions of today’s plastics manufacturing to tomorrow’s disruptive technologies, to inform the development of bio-based plastics and the circular economy for synthetic polymers.
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- 2021
9. Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid
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Marykate H. O’Brien, Gara N. Dexter, Joshua R. Elmore, Darren J. Peterson, Joshua K. Michener, Kent Gorday, Gregg T. Beckham, Adam M. Guss, Davinia Salvachúa, and Dawn M. Klingeman
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Arabinose ,Coumaric Acids ,Lignocellulosic biomass ,Bioengineering ,Xylose ,Lignin ,Zea mays ,Applied Microbiology and Biotechnology ,Hydrolysate ,03 medical and health sciences ,chemistry.chemical_compound ,Hemicellulose ,Food science ,Cellulose ,Acetic Acid ,030304 developmental biology ,0303 health sciences ,biology ,Pseudomonas putida ,030306 microbiology ,biology.organism_classification ,Glucose ,Corn stover ,chemistry ,Fermentation ,Biotechnology - Abstract
Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h−1 and 0.38 h−1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.
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- 2020
10. Gene amplification, laboratory evolution, and biosensor screening reveal MucK as a terephthalic acid transporter in Acinetobacter baylyi ADP1
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Ramesh K. Jha, Molly Gaddis, Emily A. McIntyre, Ellen L. Neidle, Felicia Bratti, William E. Michener, Christopher W. Johnson, Ryan E. Bermel, Taraka Dale, Isabel Pardo, and Gregg T. Beckham
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Operon ,Phthalic Acids ,Repressor ,Heterologous ,Bioengineering ,Biosensing Techniques ,Applied Microbiology and Biotechnology ,Metabolic engineering ,03 medical and health sciences ,Rhodococcus ,Gene ,030304 developmental biology ,0303 health sciences ,Acinetobacter ,integumentary system ,biology ,030306 microbiology ,Chemistry ,Gene Amplification ,biology.organism_classification ,Major facilitator superfamily ,Biochemistry ,Heterologous expression ,Laboratories ,Bacteria ,Biotechnology - Abstract
Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.
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- 2020
11. Corrigendum to 'Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440' [Metab. Eng. 59 (2020) 64–75]
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Gayle J. Bentley, Niju Narayanan, Ramesh K. Jha, Davinia Salvachúa, Joshua R. Elmore, George L. Peabody, Brenna A. Black, Kelsey Ramirez, Annette De Capite, William E. Michener, Allison Z. Werner, Dawn M. Klingeman, Heidi S. Schindel, Robert Nelson, Lindsey Foust, Adam M. Guss, Taraka Dale, Christopher W. Johnson, and Gregg T. Beckham
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Bioengineering ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2022
12. Corrigendum to 'Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization' [Metab. Eng. 48 (2018) 197–207]
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Mary Ann Franden, Nicholas S. Cleveland, William E. Michener, Lars M. Blank, Neil J. Wagner, Bernhard Hauer, Janosch Klebensberger, Lahiru N. Jayakody, Gregg T. Beckham, Nick Wierckx, and Wing-Jin Li
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chemistry.chemical_compound ,chemistry ,biology ,Organic chemistry ,Bioengineering ,ddc:610 ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Ethylene glycol ,Pseudomonas putida ,Biotechnology - Abstract
The sequence of the promoter used to drive expression of glcDEF operon in the engineered strain MFL185 in the original article was incorrect. As described here, we performed additional experiments that indicate expression of this operon was increased in MFL185, as intended. Ultimately, this error is immaterial with respect to the findings and conclusions reported in the original article.
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- 2021
13. The hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function
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Gregg T. Beckham, Jerry Ståhlberg, Michael F. Crowley, Brandon C. Knott, and Vivek S. Bharadwaj
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0301 basic medicine ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Stereochemistry ,In silico ,Cell Biology ,Cellulase ,Biochemistry ,03 medical and health sciences ,Expansin ,Hydrolysis ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,chemistry ,Enzymology ,biology.protein ,Glycoside hydrolase ,Hydroxymethyl ,Molecular Biology ,Function (biology) - Abstract
Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.
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- 2020
14. Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate
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Jacob A. Fenster, Allison Z. Werner, Jian Wei Tay, Matthew Gillen, Leo Schirokauer, Nicholas C. Hill, Audrey Watson, Kelsey J. Ramirez, Christopher W. Johnson, Gregg T. Beckham, Jeffrey C. Cameron, and Carrie A. Eckert
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Endocrinology, Diabetes and Metabolism ,Biomedical Engineering - Published
- 2022
15. Molecular simulation of lignin-related aromatic compound permeation through gram-negative bacterial outer membranes
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Josh V. Vermaas, Michael F. Crowley, and Gregg T. Beckham
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Diffusion ,Bacterial Outer Membrane ,Biological Transport ,Cell Biology ,Molecular Dynamics Simulation ,Lignin ,Molecular Biology ,Biochemistry ,Bacterial Outer Membrane Proteins - Abstract
Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.
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- 2022
16. Quantification and evaluation of plastic waste in the United States
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Anelia Milbrandt, Kamyria Coney, Alex Badgett, and Gregg T. Beckham
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Economics and Econometrics ,Waste Management and Disposal - Published
- 2022
17. Enhanced Catalyst Durability for Bio-Based Adipic Acid Production by Atomic Layer Deposition
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Karthikeyan K. Ramasamy, Ryon W. Tracy, Amy E. Settle, Reuben Sarkar, Watson Michael John, Katherine E. Hurst, Ryan M. Richards, Michael B. Griffin, Derek R. Vardon, Arrelaine A. Dameron, Davis R. Conklin, Xiangchen Huo, Gregg T. Beckham, Arun Devaraj, Kinga A. Unocic, Eric C. D. Tan, Nicholas S. Cleveland, Allyson M. York, Carrie A. Farberow, Steven T. Christensen, and Elizabeth J. Kautz
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endocrine system ,Muconic acid ,Adipic acid ,Materials science ,endocrine system diseases ,Oxide ,02 engineering and technology ,engineering.material ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,Catalysis ,chemistry.chemical_compound ,Atomic layer deposition ,General Energy ,chemistry ,Chemical engineering ,Coating ,engineering ,Thermal stability ,Leaching (metallurgy) ,0210 nano-technology - Abstract
Summary Atomic layer deposition (ALD) improves the durability of metal catalysts using nanoscale metal oxide coatings. However, targeted coating strategies and economic models are lacking for process-specific deactivation challenges that account for implications at scale. Herein, we apply Al2O3 ALD to Pd/TiO2 to increase durability during hydrogenation of muconic acid, a bio-based platform chemical, to adipic acid. Initial coating development and characterization are performed on the milligram scale using stop-flow ALD. Subsequently, ALD coating scale is increased by 3 orders of magnitude using fluidized bed ALD. Activity, leaching resistance, and thermal stability are evaluated at each synthesis scale. ALD-coated catalysts retain up to 2-fold greater muconic acid hydrogenation activity and undergo significantly less physical restructuring than uncoated Pd/TiO2 after high-temperature treatments, while reducing Pd leaching by over 4-fold. Techno-economic analysis for an adipic acid biorefinery supports increased ALD material costs through catalyst lifetime extension, underscoring the potential viability of this technology.
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- 2019
18. Innovative Chemicals and Materials from Bacterial Aromatic Catabolic Pathways
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Peter C. St. John, Nicholas S. Cleveland, Graham Dominick, Priyanka Singh, William E. Michener, Davinia Salvachúa, Xiunan Yi, Brenna A. Black, Derek R. Vardon, Kelsey J. Ramirez, Chelsea R. Martinez, Adam M. Guss, A. Nolan Wilson, Gregg T. Beckham, Nicholas J. Grundl, Todd A. VanderWall, Nicholas A. Rorrer, Christopher W. Johnson, Payal Khanna, Joshua R. Elmore, Darren J. Peterson, Mary J. Biddy, and Yannick J. Bomble
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Muconic acid ,biology ,Catabolism ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,biology.organism_classification ,01 natural sciences ,Decomposition ,Combinatorial chemistry ,Pseudomonas putida ,0104 chemical sciences ,Metabolic pathway ,chemistry.chemical_compound ,General Energy ,Petrochemical ,chemistry ,Bioreactor ,Molecule ,0210 nano-technology - Abstract
Summary To drive innovation in chemical and material applications beyond what has been afforded by the mature petrochemical industry, new molecules that possess diverse chemical functionality are needed. One source of such molecules lies in the varied metabolic pathways that soil microbes utilize to catabolize aromatic compounds generated during plant decomposition. Here, we have engineered Pseudomonas putida KT2440 to convert these aromatic compounds to 15 catabolic intermediates that exhibit substantial chemical diversity. Bioreactor cultivations, analytical methods, and bench-scale separations were developed to enable production (up to 58 g/L), detection, and purification of each target molecule. We further engineered strains for production of a subset of these molecules from glucose, achieving a 41% molar yield of muconic acid. Finally, we produce materials from three compounds to illustrate the potential for realizing performance-advantaged properties relative to petroleum-derived analogs.
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- 2019
19. Combining Reclaimed PET with Bio-based Monomers Enables Plastics Upcycling
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Gregg T. Beckham, Mary J. Biddy, Nicholas A. Rorrer, Alberta Carpenter, Scott R. Nicholson, and Nicholas J. Grundl
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Waste management ,Annual production ,Bio based ,Biomass ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,0104 chemical sciences ,chemistry.chemical_compound ,Upcycling ,General Energy ,chemistry ,Land reclamation ,Polyethylene terephthalate ,Environmental science ,Plastic waste ,0210 nano-technology ,Embodied energy - Abstract
Summary Polyethylene terephthalate (PET) is the largest produced polyester globally with an annual production exceeding 26 million tons for use in carpet, clothing, and single-use beverage bottles, among others. Today, only PET bottles are reclaimed for recycling, albeit at a low reclamation rate, with most of the waste PET accumulating in landfills or the environment. In this study, PET is upcycled to higher-value, long-lifetime materials, namely two types of fiber-reinforced plastics (FRPs), via combination with renewably sourceable monomers. By harnessing the embodied energy in reclaimed PET (rPET) and implementing renewably sourceable monomers with distinct chemical functionality relative to petroleum building blocks, the resultant rPET-FRPs exhibit considerably improved material properties and are predicted to save 57% in the total supply chain energy and reduce greenhouse gas emissions by 40% over standard petroleum-based FRPs. Overall, this study enables a route to PET upcycling via bio-based monomers that could incentivize both improved plastics reclamation and acceleration of the bioeconomy.
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- 2019
20. Production of β-ketoadipic acid from glucose in Pseudomonas putida KT2440 for use in performance-advantaged nylons
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Nicholas A. Rorrer, Sandra F. Notonier, Brandon C. Knott, Brenna A. Black, Avantika Singh, Scott R. Nicholson, Christopher P. Kinchin, Graham P. Schmidt, Alberta C. Carpenter, Kelsey J. Ramirez, Christopher W. Johnson, Davinia Salvachúa, Michael F. Crowley, and Gregg T. Beckham
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General Energy ,General Engineering ,General Physics and Astronomy ,General Materials Science ,General Chemistry - Published
- 2022
21. Corrigendum to 'Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound' [Metab. Eng. 65 (2021) 1–10]
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Joshua K. Michener, Kelsey J. Ramirez, Rui Katahira, Allison Z. Werner, Gregg T. Beckham, Stefan J. Haugen, David C. Garcia, Gerald N. Presley, and Richard J. Giannone
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chemistry.chemical_compound ,chemistry ,Stereochemistry ,Lignin ,Bioengineering ,Cleavage (embryo) ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2021
22. A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution
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Christopher W. Johnson, Payal Khanna, Jeremy M. Bingen, Taraka Dale, Gregg T. Beckham, Theresa L. Kern, Ramesh K. Jha, Daniel S. Trettel, and Charlie E. M. Strauss
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0301 basic medicine ,lcsh:Biotechnology ,Endocrinology, Diabetes and Metabolism ,Allosteric regulation ,ved/biology.organism_classification_rank.species ,Biomedical Engineering ,macromolecular substances ,Computational biology ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Protein evolution ,03 medical and health sciences ,Synthetic biology ,lcsh:TP248.13-248.65 ,medicine ,Model organism ,lcsh:QH301-705.5 ,Escherichia coli ,biology ,Chemistry ,ved/biology ,technology, industry, and agriculture ,Limiting ,biology.organism_classification ,Pseudomonas putida ,0104 chemical sciences ,030104 developmental biology ,lcsh:Biology (General) ,Biosensor - Abstract
Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate
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- 2018
23. Flowthrough Reductive Catalytic Fractionation of Biomass
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Michelle Reed, Eric M. Anderson, Gregg T. Beckham, Yuriy Román-Leshkov, Michael L. Stone, and Rui Katahira
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010405 organic chemistry ,Continuous reactor ,Biomass ,Fractionation ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Catalysis ,chemistry.chemical_compound ,General Energy ,chemistry ,Chemical engineering ,Hydrogenolysis ,Lignin ,Organic chemistry ,Leaching (metallurgy) ,Solvolysis - Abstract
Summary Reductive catalytic fractionation (RCF) has emerged as a leading biomass fractionation and lignin valorization strategy. Here, flowthrough reactors were used to investigate RCF of poplar. Most RCF studies to date have been conducted in batch, but a flow-based process enables the acquisition of intrinsic kinetic and mechanistic data essential to accelerate the design, optimization, and scale-up of RCF processes. Time-resolved product distributions and yields obtained from experiments with different catalyst loadings were used to identify and deconvolute events during solvolysis and hydrogenolysis. Multi-bed RCF experiments provided unique insights into catalyst deactivation, showing that leaching, sintering, and surface poisoning are causes for decreased catalyst performance. The onset of catalyst deactivation resulted in higher concentrations of unsaturated lignin intermediates and increased occurrence of repolymerization reactions, producing high-molecular-weight species. Overall, this study demonstrates the concept of flowthrough RCF, which will be vital for realistic scale-up of this promising approach.
- Published
- 2017
24. Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases
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Gregg T. Beckham, Brent Harrison, Japheth E. Gado, Mats Sandgren, Christina M. Payne, and Jerry Ståhlberg
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Glycoside Hydrolases ,Trichoderma reesei ,Cellulase ,Molecular Dynamics Simulation ,Biology ,Machine learning ,computer.software_genre ,CBH, cellobiohydrolase ,KNN, k-nearest neighbor ,Biochemistry ,k-nearest neighbors algorithm ,Machine Learning ,GH, glycoside hydrolase ,Catalytic Domain ,CBM, carbohydrate-binding module ,tryptophan ,glycoside hydrolase ,Glycoside hydrolase ,ML, machine learning ,Cellulose ,Molecular Biology ,Sequence (medicine) ,chemistry.chemical_classification ,cellulase ,Multiple sequence alignment ,Chemistry ,business.industry ,Biochemistry and Molecular Biology ,CD, catalytic domain ,Active site ,bioinformatics ,Cell Biology ,EG, endoglucanase ,Amino acid ,MSA, multiple sequence alignment ,Kinetics ,statistics ,HMM, hidden Markov model ,LPMO, lytic polysaccharide monooxygenase ,GH7, family 7 glycoside hydrolase ,biology.protein ,Artificial intelligence ,Carbohydrate-binding module ,business ,Linker ,computer ,Function (biology) ,Research Article - Abstract
Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These important enzymes are often bimodular, comprised of a catalytic domain attached to a carbohydrate binding module (CBM) via a flexible linker, and exhibit a long active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical biological and industrial importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, using the number of residues in the active-site loops as features, were able discriminate GH7 CBHs and EGs with up to 99% accuracy. The lengths of the A4, B2, B3, and B4 loops were strongly correlated with functional subtype across the GH7 family. Position-specific classification rules were derived such that specific amino acids at 42 different sequence positions predicted the functional subtype with accuracies greater than 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. We propose these positions play vital roles in the functional variation of GH7 cellulases. Taken together, our results complement numerous experimental findings and present functional relationships that can be applied when prospecting GH7 cellulases from nature, for sequence annotation, and to understand or manipulate function.
- Published
- 2021
25. Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by Pseudomonas putida KT2440
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Mary Ann Franden, Gregg T. Beckham, Michael T. Guarnieri, and Christopher W. Johnson
- Subjects
0301 basic medicine ,integumentary system ,biology ,lcsh:Biotechnology ,Endocrinology, Diabetes and Metabolism ,Cupriavidus basilensis ,Biomedical Engineering ,biology.organism_classification ,Furfural ,Article ,Pseudomonas putida ,Hydrolysate ,Furfuryl alcohol ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,Biochemistry ,lcsh:TP248.13-248.65 ,Organic chemistry ,Hydroxymethyl ,Energy source ,Sugar ,lcsh:QH301-705.5 - Abstract
The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment., Highlights • HMF and furfural are common microbial inhibitors in biomass conversion. • HMF and furfural gene cluster was isolated from Burkholderia phytofirmans.. • We heterologously express the HMF/furfural gene cluster in Pseudomonas putida.. • Expression enables cultivation on HMF and furfural as a sole carbon source. • Expression also enables enhanced conversion on lignocellulosic hydrolysate.
- Published
- 2017
26. CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli
- Author
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Violeta Sànchez i Nogué, Ryan T. Gill, Gregg T. Beckham, Rongming Liu, Thomas Lee, Andrew D. Garst, and Liya Liang
- Subjects
0106 biological sciences ,0301 basic medicine ,Bioengineering ,Computational biology ,Biology ,medicine.disease_cause ,01 natural sciences ,Applied Microbiology and Biotechnology ,Genome ,Genome engineering ,2-Propanol ,03 medical and health sciences ,Synthetic biology ,Eukaryotic translation ,010608 biotechnology ,Escherichia coli ,medicine ,CRISPR ,Gene ,Gene Editing ,Genetics ,Strain (chemistry) ,030104 developmental biology ,Metabolic Engineering ,CRISPR-Cas Systems ,Biotechnology - Abstract
Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5 g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40 g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these variants, we constructed a combinatorial library containing 256 total variants from four different RBS levels for each gene. The best producing variant, PA14, produced isopropanol at titers of 7.1 g/L at 24 h, with a yield of 0.75 (mol/mol), and maximum productivity of 0.62 g/L/h (which was 0.22 g/L/h above the parent strain PA07). We demonstrate the ability to rapidly construct and test close to ~1000 designer strains and identify superior performers.
- Published
- 2017
27. The Dissociation Mechanism of the Processive Cellulase TrCel7A
- Author
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Mikael Gudmundsson, Josh V. Vermaas, Jerry Ståhlberg, Mats Sandgren, Priit Väljamäe, Gregg T. Beckham, Brandon C. Knott, Michael F. Crowley, and Riin Kont
- Subjects
biology ,Chemistry ,Biophysics ,biology.protein ,Cellulase ,Dissociation (chemistry) - Published
- 2020
28. Comparing Inner and Outer Membrane Permeabilities for Lignin Related Aromatic Compounds
- Author
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Gregg T. Beckham, Josh V. Vermaas, and Michael F. Crowley
- Subjects
chemistry.chemical_compound ,Chemical engineering ,Chemistry ,Biophysics ,Lignin ,Bacterial outer membrane - Published
- 2021
29. Succinic acid production from lignocellulosic hydrolysate by Basfia succiniciproducens
- Author
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Nancy Dowe, Darren J. Peterson, Gregg T. Beckham, Ali Mohagheghi, Peter C. St. John, Davinia Salvachúa, Holly Smith, and Brenna A. Black
- Subjects
0106 biological sciences ,0301 basic medicine ,Environmental Engineering ,Succinic Acid ,Biomass ,Bioengineering ,Xylose ,Lignin ,Zea mays ,01 natural sciences ,Hydrolysate ,03 medical and health sciences ,chemistry.chemical_compound ,010608 biotechnology ,Food science ,Sugar ,Waste Management and Disposal ,biology ,Renewable Energy, Sustainability and the Environment ,Hydrolysis ,Actinobacillus ,General Medicine ,Sulfuric Acids ,Biorefinery ,biology.organism_classification ,Actinobacillus succinogenes ,Glucose ,030104 developmental biology ,Corn stover ,chemistry ,Biochemistry ,Batch Cell Culture Techniques ,Succinic acid ,Fermentation ,Metabolic Networks and Pathways - Abstract
The production of chemicals alongside fuels will be essential to enhance the feasibility of lignocellulosic biorefineries. Succinic acid (SA), a naturally occurring C4-diacid, is a primary intermediate of the tricarboxylic acid cycle and a promising building block chemical that has received significant industrial attention. Basfia succiniciproducens is a relatively unexplored SA-producing bacterium with advantageous features such as broad substrate utilization, genetic tractability, and facultative anaerobic metabolism. Here B. succiniciproducens is evaluated in high xylose-content hydrolysates from corn stover and different synthetic media in batch fermentation. SA titers in hydrolysate at an initial sugar concentration of 60 g/L reached up to 30 g/L, with metabolic yields of 0.69 g/g, and an overall productivity of 0.43 g/L/h. These results demonstrate that B. succiniciproducens may be an attractive platform organism for bio-SA production from biomass hydrolysates.
- Published
- 2016
30. Reaction: Proteins from Chemocatalysis; It’s What’s for Dinner
- Author
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Lucas D. Ellis and Gregg T. Beckham
- Subjects
Engineering ,business.industry ,General Chemical Engineering ,Biochemistry (medical) ,Biomass ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Renewable energy ,Upcycling ,Environmental protection ,Materials Chemistry ,Environmental Chemistry ,0210 nano-technology ,business - Abstract
Lucas D. Ellis received his BS from California Polytechnic State University, MS from Dartmouth College, and PhD focusing on heterogeneous catalysis from the University of Colorado. He is currently a Director’s Postdoctoral Fellow at the National Renewable Energy Laboratory (NREL). His research focuses on developing heterogeneous catalysts and chemical processes capable of reducing the impact of climate change. Gregg T. Beckham is a senior research fellow at NREL. He received a PhD and MSCEP from the Massachusetts Institute of Technology and a BS from Oklahoma State University. He currently leads an interdisciplinary team on the conversion of biomass to chemicals and materials and in the area of plastics upcycling.
- Published
- 2019
31. Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin
- Author
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Gregg T. Beckham and Christopher W. Johnson
- Subjects
chemistry.chemical_classification ,Catechol ,biology ,Pseudomonas putida ,Bioengineering ,biology.organism_classification ,Lignin ,Applied Microbiology and Biotechnology ,Sphingobium ,Citric acid cycle ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Metabolic Engineering ,Dioxygenase ,Yield (chemistry) ,Pyruvic Acid ,Lactic Acid ,Biotechnology - Abstract
Lignin represents an untapped feedstock for the production of fuels and chemicals, but its intrinsic heterogeneity makes lignin valorization a significant challenge. In nature, many aerobic organisms degrade lignin-derived aromatic molecules through conserved central intermediates including catechol and protocatechuate. Harnessing this microbial approach offers potential for lignin upgrading in modern biorefineries, but significant technical development is needed to achieve this end. Catechol and protocatechuate are subjected to aromatic ring cleavage by dioxygenase enzymes that, depending on the position, ortho or meta relative to adjacent hydroxyl groups, result in different products that are metabolized through parallel pathways for entry into the TCA cycle. These degradation pathways differ in the combination of succinate, acetyl-CoA, and pyruvate produced, the reducing equivalents regenerated, and the amount of carbon emitted as CO2-factors that will ultimately impact the yield of the targeted product. As shown here, the ring-cleavage pathways can be interchanged with one another, and such substitutions have a predictable and substantial impact on product yield. We demonstrate that replacement of the catechol ortho degradation pathway endogenous to Pseudomonas putida KT2440 with an exogenous meta-cleavage pathway from P. putida mt-2 increases yields of pyruvate produced from aromatic molecules in engineered strains. Even more dramatically, replacing the endogenous protocatechuate ortho pathway with a meta-cleavage pathway from Sphingobium sp. SYK-6 results in a nearly five-fold increase in pyruvate production. We further demonstrate the aerobic conversion of pyruvate to l-lactate with a yield of 41.1 ± 2.6% (wt/wt). Overall, this study illustrates how aromatic degradation pathways can be tuned to optimize the yield of a desired product in biological lignin upgrading.
- Published
- 2015
- Full Text
- View/download PDF
32. Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction
- Author
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Jerry Ståhlberg, Gustav Vaaje-Kolstad, Antoine Royant, Gregg T. Beckham, Daniel Lundberg, Seonah Kim, Majid Hadadd Momeni, Vincent G. H. Eijsink, Mats Sandgren, Takuya Ishida, Miao Wu, Mikael Gudmundsson, Department of Physics, Stockholm University, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), University of Luebeck, Biotechnology and Food Science (Department of Chemistry), Center for Molecular Microbiology, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
inorganic chemicals ,Models, Molecular ,MESH: Oxidation-Reduction ,MESH: Databases, Protein ,MESH: Enterococcus faecalis ,Inorganic chemistry ,MESH: Catalytic Domain ,Electrons ,Crystal structure ,Biochemistry ,Mixed Function Oxygenases ,Metal ,MESH: X-Rays ,Polysaccharides ,Oxidation state ,Catalytic Domain ,Enterococcus faecalis ,Molecule ,Databases, Protein ,Molecular Biology ,MESH: Electrons ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,biology ,Chemistry ,X-Rays ,Active site ,Cell Biology ,MESH: Mixed Function Oxygenases ,Photochemical Processes ,Oxygen ,MESH: Copper ,Crystallography ,Trigonal bipyramidal molecular geometry ,MESH: Polysaccharides ,visual_art ,X-ray crystallography ,Enzymology ,biology.protein ,visual_art.visual_art_medium ,Quantum Theory ,MESH: Photochemical Processes ,Oxidation-Reduction ,MESH: Quantum Theory ,Copper ,MESH: Oxygen ,MESH: Models, Molecular - Abstract
International audience; Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.
- Published
- 2014
33. Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum
- Author
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Yukishige Ito, Sophon Kaeothip, Gregg T. Beckham, Akihiro Ishiwata, Takatoshi Arakawa, Kiyotaka Fujita, Takayoshi Wakagi, Tasuku Ito, Shinya Fushinobu, Seonah Kim, and Kyo Saikawa
- Subjects
Models, Molecular ,Bifidobacterium longum ,Glycoside Hydrolases ,Stereochemistry ,Molecular Sequence Data ,Biophysics ,Glutamic Acid ,Biochemistry ,Substrate Specificity ,Catalytic Domain ,Hydrolase ,Glycoside hydrolase ,Amino Acid Sequence ,Cysteine ,Molecular Biology ,chemistry.chemical_classification ,biology ,Active site ,Glycoside ,Glycosidic bond ,Cell Biology ,biology.organism_classification ,Arabinose ,Protein Structure, Tertiary ,Amino acid ,Zinc ,chemistry ,biology.protein ,Quantum Theory ,Bifidobacterium ,Sequence Alignment - Abstract
Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β- l -arabinofuranosyl oligosaccharides in plant cells. Recently, a β- l -arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β- l -arabinooligosaccharides in the bacterium Bifidobacterium longum . Here, we report the crystal structure of HypBA1 in the ligand-free and β- l -arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn 2+ coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β- l -arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.
- Published
- 2014
34. Lignin-Cellulose Binding Affinity Dependence on Cellulose Face and Lignin Composition
- Author
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Josh V. Vermaas, Gregg T. Beckham, and Michael F. Crowley
- Subjects
chemistry.chemical_compound ,chemistry ,Biophysics ,Lignin ,Organic chemistry ,Composition (visual arts) ,Cellulose ,Cellulose binding - Published
- 2019
35. Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium
- Author
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Masahiro Samejima, Svein Jarle Horn, Christina M. Payne, Kiyohiko Igarashi, Takuya Ishida, Gregg T. Beckham, Seonah Kim, Vincent G. H. Eijsink, Bjørge Westereng, Michael E. Himmel, Jerry Ståhlberg, Anna M. Larsson, Michael F. Crowley, Miao Wu, and Mats Sandgren
- Subjects
Cellobiose ,Crystallography, X-Ray ,Phanerochaete ,Polysaccharide ,Biochemistry ,Mixed Function Oxygenases ,Fungal Proteins ,chemistry.chemical_compound ,Catalytic Domain ,Hydrolase ,Glycoside hydrolase ,Cellulose ,Molecular Biology ,chemistry.chemical_classification ,Fungal protein ,biology ,Active site ,Cell Biology ,biology.organism_classification ,chemistry ,Enzymology ,biology.protein ,Copper - Abstract
Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.
- Published
- 2013
36. Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases
- Author
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Lintao Bu, Gregg T. Beckham, Michael E. Himmel, and Michael F. Crowley
- Subjects
Glycoside Hydrolases ,Stereochemistry ,Hypocrea ,Cellulase ,Cellobiose ,Molecular Dynamics Simulation ,Biochemistry ,Catalysis ,Protein Structure, Secondary ,Fungal Proteins ,Hydrolysis ,chemistry.chemical_compound ,Organic chemistry ,Computer Simulation ,Glycoside hydrolase ,Cellulose ,Molecular Biology ,Trichoderma reesei ,chemistry.chemical_classification ,Fungal protein ,biology ,Computational Biology ,Glycosidic bond ,Cell Biology ,Hydrogen-Ion Concentration ,biology.organism_classification ,chemistry ,biology.protein - Abstract
Cellulase enzymes cleave glycosidic bonds in cellulose to produce cellobiose via either retaining or inverting hydrolysis mechanisms, which are significantly pH-dependent. Many fungal cellulases function optimally at pH ~5, and their activities decrease dramatically at higher or lower pH. To understand the molecular-level implications of pH in cellulase structure, we use a hybrid, solvent-based, constant pH molecular dynamics method combined with pH-based replica exchange to determine the pK(a) values of titratable residues of a glycoside hydrolase (GH) family 6 cellobiohydrolase (Cel6A) and a GH family 7 cellobiohydrolase (Cel7A) from the fungus Hypocrea jecorina. For both enzymes, we demonstrate that a bound substrate significantly affects the pKa values of the acid residues at the catalytic center. The calculated pK(a) values of catalytic residues confirm their proposed roles from structural studies and are consistent with the experimentally measured apparent pKa values. Additionally, GHs are known to impart a strained pucker conformation in carbohydrate substrates in active sites for catalysis, and results from free energy calculations combined with constant pH molecular dynamics suggest that the correct ring pucker is stable near the optimal pH for both Cel6A and Cel7A. Much longer molecular dynamics simulations of Cel6A and Cel7A with fixed protonation states based on the calculated pK(a) values suggest that pH affects the flexibility of tunnel loops, which likely affects processivity and substrate complexation. Taken together, this work demonstrates several molecular-level effects of pH on GH enzymes important for cellulose turnover in the biosphere and relevant to biomass conversion processes.
- Published
- 2013
37. Harnessing glycosylation to improve cellulase activity
- Author
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Michael E. Himmel, Michelle Momany, Gregg T. Beckham, Christina M. Payne, William S. Adney, James F. Matthews, Ziyu Dai, and Scott E. Baker
- Subjects
Glycan ,Glycosylation ,Glycoside Hydrolases ,Biomedical Engineering ,Bioengineering ,Cellulase ,Protein Engineering ,chemistry.chemical_compound ,Cell Wall ,Polysaccharides ,Cellulases ,Glycoside hydrolase ,Biomass ,chemistry.chemical_classification ,biology ,Fungi ,Protein engineering ,Enzyme structure ,Enzyme assay ,carbohydrates (lipids) ,Enzyme ,chemistry ,Biochemistry ,Biofuels ,biology.protein ,Biotechnology - Abstract
Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.
- Published
- 2012
38. Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-binding Module on Cellulose
- Author
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Lintao Bu, Gregg T. Beckham, Michael E. Himmel, Michael F. Crowley, James F. Matthews, and Mark R. Nimlos
- Subjects
Protein Structure ,Molecular model ,Trichoderma reesei ,Molecular Modeling ,Cellulase ,Molecular Dynamics Simulation ,Molecular Dynamics ,Biochemistry ,Carbohydrate-binding Module ,Fungal Proteins ,Protein Dynamics ,chemistry.chemical_compound ,Molecular dynamics ,Protein structure ,Organic chemistry ,Cellulose ,Molecular Biology ,Trichoderma ,Fungal protein ,biology ,Cel7A ,Computational Biology ,Hypocrea jecorina ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Models, Chemical ,chemistry ,Biophysics ,biology.protein ,Carbohydrate-binding module ,Hydrophobic and Hydrophilic Interactions - Abstract
Background: Family 1 carbohydrate-binding modules (CBMs) bind selectively to the hydrophobic surfaces of cellulose. Results: Simulations have shown that the planar face of the CBM binds preferentially to the hydrophobic face. Conclusion: Thermodynamic driving forces enable transfer of the CBM from the hydrophilic to hydrophobic surfaces. Significance: Selectivity of CBM provides access of cellulases to active surfaces of cellulose., Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.
- Published
- 2012
39. Probing Carbohydrate Product Expulsion from a Processive Cellulase with Multiple Absolute Binding Free Energy Methods
- Author
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Gregg T. Beckham, Mark R. Nimlos, Michael E. Himmel, Lintao Bu, William S. Adney, Michael R. Shirts, and Michael F. Crowley
- Subjects
Carbohydrate ,Cellobiose ,Biophysics ,Carbohydrates ,Enzyme Mechanisms ,Cellulase ,Bioenergetics ,Biochemistry ,Fungal Proteins ,Free energy perturbation ,chemistry.chemical_compound ,Molecular dynamics ,Computational chemistry ,Organic chemistry ,Enzyme Inhibitors ,Molecular Biology ,Trichoderma reesei ,Trichoderma ,Fungal protein ,biology ,Chemistry ,Computational Biology ,Cell Biology ,biology.organism_classification ,Glucose binding ,Binding Free Energy ,Product inhibition ,Computation ,biology.protein ,Cellobiohydrolase ,Thermodynamics ,Computer Modeling ,Protein Binding - Abstract
Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, -14.4 kcal/mol using SMD and -11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are -13.1, -6.0, -11.5, -7.5, and -8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion.
- Published
- 2011
40. The O-Glycosylated Linker from the Trichoderma reesei Family 7 Cellulase Is a Flexible, Disordered Protein
- Author
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Malin Bergenstråhle, Courtney B. Taylor, Xiongce Zhao, John W. Brady, Yannick J. Bomble, Lintao Bu, James F. Matthews, John M. Yarbrough, Jakob Wohlert, Gregg T. Beckham, Stephen R. Decker, William S. Adney, Clare McCabe, Michael F. Crowley, Michael E. Himmel, and Michael G. Resch
- Subjects
Models, Molecular ,Glycosylation ,Molecular Sequence Data ,Biophysics ,Cellulase ,Serine ,chemistry.chemical_compound ,Amino Acid Sequence ,Cellulose ,Threonine ,Trichoderma reesei ,chemistry.chemical_classification ,Trichoderma ,biology ,Protein ,biology.organism_classification ,Protein Structure, Tertiary ,carbohydrates (lipids) ,Kinetics ,chemistry ,Biochemistry ,biology.protein ,Thermodynamics ,Glycoprotein ,Linker - Abstract
Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.
- Published
- 2010
- Full Text
- View/download PDF
41. Computational Insights into Fuels and Chemicals Extraction from Microbial Biorefineries
- Author
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Josh V. Vermaas, Michael F. Crowley, and Gregg T. Beckham
- Subjects
Extraction (chemistry) ,Biophysics ,Environmental science ,Biochemical engineering - Published
- 2018
42. Systematic Parameterization of Lignin for the Charmm Force Field
- Author
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Loukas Petridis, Gregg T. Beckham, Michael F. Crowley, and Josh V. Vermaas
- Subjects
Materials science ,010304 chemical physics ,Biophysics ,Force field parameterization ,010402 general chemistry ,01 natural sciences ,Force field (chemistry) ,0104 chemical sciences ,chemistry.chemical_compound ,Molecular dynamics ,chemistry ,0103 physical sciences ,Fungal enzymes ,Lignin ,Molecule ,Biomass fuels ,Biological system - Abstract
Lignin is an abundant aromatic biopolymer within plant cell walls formed through radical coupling chemistry, whose composition and topology can vary greatly depending on the biomass source. Computational modeling provides a complementary approach to traditional experimental techniques to probe lignin interactions, lignin structure, and lignin material properties. However, current modeling approaches are limited based on the subset of lignin chemistries covered by existing lignin force fields. To fill the gap, we developed a comprehensive lignin force field that accounts for more lignin–lignin and lignin–carbohydrate interlinkages than existing lignin force fields, and also greatly expands the lignin monomer chemistries that can be modeled beyond simple alcohols and into the rich mixture of natural lignin varieties. The development of this force field utilizes recent developments in parameterization methodology, and synthesizes them into a workflow that combines target data from multiple molecules simultaneously into a single consistent and comprehensive parameter set. The parameter set represents a significant improvement to alternatives for atomic modeling of diverse lignin topologies, more accurately reproducing experimental observables while also significantly reducing the error relative to quantum calculations. The improved energetics, as well as the rigid adherence to CHARMM parameterization philosophy, enables simulation of lignin within its biological context with greater accuracy than was previously possible. The lignin force field presented here is therefore a crucial first step towards modeling lignin structure across a broad range of environments, including within plant cell walls where lignin is complexed with carbohydrates and deconstructed by bacterial or fungal enzymes, or as it exists within industrial solvent mixtures. Future simulations enabled by this updated lignin force field will thus lead to better chemical and structural understanding of lignin, providing new insight into its role in biomass recalcitrance or probing the potential for lignin to be used within industrial processes.
- Published
- 2017
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