Multi-enzym Screening Ved hjælp af en High-throughput Genetic Enzyme Screening System
Summary
This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate).
Abstract
The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.
Introduction
En nyligt udviklet high-throughput enkelt-celleassay teknik muliggør hidtil ukendte enzymer, der skal screenes fra en storstilet genetisk bibliotek baseret på deres funktionelle aktiviteter 1. På enkelt celle niveau, er proteiner, der regulerer transkription anvendes til at udløse reportergenekspression ved at registrere små molekyler, der er produceret som et resultat af et mål-enzymaktivitet. En tidlig fremgangsmåde involverede isolering af en phenol-nedbrydende operon fra Ralstonia eutropha E2 anvendelse af substratet-inducerede genetiske ekspression screening (SIGEX) fremgangsmåde, ved hvilken substratet inducerer ekspressionen af et reporter-protein 2. Nhar af Pseudomonas putida blev anvendt til at vælge benzaldehyd dehydrogenase 3, og LysG fra Corynebacterium glutamicum blev anvendt for high-throughput screening af en ny L-lysin-producerende stamme fra forskellige mutantbiblioteker 4.
Tidligere en genetisk enzym flotationng-system (GESS) blev foreslået som en generelt gældende screening platform 5. Dette system bruger phenolen-genkendende dimethylphenol regulator, DmpR, af P. putida. DmpR (E135K), og en mutant af DmpR, kan også anvendes i GESS (pNP-GESS) til påvisning af p-nitrophenol (pNP). I nærvær af målenzymer producerer phenolforbindelser, GESS i E. coli celler udsender et fluorescenssignal, giver mulighed for hurtig isolering af enkelte celler under anvendelse af en fluorescens-aktiveret cellesorterer (FACS). Men ekspressionen af metagenomisk enzym synes at være svagere end i konventionelle rekombinante enzymer; derfor blev GESS designet til at detektere phenolforbindelser med maksimal følsomhed ved at undersøge kombinationen af ribosomale bindingssted (RBS) og terminatorsekvenser sammen med optimal driftstilstand 5.
Et af de grundlæggende fordele ved GESS er, at denne enkelt metode teoretisk muliggør screening af ovis end 200 forskellige typer af enzymer i BRENDA databasen (tabel 1, http: // www.brenda-enzymes.info, 2013.7) ved blot at ansætte forskellige substrater. Det blev vist, cellulase, lipase, og kan påvises methyl parathion hydrolase (MPH) ved anvendelse pNP-GESS med passende substrater af p-nitrophenyl-butyrat, p-nitrophenyl-cellotrioside og methyl parathion, henholdsvis 5. For nylig blev det vist, at et alkalisk phosphatase (AP), som er en af de hidtil ukendte enzymer identificeret ved anvendelse pNP-GESS, er den første termolabile AP findes i koldadapterede marine metagenomes 6.
Her er detaljerne i screeningsprocessen præsenteret med pNP-GESS afsløre aktiviteter tre forskellige typer af enzymes- lipase, cellulase, og alkalisk fosfatase -og hurtigt at identificere nye kandidatlande enzymer fra en metagenomisk bibliotek 5,6. Processerne omfatter metagenomet forbehandling med pNP-GESS og drift af en flow cytometri sorteringsanlæg. Mens de opnåede hits vil skulle sekventeret for yderligere identifikation, denne protokol dækker proceduren indtil trinnene enzymaktivitet bekræftelse ved anvendelse af flowcytometri.
Protocol
Representative Results
Discussion
Øget produktion effektivitet biokatalysatorer er nøglen til succes for bio-kemiske industri 9 og metagenomet betragtes som en af de bedste naturlige enzym kilde. I denne forbindelse er det vigtigt at screene hidtil ukendte enzymer fra metagenomet hvor størstedelen af de genetiske ressourcer ikke er blevet udforsket 10. Adskillige screeningsmetoder er blevet udviklet som direkte opdage enzymprodukter hjælp transkriptionelle aktivatorer 11, 12, men disse teknikker kræver specifik meta…
Declarações
The authors have nothing to disclose.
Acknowledgements
This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.
Materials
| CopyControl | Epicentre | CCFOS110 | Fosmid library production kit |
| CopyControl Induction Solution | Epicentre | CCIS125 | Fosmid copy induction solution |
| EPI300 | Epicentre | EC300110 | Electrocompetent cell |
| pCC1FOS | Epicentre | CCFOS110 | Fosmid vector |
| Gene Pulser Mxcell | Bio-Rad | Electroporation cuvette and electroporate system | |
| FACSAria III | Becton Dickinson | Flow Cytometry (FACS machine) | |
| AZ100M | Nikon | Microscope | |
| UltraSlim | Maestrogen | LED illuminator | |
| 50-mL conical tube | BD Falcon | ||
| 14-mL round-bottom tube | BD Falcon | ||
| 5-mL round-bottom tube | BD Falcon | ||
| p-nitrophenyl phosphate | Sigma-Aldrich | N7653 | Substrate |
| p-nitrophenyl β-D-cellobioside | Sigma-Aldrich | N5759 | Substrate |
| p-nitrophenyl butylate | Sigma-Aldrich | N9876 | Substrate |
| Luria- Bertani (LB) | BD Difco | 244620 | Tryptone 10g/L, Yeast extract 5g/L, Sodium Chloride 10g/L |
| Super Optimal broth (SOB) | BD Difco | 244310 | Tryptone 20g/L, Yeast extract 5g/L, Sodium Chloride 0.5g/L, Magnesium Sulfate 2.4g/L, Potassium Chloride 186mg/L |
| Super Optimal broth with Catabolite repression (SOC) | SOB, 0.4 % glucose | ||
| 2x Yeast Extract Tryptone (2xYT) | BD Difco | 244020 | Pancreatic digest of Casein 16g/L, Yeast extract 10g/L, Yeast extract 5g/L |
| Cell storage media | 2xYT broth, 15 % Glycerol, 2 % Glucose | ||
| pGESS(E135K) | A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator. See the reference 5 in the manuscript for more details. |
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| Chloramphenicol | Sigma | C0378 | |
| Ampicillin | Sigma | A9518 | |
| BD FACSDiva | Becton Dickinson | Flow Cytometry Software Version 7.0 | |
| PBS | Gibco | 70011-044 | 0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4 |
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