The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

Angewandte Chemie International Edition, Journal Year: 2019, Volume and Issue: 59(32), P. 13204 - 13231

Published: July 3, 2019

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry biotechnology. In order this protein engineering technique be efficient, fast, reliable, also relevance synthetic chemistry, methodology development was still is necessary. Following description early key contributions, review focuses on recent developments. It includes optimization molecular biological methods gene mutagenesis the design efficient strategies their application, resulting notable reduction screening effort (bottleneck directed evolution). When aiming laboratory selectivity activity, second-generation versions Combinatorial Active-Site Saturation Test (CAST) Iterative Mutagenesis (ISM), both involving saturation (SM) at sites lining binding pocket, have emerged as preferred approaches, aided by silico such machine learning. The recently proposed Focused Rational Site-specific (FRISM) fusion rational evolution. On-chip solid-phase chemical synthesis rapid library construction enhances quality notably eliminating undesired amino acid bias, future evolution?

Language: Английский

---

Power of Biocatalysis for Organic Synthesis

ACS Central Science, Journal Year: 2021, Volume and Issue: 7(1), P. 55 - 71

Published: Jan. 14, 2021

Biocatalysis, using defined enzymes for organic transformations, has become a common tool in synthesis, which is also frequently applied industry. The generally high activity and outstanding stereo-, regio-, chemoselectivity observed many biotransformations are the result of precise control reaction active site biocatalyst. This achieved by exact positioning reagents relative to each other fine-tuned 3D environment, specific activating interactions between protein, subtle movements catalyst. Enzyme engineering enables one adapt catalyst desired process. A well-filled biocatalytic toolbox ready be used various reactions. Providing nonnatural conditions evolving biocatalysts play with myriad options creating novel transformations thereby opening new, short pathways target molecules. Combining several pot perform reactions concurrently increases efficiency biocatalysis even further.

Language: Английский

---

Recent trends in biocatalysis

Chemical Society Reviews, Journal Year: 2021, Volume and Issue: 50(14), P. 8003 - 8049

Published: Jan. 1, 2021

Technological developments enable the discovery of novel enzymes, advancement enzyme cascade designs and pathway engineering, moving biocatalysis into an era technology integration, intelligent manufacturing enzymatic total synthesis.

Language: Английский

---

Deep learning-based kcat prediction enables improved enzyme-constrained model reconstruction

Nature Catalysis, Journal Year: 2022, Volume and Issue: 5(8), P. 662 - 672

Published: June 16, 2022

Abstract Enzyme turnover numbers ( k cat ) are key to understanding cellular metabolism, proteome allocation and physiological diversity, but experimentally measured data sparse noisy. Here we provide a deep learning approach (DLKcat) for high-throughput prediction metabolic enzymes from any organism merely substrate structures protein sequences. DLKcat can capture changes mutated identify amino acid residues with strong impact on values. We applied this predict genome-scale values more than 300 yeast species. Additionally, designed Bayesian pipeline parameterize enzyme-constrained models predicted The resulting outperformed the corresponding original previous pipelines in predicting phenotypes proteomes, enabled us explain phenotypic differences. model construction valuable tools uncover global trends of enzyme kinetics further elucidate metabolism large scale.

Language: Английский

---

Enzyme discovery and engineering for sustainable plastic recycling

Trends in biotechnology, Journal Year: 2021, Volume and Issue: 40(1), P. 22 - 37

Published: March 3, 2021

Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment recycling, which aims to reduce adverse environmental effects recover valuable components from waste.Metagenomic proteomic approaches can be harnessed powerful tools in mining capable of a wide variety environments ecosystems.Plastic-degrading optimized protein engineering improved performance, including enhancement enzyme thermostability, reinforcement the binding substrate active site, interaction between surface, refinement catalytic capacity. The drastically increasing amount waste is causing crisis that requires innovative technologies recycling post-consumer plastics achieve valorization while meeting quality goals. recycling. A plastic-degrading have been discovered microbial sources. Meanwhile, exploited modify optimize enzymes. This review highlights recent trends up-to-date advances novel through state-of-the-art omics-based techniques improving efficiency stability via various strategies. Future research prospects challenges are also discussed. Plastic materials play revolutionary role modern world, although enormous manufacture extensive use commodities inevitably generate extraordinary waste. Around 12 000 million metric tons predicted accumulate landfills natural environment 2050 [1.Geyer R. et al.Production, use, fate all ever made.Sci. Adv. 2017; 3e1700782Crossref PubMed Scopus (3209) Google Scholar]. Improper handling caused grand challenge. debris waste, especially microplastics (see Glossary), impose hazardous on organisms eventually threaten human well-being [2.Redondo-Hasselerharm P.E. al.Nano- affect composition freshwater benthic communities long term.Sci. 2020; 6eaay4054Crossref (14) Scholar, 3.Koelmans A.A. al.Microplastics freshwaters drinking water: critical assessment data quality.Water Res. 2019; 155: 410-422Crossref (256) 4.Seeley M.E. sedimentary nitrogen cycling.Nat. Commun. 11: 2372Crossref (27) 5.Boots B. al.Effects soil ecosystems: above below ground.Environ. Sci. Technol. 53: 11496-11506Crossref (63) In addition, degradation resistance further escalates their impacts [6.Chamas A. al.Degradation rates environment.ACS Sustain. Chem. Eng. 8: 3494-3511Crossref (230) Therefore, it urgent develop plastics, both protection. Enzymatic biocatalysis gained attention eco-friendly conventional methods (Box 1) [7.Wei al.Possibilities limitations biotechnological recycling.Nat. Catal. 3: 867-871Crossref To date, discovered, representing promising biocatalyst candidates depolymerization. Considering ubiquity different ecosystems tremendous metabolic genetic diversity microorganisms, habitats likely evolved capabilities decomposition utilization. identified so far might only account small portion relevant environment. ever-growing interest explore diverse discover new with desirable properties functionalities. However, naturally occurring not well suited synthetic industrial applications due poor thermostability low activity. Particularly, usually possess distinct physical chemical (e.g., high crystallinity) render them more resistant enzymatic attack than biogenic polymers. increasingly utilized construct better stability. Recent efforts made significant discovering enzymes, showing great promise progress discovery using optimization article timely provides holistic view current stage emerging obtaining effective biocatalysts degradation, will inspire future address Metagenomics demonstrated potential facilitate ecological habitats. culture-dependent method applied most known [8.Satti S.M. Shah Polyester-based biodegradable plastics: approach towards development.Lett. Appl. Microbiol. 70: 413-430Crossref (3) Scholar,9.Wierckx N. al.Plastic biodegradation: opportunities.in: Steffan Consequences Microbial Interactions Hydrocarbons, Oils, Lipids: Biodegradation Bioremediation. Springer International Publishing, 2018: 1-29Crossref method, microorganisms expressing desired first enriched isolated under proper cultivation conditions, followed strain taxonomical classification, identification putative molecular biological or computational (Figure 1A ) [10.Kawai F. al.A Ca2+-activated, thermostabilized polyesterase hydrolyzing polyethylene terephthalate Saccharomonospora viridis AHK190.Appl. Biotechnol. 2014; 98: 10053-10064Crossref (112) 11.Taniguchi I. al.Biodegradation PET: status application aspects.ACS 9: 4089-4105Crossref (106) 12.Yoshida S. bacterium degrades assimilates poly(ethylene terephthalate).Science. 2016; 351: 1196-1199Crossref (705) seriously limits scope finding because estimated less 1% total planet cultured. By contrast, culture-independent metagenomic tool vast majority As summarized Table 1, many genes encoding depolymerizing retrieved wealth metagenome samples. this section we discuss deciphering huge reservoir techniques. overall workflow metagenomics illustrated Figure 1B. Among these steps, selecting appropriate screening pivotal mining. Generally, there two commonly used screen library, sequence-based function-based [13.Ufarte L. al.Metagenomics pollutant degrading enzymes.Biotechnol. 2015; 33: 1845-1854Crossref (0) Scholar,14.Sankara Subramanian S.H. al.RemeDB: rapid prediction involved bioremediation high-throughput sets.J. Comput. Biol. 27: 1020-1029Crossref (2) Sequence-based takes advantage sequence similarity comparison functional gene annotation searching bioinformatic databases [14.Sankara For example, terephthalate) (PET) hydrolytic (PET2) was uncovered silico search algorithm powered hidden Markov model [15.Danso D. al.New insights into function global distribution (PET)-degrading bacteria marine terrestrial metagenomes.Appl. Environ. 2018; 84: e02773-e02817Crossref (50) More recently, number sequences similar ones activity degrade polyurethane (PU) were landfill-derived metagenomes [16.Gaytan recalcitrant xenobiotic additives selected landfill community its biodegradative revealed proximity lgation-based analysis.Front. 10: 2986Crossref (8) relatively cost-effective success limited size could miss families previously characterized ones. similarities do guarantee activity, characterization validation functionality needed [17.Muller C.A. al.Discovery polyesterases moss-associated microorganisms.Appl. 83: e02641-e02716Crossref Alternatively, uses assays phenotypes libraries 1B). particularly advantageous over screening, completely groups divergent existing homologous multiple phylogenetically belonging entirely esterase screened agar plate assays, exhibited polyesters, poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(butylene succinate-co-adipate) (PBSA) [18.Hajighasemi M. al.Screening against polyesters.Environ. 52: 12388-12401Crossref (11) Scholar] (Table 1). Traditional capability large-sized libraries. studies developing accelerate microbes [19.Weinberger al.High throughput fungal polyester enzymes.Front. 554Crossref Scholar,20.Bunzel H.A. al.Speeding up ultrahigh-throughput methods.Curr. Opin. Struct. 48: 149-156Crossref (54) When approach, important select host cell constructing heterologous expression level library representativeness. Escherichia coli widely convenient manipulation [21.Lorenz P. Eck J. applications.Nat. Rev. 2005; 510-516Crossref (370) systems employed ensure expression. instance, eukaryotic cells, such yeast Pichia pastoris, disulfide bonds, they unsuitably expressed common E. [22.Fecker T. al.Active site flexibility hallmark PET sakaiensis PETase.Biophys. 114: 1302-1312Abstract Full Text PDF (84) 23.Urbanek A.K. al.Biochemical polyester-type plastics.Biochim. Biophys. Acta Proteins Proteom. 1868140315Crossref (13) 24.Chen al.Contribution bond Thermobifida fusca cutinase.Food Biosci. 6-10Crossref It type successful screening. chosen determined factors; coverage. Due short length insert plasmid harbor, plasmid-based large but coverage, unfavorable longer DNA fragments inserted phage fosmid Moreover, phage-based some toxic target concomitant lysis cells directly plaques. Besides methods, sampling sources determining discovery. Most investigated showed hit rate related 1), major challenge analysis worldwide broad extremely frequency indicating slow evolution indigenous utilize anthropogenic likelihood greater abundant biopolymeric substances. thermostable cutinase homologue, leaf branch compost (LCC), PCL leaf-branch copious plant-derived polymers [25.Sulaiman al.Isolation homolog terephthalate-degrading approach.Appl. 2012; 78: 1556-1562Crossref (155) Likewise, esterases poly(diethylene glycol adipate) (poly DEGA) copolyester adipate-co-terephthalate) (PBAT) constructed Sphagnum moss, respectively Scholar,26.Kang C.H. family VII library.Microb. Cell Factories. 2011; 41Crossref (38) plastisphere source compounds survival growth [27.Roager Sonnenschein E.C. Bacterial colonization debris.Environ. 11636-11643Crossref (25) 28.Jacquin al.Microbial ecotoxicology debris: biodegradation ‘plastisphere.Front. 865Crossref 29.Amaral-Zettler L.A. al.Ecology plastisphere.Nat. 18: 139-151Crossref currently underexplored growing Techniques targeted stable-isotope probing (SIP) helpful increase Targeted stimulate presence functions before extraction, situ habitat. pre-incubation native activated prevalence species raised [30.Mayumi al.Identification poly(DL-lactic depolymerases metagenome.Appl. 2008; 79: 743-775Crossref (34) Additionally, SIP technique integrated [31.Coyotzi al.Targeted populations probing.Curr. 41: 1-8Crossref (39) Scholar,32.Chen Y. Murrell J.C. meets probing: perspectives.Trends 2010; 157-163Abstract Recently, 13C-labeled developed [33.Sander al.Assessing transformation nanoplastic 13C-labelled polymers.Nat. Nanotechnol. 14: 301-303Crossref (7) Scholar,34.Zumstein M.T. soils: tracking carbon CO2 biomass.Sci. 4eaas9024Crossref (52) Using would help pinpoint participating processes. proteomics-based detects quantifies proven repertoire [35.Bers K. hydrolase genomic-proteomic phenylurea herbicide mineralization Variovorax sp. SRS16.Appl. 77: 8754-8764Crossref (48) Scholar,36.Sturmberger al.Synergism proteomics mRNA sequencing discovery.J. 235: 132-138Crossref (9) 1C shows First, pure consortia grown without substrate, differentially induce express produced cultures extracted digested peptides, subjected sequencing, analysis. Typically, exoproteome principal when insoluble unable enter engaged secreted extracellularly [23.Urbanek effectiveness already identifying plant biopolymer inspiring implementation [37.Schneider al.Proteome bacterial involvement litter decomposition.Proteomics. 1819-1830Crossref (64) Comparative frequently based presumption incubation comparatively analyzing Pseudomonas pseudoalcaligenes fungus Knufia chersonesos, several PBAT identified, demonstrating unavailable annotated genomic [38.Tesei al.Shotgun reveals secretome rock-inhabiting chersonesos.Sci. Rep. 9770Crossref (1) Scholar,39.Wallace P.W. al.PpEst pseudoalcaligenes.Appl. 101: 2291-2303Crossref (16) another study, polyhydroxybutyrate (PHB) depolymerase ALC24_4107 Alcanivorax 24 comparative exoproteomic [40.Zadjelovic V. al.Beyond oil degradation: 22: 1356-1369Crossref Proteomics-guided still infancy, reported conducted cultures. Direct metaproteomics complex samples challenging, difficulty high-quality extraction availability downstream [41.Biswas Sarkar ‘Omics’ microbiology: state art.in: Adhya T.K. Advances Soil Microbiology: Trends Prospects. Singapore, 35-64Crossref Leveraging improve performance recently topic. Protein categories general; rational design directed evolution. Rational modifies knowledge structure mechanistic characteristics, simulation, modeling. Almost reports available structural information lack main barrier attempt far, employing direct engineer PHB Ralstonia pickettii T1, failed acquire any variant [42.Tan L.T. al.Directed poly[(R)-3-hydroxybutyrate] surface display system: importance asparagine at position 285.Appl. 2013; 97: 4859-4871Crossref focus discussing strategies 2 examples 2. Thermostability highly depolymerization, glass transition temperature (Tg) ~65–70°C PET). reaction gets close Tg polymeric chains considerably increased mobility, facilitating accessibility [43.Wei Zimmermann W. petroleum-based how we?.Microb. 1308-1322Crossref (208) one bottleneck practical applications. Inspired unique features thermophilic proteins, designed detailed later. Introduction bonds salt bridges beneficial 2A) [44.Rigoldi al.Review: applications.APL Bioeng. 2011501Crossref 45.Son H.F. al.Structural bioinformatics-based thermo-stable PETase Ideonella sakaiensis.Enzym. Microb. 141109656Crossref 46.Oda al.Enzymatic hydrolysis roles three Ca2+ ions bound cutinase-like enzyme, Cut190*, activity.Appl. 102: 10067-10077Crossref (17) 47.Zhong-Johnson E.Z.L. al.An absorbance kinetics films.Sci. 2021; 928Crossref Disulfide crucial folding correct local conformation confer thermal resistance. residues metal responsible replaced introduce bond. D204C E253C mutations calcium TfCut2 formed bond, melting [48.Then bridge increases terephthalate.FEBS Open Bio. 6: 425-432Crossref (47) formation negatively-charged N246D residue positively-charged Arg280 contribute engineered PETaseN246D [45.Son construction work synergistically benefit ag

Language: Английский

---

The road to fully programmable protein catalysis

Nature, Journal Year: 2022, Volume and Issue: 606(7912), P. 49 - 58

Published: June 1, 2022

Language: Английский

---

Embracing Nature’s Catalysts: A Viewpoint on the Future of Biocatalysis

ACS Catalysis, Journal Year: 2020, Volume and Issue: 10(15), P. 8418 - 8427

Published: June 4, 2020

ADVERTISEMENT RETURN TO ISSUEPREVViewpointNEXTEmbracing Nature's Catalysts: A Viewpoint on the Future of BiocatalysisBernhard Hauer*Bernhard HauerInstitute Biochemistry and Technical Biochemistry, Department Universitaet Stuttgart, Allmandring 31, 70569 Germany*Email: [email protected]More by Bernhard Hauerhttp://orcid.org/0000-0001-6259-3348Cite this: ACS Catal. 2020, 10, 15, 8418–8427Publication Date (Web):June 4, 2020Publication History Received16 April 2020Published online4 June inissue 7 August 2020https://pubs.acs.org/doi/10.1021/acscatal.0c01708https://doi.org/10.1021/acscatal.0c01708article-commentaryACS PublicationsCopyright © 2020 American Chemical Society. This publication is available under these Terms Use. Request reuse permissions free to access through this site. Learn MoreArticle Views15099Altmetric-Citations190LEARN ABOUT THESE METRICSArticle Views are COUNTER-compliant sum full text article downloads since November 2008 (both PDF HTML) across all institutions individuals. These metrics regularly updated reflect usage leading up last few days.Citations number other articles citing article, calculated Crossref daily. Find more information about citation counts.The Altmetric Attention Score a quantitative measure attention that research has received online. Clicking donut icon will load page at altmetric.com with additional details score social media presence for given article. how calculated. Share Add toView InAdd Full Text ReferenceAdd Description ExportRISCitationCitation abstractCitation referencesMore Options onFacebookTwitterWechatLinked InRedditEmail (1 MB) Get e-AlertscloseSUBJECTS:Biocatalysis,Catalysts,Chemical reactions,Industrial manufacturing,Peptides proteins e-Alerts

Language: Английский

---

Synthetic biology 2020–2030: six commercially-available products that are changing our world

Nature Communications, Journal Year: 2020, Volume and Issue: 11(1)

Published: Dec. 11, 2020

Synthetic biology will transform how we grow food, what eat, and where source materials medicines. Here I have selected six products that are now on the market, highlighting underlying technologies projecting forward to future can be expected over next ten years.

Language: Английский

---

Current progress and open challenges for applying deep learning across the biosciences

Nature Communications, Journal Year: 2022, Volume and Issue: 13(1)

Published: April 1, 2022

Deep Learning (DL) has recently enabled unprecedented advances in one of the grand challenges computational biology: half-century-old problem protein structure prediction. In this paper we discuss recent advances, limitations, and future perspectives DL on five broad areas: prediction, function genome engineering, systems biology data integration, phylogenetic inference. We each application area cover main bottlenecks approaches, such as training data, scope, ability to leverage existing architectures new contexts. To conclude, provide a summary subject-specific general for across biosciences.

Language: Английский

---

Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses

Nature Chemistry, Journal Year: 2022, Volume and Issue: 14(5), P. 489 - 499

Published: May 1, 2022

Language: Английский

---