497 research outputs found
Microfluidic droplets as tools for high-throughput biology: Enzyme evolution, recruitment and discovery based on catalytic promiscuity
âPromiscuousâ enzymes possess additional activities in addition to their native ones, challenging the textbook adage âone enzyme â one activityâ. The observation of strong promiscuous activities in the alkaline phosphatase (AP) superfamily - where one active site can catalyse up to six chemically distinct hydrolytic reactions with promiscuous second order rate accelerations between 10e9 and 10e17 - suggests that even broadly promiscuous catalysis can be rather efficient. To efficiently explore the interconversion of promiscuous enzyme, we use picoliter water-inâoil emulsion droplets produced in microfluidic devices as high-throughput screening reactors. We present new workflows that allow screening of \u3e10e6 clones and permit successful selections from single protein and metagenomic libraries, where lower throughput approaches have failed.
⢠Ultrahigh-throughput Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics. Colin, P.-Y.; Kintses, B.; Gielen, F.; Miton, C. M.; Mohamed, M. F.; Fischer, G.; Hyvonen, M.; Morgavi, D. P.; Janssen, D. B.; Hollfelder, F., Nature Communications 2015, 6:10008. doi: 10.1038/ncomms10008.
⢠Enzyme engineering in biomimetic compartments. Colin, P. Y, Zinchenko, A & Hollfelder, F. Curr Opin Struct Biol. 2015, 33:42-51(doi: 10.1016/j.sbi.2015.06.001)
⢠Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Fischlechner, M.; Schaerli, Y.; Mohamed, M. F.; Patil, S.; Abell, C.; Hollfelder, F., Nat Chem 2014, 6 (9), 791-6
⢠Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F. Proc Natl Acad Sci U S A. 2016 Nov 22;113(47):E7383-E7389
Where do we find new enzymes? â Rules and tools
Enzymes for a variety of useful applications are required, but currently not known. Functional metagenomics and directed evolution promise access to such new catalysts, but the chances of finding them is low. Therefore high-throughput technologies are crucial to fight the odds. We have implemented workflows that allow screening of \u3e10e6 clones and permit successful selections in picoliter water-inâoil emulsion droplets produced in microfluidic devices. While potentially faster, the vastness of sequence space (and the scarcity of âsolutionsâ in it) require strategies and rules for the identification and interconversion of enzymes. In this context âpromiscuousâ enzymes have prominent roles: these catalysts possess additional activities in addition to their native ones, challenging the textbook adage âone enzyme â one activityâ, yet facilitating âon-the-spotâ utility for newly encountered challenges and also making future evolutionary transitions easier. Their molecular and mechanistic understanding may help us thus to chart routes towards new catalysts. Recent results of directed evolution, mechanistic investigation by kinetic and structural analysis and technology development are discussed in this context.
⢠van Loo, B.; Bayer, C. D.; Fischer, G.; Jonas, S.; Valkov, E.; Mohamed, M. F.; Vorobieva, A.; Dutruel, C.; Hyvonen, M.; Hollfelder, F., Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily. J Am Chem Soc 2019, 141 (1), 370-387.
⢠van Loo, B.; Berry, R.; Boonyuen, U.; Mohamed, M. F.; Golicnik, M.; Hengge, A. C.; Hollfelder, F., Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase. Biochemistry 2019, 58 (10), 1363-1378.
⢠Miton, C. M.; Jonas, S.; Fischer, G.; Duarte, F.; Mohamed, M. F.; van Loo, B.; Kintses, B.; Kamerlin, S. C. L.; Tokuriki, N.; Hyvonen, M.; Hollfelder, F., Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset. Proc Natl Acad Sci U S A 2018, 115 (31), E7293-E7302.
⢠Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F. Proc Natl Acad Sci U S A. 2016;113(47):E7383-E7389.
⢠Colin, P.-Y.; Kintses, B.; Gielen, F.; Miton, C. M.; Mohamed, M. F.; Fischer, G.; Hyvonen, M.; Morgavi, D. P.; Janssen, D. B.; Hollfelder, F., Ultrahigh-throughput Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics. Nature Communications 2015, 6:10008. doi: 10.1038/ncomms10008.
⢠Enzyme engineering in biomimetic compartments. Curr Opin Struct Biol. 2015, 33:42-51(doi: 10.1016/j.sbi.2015.06.001)
⢠Fischlechner, M.; Schaerli, Y.; Mohamed, M. F.; Patil, S.; Abell, C.; Hollfelder, F., Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat Chem 2014, 6 (9), 791-
An efficient method to assemble linear DNA templates for in vitro screening and selection systems.
A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl. The method is exemplified with the gene coding for a mutant version of O(6)-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles
Enzyme evolution and engineering using insertions and deletions
In Nature, proteins evolve and acquire new functions by accumulating mutations. Substitutions and InDels (Insertions and Deletions), as well as circular permutations and rearrangement of protein domains, account for the majority of evolutionary changes. While the effects of substitutions have been extensively studied and documented, understanding the structural and functional effects of InDels still remains a challenge. InDels are assumed to be highly deleterious mutations because they are more likely to disrupt the structural integrity of proteins than are substitutions. On the other hand, they may induce significant structural changes that substitutions alone cannot cause and thus are believed to be key players in many natural evolutionary processes, such as the modification of active site loops to generate new enzyme functions1 or the emergence of new protein structures2.
We aimed at performing directed evolution by randomly incorporating InDels to investigate how they would be tolerated and whether they could be selected for functional improvements. Starting from a previously reported methodology3, we developed a library construction approach to randomly incorporate InDels within a DNA sequence of interest and applied it to generate InDel variant libraries of a promiscuous enzyme (phosphotriesterase4). We screened the resulting libraries (i) to compare the impact of InDels to that of substitutions on the enzyme, (ii) to identify adaptive InDels improving a new (or promiscuous) activity and (iii) to investigate the interaction between InDels and substitutions in an adaptive process. Our results show that, while being generally more deleterious than substitutions, InDels can also lead to functional improvements and may allow access to alternative evolutionary trajectories.
References
1 Park et al. (2006). Science 311, 535â538.
2 Grishin (2001). J Struct Biol 134:167â185.
3 Jones et al. (2014). Methods in Mol Biol 1179:159-72.
4 Tokuriki et al. (2012). Nature Comm 3:1257
Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.
The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Discovery Grant RGPIN 418262- 12, http://www.nserc-crsng.gc.ca/), the Biotechnology and Biological Sciences Research Council (BBSRC, Grant BB/L002469/1, http://www. bbsrc.ac.uk/), the European Research Council (ERC, Advanced Investigator Grant 695669, https:// erc.europa.eu/), and the Human Frontiers Science Program (Grant RGP0006/2013, http://www.hfsp. org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip
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Enzyme engineering in biomimetic compartments.
The success of a directed evolution approach to creating custom-made enzymes relies in no small part on screening as many clones as possible. The miniaturisation of assays into pico to femtoliter compartments (emulsion droplets, vesicles or gel-shell beads) makes directed evolution campaigns practically more straightforward than current large scale industrial screening that requires liquid handling equipment and much manpower. Several recent experimental formats have established protocols to screen more than 10 million compartments per day, representing unprecedented throughput at low cost. This review introduces the emerging approaches towards making biomimetic man-made compartments that are poised to be adapted by a wider circle of researchers. In addition to cost and time saving, control of selection pressures and conditions, the quantitative readout that reports on every library members and the ability to develop strategies based on these data will increase the degrees of freedom in designing and testing strategies for directed evolution experiments.Our research was funded by the Biological and Biotechnological Research Council (BBSRC) and the Engineering and Physical Sciences Research Council (EPSRC). FH is an ERC Starting Investigator, PYC and AZ were supported by the EU Marie-Curie training network PhosChemRec. AZ received further support from the BBSRC and the Cambridge European Trust.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.sbi.2015.06.00
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Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput.
Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme
Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.
In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.G.H. was supported by a CASE studentship from the Engineering and Physical Sciences Research Council and MedImmune and the Marie-Curie Research Training Network ENEFP. F.H. is a starting investigator of the European Research Council. Funding to pay the Open Access publication charges for this article was provided by the Engineering and Physical Sciences Research Council.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/protein/gzv02
Konfliktpotenziale zwischen Alt und Jung im deutschen Wohlfahrtsstaat: eine quantitative Analyse des European Social Survey (ESS)
Bedingt durch eine niedrige Geburtenziffer und eine steigende Lebenserwartung, fßhrt der demografische Wandel in Deutschland zu Finanzierungslßcken in den sozialen Sicherungssystemen und zu einer politischen Machtverschiebung zu Gunsten der älteren Jahrgänge. Daraus wird oft vorschnell ein Konflikt
der Generationen abgeleitet, ohne dass eine empirische Prßfung der dafßr notwendigen sozialen Grundlagen stattgefunden hätte.
In dieser Arbeit wird deshalb ein Modell entwickelt, das die Entstehung eines Konfliktes
zwischen Alt und Jung skizziert, um anschlieĂend einzelne Aspekte der Genese des Konfliktes
empirisch zu ĂźberprĂźfen. Dazu werden drei Einstellungsvariablen analysiert: die Verantwortlichkeit
des Staates fßr die Alterssicherung, die Einschätzung des Lebensstandards im Ruhestand
und die erwartete Entwicklung des
Rentenniveaus. Als Datengrundlage dient die deutsche Teilstudie der 4. Welle des European Social Survey. Die Analysen zeigen, dass bezĂźglich der untersuchten wohlfahrtsstaatlichen Einstellungen kein nennenswertes Konfliktpotenzial zwischen Alt und Jung festgestellt werden
kann. MĂśgliche Konfliktlinien lassen sich vielmehr entlang des Bildungsstatus oder der
subjektiven Lebenszufriedenheit ausmachen. Zudem sind, in schwächerem MaĂe, altersunabhängige subjektive Wertorientierungen und
politische Einstellungen konstitutiv fĂźr die
Einstellungsbildung. Aus den Ergebnissen lässt sich schlieĂen, dass in der BevĂślkerung gegenwärtig keine Grundlage fĂźr einen Konflikt zwischen Alt und Jung auf Gesellschaftsebene
existiert
Bioinspired genotype-phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins.
The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments-water-in-oil droplets and gel-shell beads-for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components
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