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  • 1
    Online Resource
    Online Resource
    Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host
    eLife Sciences Publications, Ltd ; 2022
    In:  eLife Vol. 11 ( 2022-03-04)
    Details
    In: eLife, eLife Sciences Publications, Ltd, Vol. 11 ( 2022-03-04)
    Abstract: Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S -adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli . Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.
    Type of Medium: Online Resource
    ISSN: 2050-084X
    URL: Article
    DOI: 10.7554/eLife.70936
    Language: English
    Publisher: eLife Sciences Publications, Ltd
    Publication Date: 2022
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  • 2
    Online Resource
    Online Resource
    Supplementation of Intracellular XylR Leads to Coutilization of Hemicellulose Sugars
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 7 ( 2012-04), p. 2221-2229
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 7 ( 2012-04), p. 2221-2229
    Abstract: Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy of xylR was inserted into the araBAD operon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of an E. coli biocatalyst that can simultaneously convert all biomass sugars.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.06761-11
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Dynamic binding orientations direct activity of HIV reverse transcriptase
    Springer Science and Business Media LLC ; 2008
    In:  Nature Vol. 453, No. 7192 ( 2008-5), p. 184-189
    Details
    In: Nature, Springer Science and Business Media LLC, Vol. 453, No. 7192 ( 2008-5), p. 184-189
    Type of Medium: Online Resource
    ISSN: 0028-0836 , 1476-4687
    URL: Article
    DOI: 10.1038/nature06941
    RVK:
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    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2008
    detail.hit.zdb_id: 120714-3
    detail.hit.zdb_id: 1413423-8
    SSG: 11
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  • 4
    Online Resource
    Online Resource
    Single-Molecule RNA Folding
    Wiley ; 2005
    In:  ChemInform Vol. 36, No. 39 ( 2005-09-27)
    Details
    In: ChemInform, Wiley, Vol. 36, No. 39 ( 2005-09-27)
    Type of Medium: Online Resource
    ISSN: 0931-7597 , 1522-2667
    URL: Issue
    URL: Journal
    URL: Article
    DOI: 10.1002/chin.v36:39
    DOI: 10.1002/(ISSN)1522-2667
    DOI: 10.1002/chin.200539283
    Language: English
    Publisher: Wiley
    Publication Date: 2005
    detail.hit.zdb_id: 2110203-X
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  • 5
    Online Resource
    Online Resource
    Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance
    Proceedings of the National Academy of Sciences ; 2004
    In:  Proceedings of the National Academy of Sciences Vol. 101, No. 27 ( 2004-07-06), p. 10066-10071
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 101, No. 27 ( 2004-07-06), p. 10066-10071
    Abstract: The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0403575101
    RVK:
    TA 1000
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2004
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 6
    Online Resource
    Online Resource
    Single-molecule transition-state analysis of RNA folding
    Proceedings of the National Academy of Sciences ; 2003
    In:  Proceedings of the National Academy of Sciences Vol. 100, No. 16 ( 2003-08-05), p. 9302-9307
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 100, No. 16 ( 2003-08-05), p. 9302-9307
    Abstract: How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1133280100
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2003
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli
    Proceedings of the National Academy of Sciences ; 2011
    In:  Proceedings of the National Academy of Sciences Vol. 108, No. 50 ( 2011-12-13), p. 19949-19954
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 50 ( 2011-12-13), p. 19949-19954
    Abstract: One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli , to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1106958108
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2011
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 8
    Online Resource
    Online Resource
    Posttranslational Control of PlsB Is Sufficient To Coordinate Membrane Synthesis with Growth in Escherichia coli
    American Society for Microbiology ; 2020
    In:  mBio Vol. 11, No. 4 ( 2020-08-25)
    Details
    In: mBio, American Society for Microbiology, Vol. 11, No. 4 ( 2020-08-25)
    Abstract: Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis. IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli . Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.02703-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 2557172-2
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  • 9
    Online Resource
    Online Resource
    Microbial production of fatty-acid-derived fuels and chemicals from plant biomass
    Springer Science and Business Media LLC ; 2010
    In:  Nature Vol. 463, No. 7280 ( 2010-01-28), p. 559-562
    Details
    In: Nature, Springer Science and Business Media LLC, Vol. 463, No. 7280 ( 2010-01-28), p. 559-562
    Type of Medium: Online Resource
    ISSN: 0028-0836 , 1476-4687
    URL: Article
    DOI: 10.1038/nature08721
    RVK:
    TA 1000
    RVK:
    UA 1000
    RVK:
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    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2010
    detail.hit.zdb_id: 120714-3
    detail.hit.zdb_id: 1413423-8
    SSG: 11
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  • 10
    Online Resource
    Online Resource
    Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need
    MDPI AG ; 2021
    In:  Molecules Vol. 26, No. 22 ( 2021-11-17), p. 6930-
    Details
    In: Molecules, MDPI AG, Vol. 26, No. 22 ( 2021-11-17), p. 6930-
    Abstract: Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.
    Type of Medium: Online Resource
    ISSN: 1420-3049
    URL: Article
    DOI: 10.3390/molecules26226930
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2008644-1
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