Multi-enzymet Screening Ved hjelp av en High-throughput Genetisk 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 nylig utviklet høy gjennomstrømming encellede analysen teknikken gjør nye enzymer for å være skjermet fra en storstilt genetisk bibliotek basert på deres funksjonelle aktiviteter 1. På celle-nivået, blir proteiner som regulerer transkripsjon anvendes for å utløse reporter-gen-ekspresjon ved å avføle små molekyler som er produsert som et resultat av en target enzymaktivitet. En tidlig metode omfattet isolering av en fenol-nedbrytende operonet fra Ralstonia eutropha E2 ved hjelp av substrat-indusert ekspresjon genetisk screening (SIGEX) -metoden, hvor substratet induserer ekspresjon av et rapportørprotein 2. Nhar av Pseudomonas putida ble brukt til å velge benzaldehyd dehydrogenase 3, og LysG fra Corynebacterium glutamicum ble anvendt for high-throughput screening av en ny L-lysin-produserende stamme fra forskjellige mutante biblioteker 4.
Tidligere en genetisk enzymet maskeringsng system (GESS) ble foreslått som et generelt gjelder screening plattform 5. Dette systemet bruker fenol-gjenkjenne dimetylfenol regulator, DmpR, P. putida. DmpR (E135K) og en mutant av DmpR, kan også anvendes i GESS (PNP-GESS) for deteksjon av p-nitrofenol (PNP). I nærvær av mål-enzymer som produserer fenoliske forbindelser, GESS i E. coli-celler avgir en fluorescens-signal, slik at den hurtig isolering av enkeltceller ved hjelp av et fluorescens-aktivert cellesorterer (FACS). Men ekspresjonen av metagenomic enzym synes å være svakere enn den for konvensjonelle rekombinante enzymer; Derfor GESS er designet for å oppdage fenoliske forbindelser med maksimal følsomhet ved å undersøke kombinasjonen av ribosomale bindingssetet (RBS) og terminatorsekvenser sammen med optimal driftstilstand 5.
En av de grunnleggende fordelene ved GESS er at denne enkle metode teoretisk tillater screening av over enn 200 forskjellige typer enzymer i Brenda databasen (tabell 1, http: // www.brenda-enzymes.info, 2013.7) ved bare å bruke forskjellige underlag. Det ble vist at cellulase, lipase, og metyl-paration hydrolase (MPH) kan påvises ved hjelp av PNP-GESS med passende substrater for p-nitrofenyl butyrat, p-nitrofenyl-cellotrioside, og metyl-paration, henholdsvis 5. Nylig ble det vist at et alkalisk fosfatase (AP), som er en av de nye enzymer som er identifisert ved hjelp av pnp-GESS, er den første termolabile AP befinner seg i kuldetilpassede marine metagenomes 6.
Her er detaljene for screening prosessen presenteres med PnP-GESS påvise aktivitetene til tre ulike typer enzymes- lipase, cellulase og alkalisk fosfatase -og raskt identifisere nye kandidat enzymer fra en metagenomic bibliotek 5,6. Prosessene omfatter metagenome preprosessering med PnP-GESS og opererer en flow cytometri sorter. Mens treff som oppnås vil måtte bli sekvensert for ytterligere identifikasjon, denne protokollen omfatter fremgangsmåten opp til trinnene av enzymaktivitet bekreftelse ved hjelp av flow cytometri.
Protocol
Representative Results
Discussion
Increasing production efficiency of biocatalysts is a key for the success of bio-chemical based industry9 and metagenome is considered one of the best natural enzyme source. In this sense, it is essential to screening novel enzymes from the metagenome where majority of the genetic resources have not been explored10. Several screening methods have been developed which directly detect enzyme products using transcriptional activators11, 12 but these techniques require specific metabolite-res…
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. |
||
| 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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