450 results on '"Scrutton NS"'
Search Results
2. Building a global alliance of biofoundries (vol 10, 2040, 2019)
- Author
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Hillson, N, Caddick, M, Cai, Y, Carrasco, JA, Chang, MW, Curach, NC, Bell, DJ, Le Feuvre, R, Friedman, DC, Fu, X, Gold, ND, Herrgard, MJ, Holowko, MB, Johnson, JR, Johnson, RA, Keasling, JD, Kitney, RI, Kondo, A, Liu, C, Martin, VJJ, Menolascina, F, Ogino, C, Patron, NJ, Pavan, M, Poh, CL, Pretorius, IS, Rosser, SJ, Scrutton, NS, Storch, M, Tekotte, H, Travnik, E, Vickers, CE, Yew, WS, Yuan, Y, Zhao, H, and Freemont, PS
- Subjects
Multidisciplinary Sciences ,Science & Technology ,MD Multidisciplinary ,Science & Technology - Other Topics - Published
- 2019
3. Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects
- Author
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Jones, Alex R, Rentergent, Julius, Scrutton, NS, and Hay, S
- Subjects
Models, Molecular ,reaction mechanisms ,Kinetics ,ResearchInstitutes_Networks_Beacons/photon_science_institute ,kinetic isotope effects ,enzymes ,coenzyme B12 ,Cobamides ,Photon Science Institute ,Full Papers ,ethanolamine ammonia lyase ,Ethanolamine Ammonia-Lyase ,Catalysis - Abstract
Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.
- Published
- 2015
4. Structural basis of catalysis in the bacterial monoterpene synthases linalool synthase and 1,8-cineole synthase
- Author
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Karuppiah, Vijaykumar, Ranaghan, Kara, Leferink, Nicole G. H., Johannissen, LO, Shanmugam, Muralidharan, Cheallaigh, Aisling Ni, Bennett, Nathan J., Kearsey, Lewis J., Takano, Eriko, Gardiner, John M., Van der Kamp, Marc, Hay, Sam, Mulholland, Adrian, Leys, David, and Scrutton, NS
- Subjects
Monoterpene synthase ,Molecular dynamics simulations ,Terpenes ,Monoterpenoids ,Protein crystallography ,Sesquiterpene synthase - Abstract
Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.
- Published
- 2017
5. Cross-species analysis of protein dynamics associated with hydride and proton transfer in the catalytic cycle of the light-driven enzyme protochlorophyllide oxidoreductase
- Author
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Hoeven, Robin, Heyes, Derren, Hardman, Samantha, and Scrutton, NS
- Published
- 2016
6. Nuclear quantum tunnelling in enzymatic reactions - an enzymologist's perspective
- Author
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Johannissen, Linus, Hay, Sam, and Scrutton, NS
- Abstract
Enzyme-catalysed H-transfer reactions are ubiquitous, yet fundamental details of these reactions remain unresolved. In this perspective, we discuss the roles of nuclear quantum tunnelling and (compressive) dynamics during these reactions. Evidence for the coupling of specific substrate and/or protein vibrations to the chemical coordinate is considered and a case is made for the combination of multiple experimental and computational/theoretical approaches when studying these reactions.
- Published
- 2015
7. Does the pressure dependence of kinetic isotope effects report usefully on dynamics in enzyme H-transfer reactions?
- Author
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Hoeven, R, Heyes, D. J., Hay, S, and Scrutton, NS
- Subjects
pressure ,flavoprotein ,hydrogen transfer ,quantum tunnelling ,dynamics - Abstract
The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called ‘promoting motions/vibrations hypothesis’. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature (p-T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p-T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p-T dependencies of the KIEs in these systems. Given the complex nature of p-T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/KIEs to guide the interpretation of p-T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p-T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs.
- Published
- 2015
8. Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase RID A-4573-2009
- Author
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POLTICELLI, Fabio, Basran J, Faso C, Cona A, Minervini G, Angelini R, Federico R, Scrutton NS, TAVLADORAKI, Paraskevi, Polticelli, Fabio, Basran, J, Faso, C, Cona, A, Minervini, G, Angelini, R, Federico, R, Scrutton, N, and Tavladoraki, Paraskevi
- Abstract
Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-angstrom long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu 170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N-5 atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steadystate and stopped-flow studies with wild-type, Glu62Gln, Glul70Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pk(a) of similar to 5.8. Kinetic analysis of the Glu62Gln, Glul70Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu 170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Metspermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.
- Published
- 2005
9. Structure-function relationship of maize polyamine oxidase
- Author
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TAVLADORAKI, Paraskevi, POLTICELLI, Fabio, Basran J, Federico R, Scrutton NS, Angelini R., CONA, Alessandra, Tavladoraki, Paraskevi, Polticelli, Fabio, Basran, J, Cona, Alessandra, Federico, R, Scrutton, N, and Angelini, R.
- Published
- 2002
10. Probing the stabilizing route of C-terminal residues in trimethylamine dehydrogenase.
- Author
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Ertughrul, OWD, Errington, N, Raza, S, Sutcliffe, MJ, Rowe, AJ, and Scrutton, NS
- Published
- 1998
- Full Text
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11. Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.
- Author
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Kumar V, Agrawal D, Bommareddy RR, Islam MA, Jacob S, Balan V, Singh V, Thakur VK, Navani NK, and Scrutton NS
- Subjects
- Butylene Glycols metabolism, Lignin metabolism, Biomass, Bacteria metabolism, Fermentation, Ethanol metabolism, Lactic Acid metabolism, Sugar Alcohols metabolism, Xylitol metabolism, Arabinose metabolism
- Abstract
The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.
- Published
- 2024
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12. A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.
- Author
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Zhang L, Ye JW, Li G, Park H, Luo H, Lin Y, Li S, Yang W, Guan Y, Wu F, Huang W, Wu Q, Scrutton NS, Nielsen J, and Chen GQ
- Subjects
- Metabolic Engineering, Bacterial Proteins genetics, Bacterial Proteins metabolism, Metabolic Networks and Pathways genetics, Gene Deletion, Models, Biological, Halomonas genetics, Halomonas metabolism, Halomonas enzymology, Halomonas growth & development
- Abstract
Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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13. Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.
- Author
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Taylor A, Zhang S, Johannissen LO, Sakuma M, Phillips RS, Green AP, Hay S, Heyes DJ, and Scrutton NS
- Subjects
- Protochlorophyllide chemistry, Light, Protons, Photochemistry, Oxidoreductases Acting on CH-CH Group Donors metabolism, Cyanobacteria
- Abstract
The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2024
- Full Text
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14. Photocobilins integrate B 12 and bilin photochemistry for enzyme control.
- Author
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Zhang S, Jeffreys LN, Poddar H, Yu Y, Liu C, Patel K, Johannissen LO, Zhu L, Cliff MJ, Yan C, Schirò G, Weik M, Sakuma M, Levy CW, Leys D, Heyes DJ, and Scrutton NS
- Subjects
- Photochemistry, Biliverdine, Bacterial Proteins metabolism, Light, Bile Pigments, Photoreceptors, Microbial chemistry
- Abstract
Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B
12 ) can act as a light-sensing chromophore heralded a new field of B12 -photobiology. Although microbial genome analysis indicates that photoactive B12 -binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts., (© 2024. The Author(s).)- Published
- 2024
- Full Text
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15. Ectoine hyperproduction by engineered Halomonas bluephagenesis.
- Author
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Hu Q, Sun S, Zhang Z, Liu W, Yi X, He H, Scrutton NS, and Chen GQ
- Subjects
- Anti-Bacterial Agents, Biopolymers, Halomonas genetics, Amino Acids, Diamino genetics
- Abstract
Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
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16. PHA is not just a bioplastic!
- Author
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Park H, He H, Yan X, Liu X, Scrutton NS, and Chen GQ
- Subjects
- Biopolymers, Food, Polyhydroxyalkanoates metabolism
- Abstract
Polyhydroxyalkanoates (PHA) have evolved into versatile biopolymers, transcending their origins as mere bioplastics. This extensive review delves into the multifaceted landscape of PHA applications, shedding light on the diverse industries that have harnessed their potential. PHA has proven to be an invaluable eco-conscious option for packaging materials, finding use in films foams, paper coatings and even straws. In the textile industry, PHA offers a sustainable alternative, while its application as a carbon source for denitrification in wastewater treatment showcases its versatility in environmental remediation. In addition, PHA has made notable contributions to the medical and consumer sectors, with various roles ranging from 3D printing, tissue engineering implants, and cell growth matrices to drug delivery carriers, and cosmetic products. Through metabolic engineering efforts, PHA can be fine-tuned to align with the specific requirements of each industry, enabling the customization of material properties such as ductility, elasticity, thermal conductivity, and transparency. To unleash PHA's full potential, bridging the gap between research and commercial viability is paramount. Successful PHA production scale-up hinges on establishing direct supply chains to specific application domains, including packaging, food and beverage materials, medical devices, and agriculture. This review underscores that PHA's future rests on ongoing exploration across these industries and more, paving the way for PHA to supplant conventional plastics and foster a circular economy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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17. Nonsterile microbial production of chemicals based on Halomonas spp.
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Zhang J, Yan X, Park H, Scrutton NS, Chen T, and Chen GQ
- Subjects
- Fermentation, Bacteria, Halomonas
- Abstract
The use of extremophile organisms such as Halomomas spp. can eliminate the need for fermentation sterilization, significantly reducing process costs. Microbial fermentation is considered a pivotal strategy to reduce reliance on fossil fuel resources; however, sustainable processes continue to incur higher costs than their chemical industry counterparts. Most organisms require equipment sterilization to prevent contamination, a practice that introduces complexity and financial strain. Fermentations involving extremophile organisms can eliminate the sterilization process, relying instead on conditions that are conductive solely to the growth of the desired organism. This review discusses current challenges in pilot- and industrial-scale bioproduction when using the extremophile bacteria Halomomas spp. under nonsterile conditions., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
- Published
- 2024
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18. Chemoautotrophic production of gaseous hydrocarbons, bioplastics and osmolytes by a novel Halomonas species.
- Author
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Faulkner M, Hoeven R, Kelly PP, Sun Y, Park H, Liu LN, Toogood HS, and Scrutton NS
- Abstract
Background: Production of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions., Results: We describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH
14 CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin-Bensen-Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions., Conclusions: This proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors., (© 2023. BioMed Central Ltd., part of Springer Nature.)- Published
- 2023
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19. Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.
- Author
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Zhao J, Zhuo Y, Diaz DE, Shanmugam M, Telfer AJ, Lindley PJ, Kracher D, Hayashi T, Seibt LS, Hardy FJ, Manners O, Hedison TM, Hollywood KA, Spiess R, Cain KM, Diaz-Moreno S, Scrutton NS, Tovborg M, Walton PH, Heyes DJ, and Green AP
- Subjects
- Mixed Function Oxygenases, Oxidative Stress, Catalysis, Copper, Histidine
- Abstract
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a Cu
II -(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled ( S = 1) CuII -tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.- Published
- 2023
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20. Decoding Catalysis by Terpene Synthases.
- Author
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Whitehead JN, Leferink NGH, Johannissen LO, Hay S, and Scrutton NS
- Abstract
The review by Christianson, published in 2017 on the twentieth anniversary of the emergence of the field, summarizes the foundational discoveries and key advances in terpene synthase/cyclase (TS) biocatalysis (Christianson, D. W. Chem Rev 2017 , 117 (17), 11570-11648. DOI: 10.1021/acs.chemrev.7b00287). Here, we review the TS literature published since then, bringing the field up to date and looking forward to what could be the near future of TS rational design. Many revealing discoveries have been made in recent years, building on the knowledge and fundamental principles uncovered during those initial two decades of study. We use these to explore TS reaction chemistry and see how a combined experimental and computational approach helps to decipher the complexities of TS catalysis. Revealed are a suite of catalytic motifs which control product outcome in TSs, some obvious, some more subtle. We examine each in detail, using the most recent papers and insights to illustrate how exactly this fascinating class of enzymes takes a single acyclic substrate and turns it into the many thousands of complex terpenoids found in Nature. We then explore some of the recent strategies for TS engineering, including machine learning and other data-driven approaches. From this, rational and predictive engineering of TSs, "designer terpene synthases", will begin to emerge as a realistic goal., Competing Interests: The authors declare the following competing financial interest(s): NSS is founder and shareholder of C3 Biotechnologies Ltd., (© 2023 The Authors. Published by American Chemical Society.)
- Published
- 2023
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21. Structure-Based Design of Small Imine Reductase Panels for Target Substrates.
- Author
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Yu Y, Rué Casamajo A, Finnigan W, Schnepel C, Barker R, Morrill C, Heath RS, De Maria L, Turner NJ, and Scrutton NS
- Abstract
Biocatalysis is important in the discovery, development, and manufacture of pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here, we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing a resource-intensive laboratory-based biocatalyst screening. We selected imine reductases (IREDs) as a class of biocatalysts to illustrate the application of the computational workflow termed IREDFisher. Validation by using published data showed that IREDFisher can retrieve the best enzymes and increase the hit rate by identifying the top 20 ranked sequences. The power of IREDFisher is confirmed by computationally screening 1400 sequences for chosen reductive amination reactions with different levels of complexity. Highly active IREDs were identified by only testing 20 samples in vitro. Our speed test shows that it only takes 90 min to rank 85 sequences from user input and 30 min for the established IREDFisher database containing 591 IRED sequences. IREDFisher is available as a user-friendly web interface (https://enzymeevolver.com/IREDFisher). IREDFisher enables the rapid discovery of IREDs for applications in synthesis and directed evolution studies, with minimal time and resource expenditure. Future use of the workflow with other enzyme families could be implemented following the modification of the workflow scoring function., Competing Interests: The authors declare the following competing financial interest(s): NSS is co-founder, executive director and share holder of C3 Biotechnologies Ltd which is active in engineering biology for synthetic fuels production., (© 2023 The Authors. Published by American Chemical Society.)
- Published
- 2023
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22. Redox driven B 12 -ligand switch drives CarH photoresponse.
- Author
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Poddar H, Rios-Santacruz R, Heyes DJ, Shanmugam M, Brookfield A, Johannissen LO, Levy CW, Jeffreys LN, Zhang S, Sakuma M, Colletier JP, Hay S, Schirò G, Weik M, Scrutton NS, and Leys D
- Subjects
- Ligands, Oxidation-Reduction, Lighting, Cold Temperature, Histidine
- Abstract
CarH is a coenzyme B
12 -dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12 -binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12 -ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes., (© 2023. Springer Nature Limited.)- Published
- 2023
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23. Biosynthesis of cannabigerol and cannabigerolic acid: the gateways to further cannabinoid production.
- Author
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Kearsey LJ, Yan C, Prandi N, Toogood HS, Takano E, and Scrutton NS
- Abstract
Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press.)
- Published
- 2023
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24. Unravelling the complexity of enzyme catalysis.
- Author
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Scrutton NS
- Subjects
- Thermodynamics, Catalysis, Enzymes genetics, Enzymes chemistry
- Abstract
The study of enzymes never disappoints. Despite its long history-almost 150 years following the first documented use of the word enzyme in 1878-the field of enzymology advances apace. This long journey has witnessed landmark developments that have defined modern enzymology as a broad discipline, leading to improved understanding at the molecular level, as we aspire to discover the complex relationships between enzyme structures, catalytic mechanisms and biological function. How enzymes are regulated at the gene and post-translational levels and how catalytic activity is modulated by interactions with small ligands and macromolecules, or the broader enzyme environment, are topical areas of study. Insights from such studies guide the exploitation of natural and engineered enzymes in biomedical or industrial processes; for example, in diagnostics, pharmaceuticals manufacture and processing technologies that use immobilised enzymes and enzyme reactor-based systems. In this Focus Issue, The FEBS Journal seeks to highlight breaking science and informative reviews, as well as personal reflections, to illustrate the breadth and importance of contemporary molecular enzymology research., (© 2023 Federation of European Biochemical Societies.)
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- 2023
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25. Photoinduced Electron Transfer from a 1,4,5,6-Tetrahydro Nicotinamide Adenine Dinucleotide (Phosphate) Analogue to Oxidized Flavin in an Ene-Reductase Flavoenzyme.
- Author
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Speirs M, Hardman SJO, Iorgu AI, Johannissen LO, Heyes DJ, Scrutton NS, Sazanovich IV, and Hay S
- Subjects
- NADP, Oxidation-Reduction, Electrons, Flavins chemistry, Phosphates, Kinetics, Oxidoreductases chemistry, NAD chemistry
- Abstract
Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H
4 to FMN, generating a NAD(P)H4 cation radical and anionic FMN semiquinone. This electron transfer occurs in ∼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over ∼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.- Published
- 2023
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26. Co-production of biofuel, bioplastics and biochemicals during extended fermentation of Halomonas bluephagenesis.
- Author
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Park H, Toogood HS, Chen GQ, and Scrutton NS
- Subjects
- Fermentation, Biofuels, Propane, Hydroxybutyrates, Polyesters metabolism, Biopolymers, Halomonas genetics, Halomonas metabolism
- Abstract
Halomonas bluephagenesis TD1.0 was engineered to produce the biofuel propane, bioplastic poly-3-hydroxybutyrate (PHB), and biochemicals mandelate and hydroxymandelate in a single, semi-continuous batch fermentation under non-sterile conditions. Multi-product separation was achieved by segregation of the headspace gas (propane), fermentation broth ([hydroxy]mandelate) and cellular biomass (PHB). Engineering was performed by incorporating the genes encoding fatty acid photodecarboxylase (CvFAP) and hydroxymandelic acid synthase (SyHMAS) into a H. bluephagenesis hmgCAB cassette knockout to channel flux towards (hydroxy)mandelate. Design of Experiment strategies were coupled with fermentation trials to simultaneously optimize each product. Propane and mandelate titres were the highest reported for H. bluephagenesis (62 g/gDCW and 71 ± 10 mg/L respectively) with PHB titres (69% g/gDCW) comparable to other published studies. This proof-of-concept achievement of four easily separated products within one fermentation is a novel achievement probing the versatility of biotechnology, further elevating H. bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis by producing highly valued products at a reduced cost., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)
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- 2023
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27. Mechanism of Action of Flavin-Dependent Halogenases.
- Author
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Barker RD, Yu Y, De Maria L, Johannissen LO, and Scrutton NS
- Abstract
To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an -NH
2 Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory "cluster" models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)- Published
- 2022
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28. Mutation in a chlorophyll-binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis.
- Author
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Liu M, Ma W, Su X, Zhang X, Lu Y, Zhang S, Yan J, Feng D, Ma L, Taylor A, Ge Y, Cheng Q, Xu K, Wang Y, Li N, Gu A, Zhang J, Luo S, Xuan S, Chen X, Scrutton NS, Li C, Zhao J, and Shen S
- Subjects
- Heme, Chlorophyll A, Mutation genetics, Ferrochelatase genetics, Brassica genetics
- Abstract
The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2022
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29. Editorial overview: Catalysis and regulation: The chemistry of catalysis.
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Scrutton NS and Fujimori DG
- Subjects
- Catalysis
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- 2022
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30. Catalysis by Nature's photoenzymes.
- Author
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Taylor A, Heyes DJ, and Scrutton NS
- Subjects
- Humans, Catalysis, Flavins chemistry
- Abstract
Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biological photocatalysis., Competing Interests: Declaration of competing interest The authors confirm there are no conflicts of interest with this submission., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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31. How a 10- epi -Cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity.
- Author
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Whitehead JN, Leferink NGH, Komati Reddy G, Levy CW, Hay S, Takano E, and Scrutton NS
- Abstract
Terpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10- epi -cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature's most difficult chemistry. 10- epi -Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10- epi -cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
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- 2022
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32. Combined Pulsed Electron Double Resonance EPR and Molecular Dynamics Investigations of Calmodulin Suggest Effects of Crowding Agents on Protein Structures.
- Author
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Stewart AM, Shanmugam M, Kutta RJ, Scrutton NS, Lovett JE, and Hay S
- Subjects
- Calcium metabolism, Electrons, Protein Binding, Protein Conformation, Spin Labels, Calmodulin chemistry, Molecular Dynamics Simulation
- Abstract
Calmodulin (CaM) is a highly dynamic Ca
2+ -binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+ -replete form has been highly debated. In this study, we use PELDOR (pulsed electron-electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+ -bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+ -replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+ -replete CaM similar to the one observed in early X-ray crystal structures.- Published
- 2022
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33. Baseline proteomics characterisation of the emerging host biomanufacturing organism Halomonas bluephagenesis.
- Author
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Russell M, Currin A, Rowe W, Chen GQ, Barran P, and Scrutton NS
- Subjects
- Escherichia coli, Gene Library, Proteomics, Halomonas genetics, Halomonas metabolism, Proteome genetics
- Abstract
Despite its greener credentials, biomanufacturing remains financially uncompetitive compared with the higher carbon emitting, hydrocarbon-based chemical industry. Replacing traditional chassis such as E. coli with novel robust organisms, are a route to cost reduction for biomanufacturing. Extremophile bacteria such as the halophilic Halomonas bluephagenesis TD01 exemplify this potential by thriving in environments inherently inimical to other organisms, so reducing sterilisation costs. Novel chassis are inevitably less well annotated than established organisms. Rapid characterisation along with community data sharing will facilitate adoption of such organisms for biomanufacturing. The data record comprises a newly sequenced genome for the organism and evidence via LC-MS based proteomics for expression of 1160 proteins (30% of the proteome) including baseline quantification of 1063 proteins (27% of the proteome), and a spectral library enabling re-use for targeted LC-MS proteomics assays. Protein data are annotated with KEGG Orthology, enabling rapid matching of quantitative data to pathways of interest to biomanufacturing., (© 2022. The Author(s).)
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- 2022
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34. Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.
- Author
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Hedison TM, Iorgu AI, Calabrese D, Heyes DJ, Shanmugam M, and Scrutton NS
- Subjects
- Nitrite Reductases chemistry, Nitrite Reductases metabolism, Oxidation-Reduction, Spectrum Analysis, Copper chemistry, Electrons
- Abstract
Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron-electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.
- Published
- 2022
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35. Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.
- Author
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Rose SL, Baba S, Okumura H, Antonyuk SV, Sasaki D, Hedison TM, Shanmugam M, Heyes DJ, Scrutton NS, Kumasaka T, Tosha T, Eady RR, Yamamoto M, and Hasnain SS
- Subjects
- Catalysis, Oxidation-Reduction, Spectrum Analysis, Copper chemistry, Nitrite Reductases chemistry, Nitrites chemistry
- Abstract
Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species ( Br
2D NiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.- Published
- 2022
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36. Bioproduction of Linalool From Paper Mill Waste.
- Author
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Rinaldi MA, Tait S, Toogood HS, and Scrutton NS
- Abstract
A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A "cookie-cutter" approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both 'hit and miss' outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development., Competing Interests: C3 Biotechnologies Ltd partially funded the work. NSS is a founding director and HST a share holder of C3 Biotechnologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rinaldi, Tait, Toogood and Scrutton.)
- Published
- 2022
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37. How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis.
- Author
-
Johannissen LO, Taylor A, Hardman SJO, Heyes DJ, Scrutton NS, and Hay S
- Abstract
The photochemical reaction catalyzed by enzyme protochlorophyllide oxidoreductase (POR), a rare example of a photoactivated enzyme, is a crucial step during chlorophyll biosynthesis and involves the fastest known biological hydride transfer. Structures of the enzyme with bound substrate protochlorophyllide (PChlide) and coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) have recently been published, opening up the possibility of using computational approaches to provide a comprehensive understanding of the excited state chemistry. Herein, we propose a complete mechanism for the photochemistry between PChlide and NADPH based on density functional theory (DFT) and time-dependent DFT calculations that is consistent with recent experimental data. In this multi-step mechanism, photoexcitation of PChlide leads to electron transfer from NADPH to PChlide, which in turn facilitates hydrogen atom transfer by weakening the breaking C-H bond. This work rationalizes how photoexcitation facilitates hydride transfer in POR and has more general implications for biological hydride transfer reactions., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)
- Published
- 2022
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38. Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches.
- Author
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Leferink NGH and Scrutton NS
- Subjects
- Catalytic Domain, Cyclization, Terpenes metabolism, Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism
- Abstract
Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence-function relationship within the TS family, making product-outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high-throughput screening, and machine-learning that will allow more predictive engineering of these fascinating enzymes in the near future., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
- Published
- 2022
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39. Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases.
- Author
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Leferink NGH, Escorcia AM, Ouwersloot BR, Johanissen LO, Hay S, van der Kamp MW, and Scrutton NS
- Subjects
- Catalytic Domain, Cyclization, Eucalyptol
- Abstract
Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α-terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation-π interactions., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
- Published
- 2022
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40. A guide to time-resolved structural analysis of light-activated proteins.
- Author
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Poddar H, Heyes DJ, Schirò G, Weik M, Leys D, and Scrutton NS
- Subjects
- Lasers, Light, Models, Molecular, Proteins chemistry, Proteins radiation effects, Time Factors, X-Ray Diffraction, Crystallography, X-Ray, Protein Conformation radiation effects, Proteins ultrastructure
- Abstract
Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2022
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41. Alternative metabolic pathways and strategies to high-titre terpenoid production in Escherichia coli .
- Author
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Rinaldi MA, Ferraz CA, and Scrutton NS
- Subjects
- Escherichia coli metabolism, Metabolic Engineering methods, Metabolic Networks and Pathways, Terpenes metabolism
- Abstract
Covering: up to 2021Terpenoids are a diverse group of chemicals used in a wide range of industries. Microbial terpenoid production has the potential to displace traditional manufacturing of these compounds with renewable processes, but further titre improvements are needed to reach cost competitiveness. This review discusses strategies to increase terpenoid titres in Escherichia coli with a focus on alternative metabolic pathways. Alternative pathways can lead to improved titres by providing higher orthogonality to native metabolism that redirects carbon flux, by avoiding toxic intermediates, by bypassing highly-regulated or bottleneck steps, or by being shorter and thus more efficient and easier to manipulate. The canonical 2- C -methyl-D-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways are engineered to increase titres, sometimes using homologs from different species to address bottlenecks. Further, alternative terpenoid pathways, including additional entry points into the MEP and MVA pathways, archaeal MVA pathways, and new artificial pathways provide new tools to increase titres. Prenyl diphosphate synthases elongate terpenoid chains, and alternative homologs create orthogonal pathways and increase product diversity. Alternative sources of terpenoid synthases and modifying enzymes can also be better suited for E. coli expression. Mining the growing number of bacterial genomes for new bacterial terpenoid synthases and modifying enzymes identifies enzymes that outperform eukaryotic ones and expand microbial terpenoid production diversity. Terpenoid removal from cells is also crucial in production, and so terpenoid recovery and approaches to handle end-product toxicity increase titres. Combined, these strategies are contributing to current efforts to increase microbial terpenoid production towards commercial feasibility.
- Published
- 2022
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42. An unusual light-sensing function for coenzyme B 12 in bacterial transcription regulator CarH.
- Author
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Poddar H, Heyes DJ, Zhang S, Hardman SJ, Sakuma M, and Scrutton NS
- Subjects
- Cobamides chemistry, Cobamides genetics, Cobamides metabolism, DNA metabolism, Phosphothreonine analogs & derivatives, Thermus thermophilus genetics, Thermus thermophilus metabolism, Vitamin B 12 metabolism, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial
- Abstract
Coenzyme B
12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12 . It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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43. GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes.
- Author
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Green L, Scrutton NS, and Currin A
- Subjects
- Codon genetics, Gene Library, Mutagenesis, Mutagenesis, Site-Directed, Directed Molecular Evolution methods, Proteins
- Abstract
Directed evolution is a powerful tool for the rapid improvement of a target protein toward a desired fitness criteria, such as activity, specificity, or stability. In order to achieve these desired improvements, it is often beneficial to subject the entirety of the protein to mutagenesis. However, the creation of such libraries by targeted methods (i.e. site-directed mutagenesis) can be a laborious and costly task. Here we outline the GeneORator method, which uses Boolean "OR" logic to introduce specific codon mutations at multiple loci in a single reaction, thereby greatly reducing the experimental workload. The method describes library synthesis using asymmetric PCR, in which mutagenic primers are designed to create OR-type mutations at multiple sites of variation in a two-step protocol. As an example, we show how this can be utilized for controlled and economical mutagenesis of every amino acid codon in a gene., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
- Full Text
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44. Combinatorial use of environmental stresses and genetic engineering to increase ethanol titres in cyanobacteria.
- Author
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Andrews F, Faulkner M, Toogood HS, and Scrutton NS
- Abstract
Current industrial bioethanol production by yeast through fermentation generates carbon dioxide. Carbon neutral bioethanol production by cyanobacteria uses biological fixation (photosynthesis) of carbon dioxide or other waste inorganic carbon sources, whilst being sustainable and renewable. The first ethanologenic cyanobacterial process was developed over two decades ago using Synechococcus elongatus PCC 7942, by incorporating the recombinant pdc and adh genes from Zymomonas mobilis. Further engineering has increased bioethanol titres 24-fold, yet current levels are far below what is required for industrial application. At the heart of the problem is that the rate of carbon fixation cannot be drastically accelerated and carbon partitioning towards bioethanol production impacts on cell fitness. Key progress has been achieved by increasing the precursor pyruvate levels intracellularly, upregulating synthetic genes and knocking out pathways competing for pyruvate. Studies have shown that cyanobacteria accumulate high proportions of carbon reserves that are mobilised under specific environmental stresses or through pathway engineering to increase ethanol production. When used in conjunction with specific genetic knockouts, they supply significantly more carbon for ethanol production. This review will discuss the progress in generating ethanologenic cyanobacteria through chassis engineering, and exploring the impact of environmental stresses on increasing carbon flux towards ethanol production., (© 2021. The Author(s).)
- Published
- 2021
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45. Flavin oxidation state impacts on nitrofuran antibiotic binding orientation in nitroreductases.
- Author
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Toogood HS and Scrutton NS
- Subjects
- Escherichia coli metabolism, Flavins, Kinetics, Nitroreductases metabolism, Oxidation-Reduction, Anti-Bacterial Agents, Nitrofurans
- Abstract
Nitroreductases catalyse the NAD(P)H-dependent nitro reduction in nitrofuran antibiotics, which activates them into cytotoxic molecules leading to cell death. The design of new effective nitrofuran antibiotics relies on knowledge of the kinetic mechanism and nitrofuran binding mode of microbial nitroreductases NfsA and NfsB. This has been hampered by multiple co-crystallisation studies revealing ligand binding in non-electron transfer competent states. In a recent study by Day et al. (2021) the authors investigated the likely reaction mechanism and mode of nitrofurantoin binding to NfsA using potentiometry, global kinetics analysis, crystallography and molecular dynamics simulations. Their findings suggest nitrofurantoin reduction proceeds via a direct hydride transfer from reduced FMN, while the crystallographic binding orientation is an inhibitory complex. Molecular dynamics simulations suggest ligand binding orientations is dependent on the oxidation state of the FMN. This study highlights the importance of utilising computational studies alongside traditional crystallographic approaches, when multiple stable ligand binding orientations can occur., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2021
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46. Blood, sweat, and tears: extraterrestrial regolith biocomposites with in vivo binders.
- Author
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Roberts AD, Whittall DR, Breitling R, Takano E, Blaker JJ, Hay S, and Scrutton NS
- Abstract
The proverbial phrase 'you can't get blood from a stone' is used to describe a task that is practically impossible regardless of how much force or effort is exerted. This phrase is well-suited to humanity's first crewed mission to Mars, which will likely be the most difficult and technologically challenging human endeavor ever undertaken. The high cost and significant time delay associated with delivering payloads to the Martian surface means that exploitation of resources in situ - including inorganic rock and dust (regolith), water deposits, and atmospheric gases - will be an important part of any crewed mission to the Red Planet. Yet there is one significant, but chronically overlooked, source of natural resources that will - by definition - also be available on any crewed mission to Mars: the crew themselves. In this work, we explore the use of human serum albumin (HSA) - a common protein obtained from blood plasma - as a binder for simulated Lunar and Martian regolith to produce so-called 'extraterrestrial regolith biocomposites (ERBs).' In essence, HSA produced by astronauts in vivo could be extracted on a semi-continuous basis and combined with Lunar or Martian regolith to 'get stone from blood', to rephrase the proverb. Employing a simple fabrication strategy, HSA-based ERBs were produced and displayed compressive strengths as high as 25.0 MPa. For comparison, standard concrete typically has a compressive strength ranging between 20 and 32 MPa. The incorporation of urea - which could be extracted from the urine, sweat, or tears of astronauts - could further increase the compressive strength by over 300% in some instances, with the best-performing formulation having an average compressive strength of 39.7 MPa. Furthermore, we demonstrate that HSA-ERBs have the potential to be 3D-printed, opening up an interesting potential avenue for extraterrestrial construction using human-derived feedstocks. The mechanism of adhesion was investigated and attributed to the dehydration-induced reorganization of the protein secondary structure into a densely hydrogen-bonded, supramolecular β-sheet network - analogous to the cohesion mechanism of spider silk. For comparison, synthetic spider silk and bovine serum albumin (BSA) were also investigated as regolith binders - which could also feasibly be produced on a Martian colony with future advancements in biomanufacturing technology., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)
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- 2021
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47. The evolving art of creating genetic diversity: From directed evolution to synthetic biology.
- Author
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Currin A, Parker S, Robinson CJ, Takano E, Scrutton NS, and Breitling R
- Subjects
- Biotechnology, Genetic Variation genetics, Phenotype, Directed Molecular Evolution, Synthetic Biology
- Abstract
The ability to engineer biological systems, whether to introduce novel functionality or improved performance, is a cornerstone of biotechnology and synthetic biology. Typically, this requires the generation of genetic diversity to explore variations in phenotype, a process that can be performed at many levels, from single molecule targets (i.e., in directed evolution of enzymes) to whole organisms (e.g., in chassis engineering). Recent advances in DNA synthesis technology and automation have enhanced our ability to create variant libraries with greater control and throughput. This review highlights the latest developments in approaches to create such a hierarchy of diversity from the enzyme level to entire pathways in vitro, with a focus on the creation of combinatorial libraries that are required to navigate a target's vast design space successfully to uncover significant improvements in function., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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48. Design and fabrication of recombinant reflectin-based multilayer reflectors: bio-design engineering and photoisomerism induced wavelength modulation.
- Author
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Wolde-Michael E, Roberts AD, Heyes DJ, Dumanli AG, Blaker JJ, Takano E, and Scrutton NS
- Abstract
The remarkable camouflage capabilities of cephalopods have inspired many to develop dynamic optical materials which exploit certain design principles and/or material properties from cephalopod dermal cells. Here, the angle-dependent optical properties of various single-layer reflectin thin-films on Si wafers are characterized within the UV-Vis-NIR regions. Following this, initial efforts to design, fabricate, and optically characterize a bio-inspired reflectin-based multilayer reflector is described, which was found to conserve the optical properties of single layer films but exhibit reduced angle-dependent visible reflectivity. Finally, we report the integration of phytochrome visible light-induced isomerism into reflectin-based films, which was found to subtly modulate reflectin thin-film reflectivity., (© 2021. The Author(s).)
- Published
- 2021
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49. Insights into the H 2 O 2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.
- Author
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Hedison TM, Breslmayr E, Shanmugam M, Karnpakdee K, Heyes DJ, Green AP, Ludwig R, Scrutton NS, and Kracher D
- Subjects
- Biocatalysis, Cellulose metabolism, Electron Spin Resonance Spectroscopy methods, Fungal Proteins genetics, Glucans metabolism, Mixed Function Oxygenases genetics, Neurospora crassa genetics, Oxidation-Reduction, Polysaccharides metabolism, Protein Binding, Recombinant Proteins metabolism, Spectrophotometry methods, Substrate Specificity, Xylans metabolism, Fungal Polysaccharides metabolism, Fungal Proteins metabolism, Hydrogen Peroxide metabolism, Mixed Function Oxygenases metabolism, Neurospora crassa enzymology
- Abstract
Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H
2 O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2 O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2 O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2 O2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H2 O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2 O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2 O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)- Published
- 2021
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50. Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase.
- Author
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Ferraz CA, Leferink NGH, Kosov I, and Scrutton NS
- Subjects
- Acyclic Monoterpenes metabolism, Amino Acid Sequence, Escherichia coli genetics, Hemiterpenes metabolism, Mevalonic Acid metabolism, Pentanols metabolism, Protein Conformation, Protein Engineering, Signal Transduction, Streptomyces enzymology, Terpenes metabolism, Transferases genetics, Acyclic Monoterpenes chemistry, Pentanols chemistry, Sesquiterpenes metabolism, Transferases metabolism
- Abstract
Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
- Published
- 2021
- Full Text
- View/download PDF
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