Multi-enzyme Screening Using a 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

A recently developed high-throughput single-cell assay technique allows novel enzymes to be screened from a large-scale genetic library based on their functional activities¹. At the single cell level, proteins regulating transcription are employed to trigger reporter gene expression by sensing small molecules that are produced as a result of a target enzyme activity. One early approach involved the isolation of a phenol-degrading operon from Ralstonia eutropha E2 using the substrate-induced genetic expression screening (SIGEX) method, in which the substrate induces the expression of a reporter protein². NhaR of Pseudomonas putida

Protocol

1. Preparing the Metagenomic Library with pNP-GESS

1. Construct a metagenomic library in E. coli with a fosmid vector using a fosmid library production kit according to the manufacturer's protocol ⁵.
2. Aliquot 100 µl of the library for storage at −70 °C, which is a source of metagenomic library cells.
   Note: The optical density of a sample measured at a wavelength of 600 nm (OD₆₀₀) of this library stock is approximately 100.
3. Thaw 100 µl of the stock metagenomic library on ice and inoculate in a 500 ml flask containing 50 ml Luria-Bertani (LB) and 12.5 µg/ml chloramphenicol fo....

Results

The three phenolic substrates were examined to identify novel metagenomic enzymes from a metagenome library of ocean-tidal flat sediments in Taean, South Korea by following the proposed protocol. For the library construction, average 30 - 40 kb metagenome sequences were inserted into fosmids, which are based on the E. coli F factor replicon and presented as a single copy in a cell. Note that fosmids have been widely used for constructing complex genomic libraries due to their sta.......

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 activators^{11, 12} but these techniques require specific metabolite-res.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 10 g/L, Yeast extract 5 g/L, Sodium Chloride 10 g/L
Super Optimal broth (SOB)	BD Difco	244310	Tryptone 20 g/L, Yeast extract 5 g/L, Sodium Chloride 0.5 g/L, Magnesium Sulfate 2.4 g/L, Potassium Chloride 186 mg/L
Super Optimal broth with Catabolite repression (SOC)		SOB, 0.4 % glucose
2x Yeast Extract Tryptone (2xYT)	BD Difco	244020	Pancreatic digest of Casein 16 g/L, Yeast extract 10 g/L, Yeast extract 5 g/L
Cell storage media			2x YT 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