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 was used to select benzaldehyde dehydrogenase³, and LysG from Corynebacterium glutamicum was utilized for the high-throughput screening of a new L-lysine-producing strain from diverse mutant libraries⁴.

Previously, a genetic enzyme screening system (GESS) was proposed as a generally applicable screening platform⁵. This system uses the phenol-recognizing dimethylphenol regulator, DmpR, of P. putida. DmpR(E135K), and a mutant of DmpR, can also be employed in GESS (pNP-GESS) for the detection of p-nitrophenol (pNP). In the presence of target enzymes producing phenolic compounds, GESS in E. coli cells emits a fluorescence signal, enabling the rapid isolation of single cells using a fluorescence-activated cell sorter (FACS). But the expression of metagenomic enzyme appears to be weaker than that of conventional recombinant enzymes; therefore, GESS was designed to detect phenolic compounds with maximum sensitivity by investigating the combination of ribosomal binding site (RBS) and terminator sequences along with optimal operating condition⁵.

One of the fundamental advantages of GESS is that this single method theoretically allows the screening of over than 200 different types of enzymes in the BRENDA database (Table 1, http:// www.brenda-enzymes.info, 2013.7) by simply employing different substrates. It was shown that cellulase, lipase, and methyl parathion hydrolase (MPH) can be detected using pNP-GESS with appropriate substrates of p-nitrophenyl butyrate, p-nitrophenyl-cellotrioside, and methyl parathion, respectively⁵. Recently, it was proved that an alkaline phosphatase (AP), which is one of the novel enzymes identified using pNP-GESS, is the first thermolabile AP found in cold-adapted marine metagenomes⁶.

Here, details of the screening process is presented with pNP-GESS detecting the activities of three different types of enzymes- lipase, cellulase, and alkaline phosphatase -and rapidly identifying novel candidate enzymes from a metagenomic library^5,6. The processes include metagenome preprocessing with pNP-GESS and operating a flow cytometry sorter. While the hits obtained will need to be sequenced for further identification, this protocol covers the procedure up to the steps of enzyme activity confirmation using flow cytometry.

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 followed by 37 °C incubation for 2 hr.
4. Harvest the cells in a 50 ml conical tube by centrifugation at 1,000 x g for 20 min at 4 °C.
5. Resuspend the pellet quickly in 50 ml ice-cold distilled water (DW) and centrifuge at 1,000 x g for 20 min at 4 °C again.
6. Resuspend the pellet in 50 µl ice-cold DW with 10% (v/v) glycerol. Use 50 µl of this cell aliquot for electroporation.
7. Place the mixture of electrocompetent cells 50 µl and pGESS(E135K)⁵ DNA (100ng) in an ice-cold electroporation cuvette and electroporate (1.8 kV/cm, 25 µF) the mixture.
8. Quickly add 1 ml super optimal broth with catabolite repression (SOC) medium and resuspend the cells gently.
9. Transfer the cells into 14 ml round-bottom tube using a pipette and incubate at 37 °C for 1 hr.
10. Spread 500 µl of the recovered cells on a LB 20 x 20 cm square plate containing 12.5 µg/ml chloramphenicol and 50 µg/ml ampicillin. Incubate plate at 30 °C for 12 hr.
11. Scrape the colonies using a cell scraper and collect cells into a 50 ml conical tube using ice-cold cell storage media.
12. Centrifuge at 1,000 x g for 20 min at 4 °C. Resuspend the pellet in 10 ml ice-cold cell storage media to reach an OD₆₀₀ of 100.
13. Aliquot 20 µl of the cells for storage at −70 °C.

2. Removing False Positives from the Metagenomic Library

1. Thaw the stock metagenomic library cells (from Step 1.13) containing metagenomic DNA fosmid and pGESS(E135K) on ice.
2. Inoculate 10 µl of the cells in 2 ml LB containing 50 µg/ml ampicillin and 12.5 µg/ml chloramphenicol in a 14-ml round-bottom tube. Incubate at 37 °C with shaking at 200 rpm for 4 hr.
3. Meanwhile, turn on the FACS machine and open the default FACS software. Use the following settings: nozzle tip diameter, 70 µm; Forward Scatter Area (FSC-A) sensitivity, 300 V-logarithmic amplification; Side Scatter Area (SSC-A) sensitivity, 350 V-logarithmic amplification; Fluorescein Isothiocyanate Area (FITC-A) sensitivity, 450 V-logarithmic amplification; threshold parameter, FSC-A value 5.
   Note: Typical settings are given for FACS device mentioned in the Table of Materials. Adjust the settings for other FACS devices.
4. Dilute the metagenomic library cells from Step 2.2 by adding 5 µl of the sample to a 5-ml round bottom tube containing 1 ml PBS.
5. Place the diluted library sample onto the loading port of the FACS instrument and click on Load button on the Acquisition Dashboard of the FACS software.
6. Adjust the event rate to 1,000 - 1,500 events/sec by clicking and controlling the Flow Rate buttons on the dashboard.
7. Create log scaled FSC-A vs. log scaled SSC-A scatter plot on a global worksheet by clicking on the Dot Plot button in the tool bar. Adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button in the tool bar.
8. Plot a histogram with cell count vs. log scaled FITC-A on the worksheet by clicking on Histogram button. Then, adjust the FITC voltage from the Cytometer Settings tab on the Inspector Control window such that the peak of the bell-shaped distribution is less than 10² of FITC-A.
9. Create a log scaled FSC-A vs. the log FITC-A plot by clicking on the Dot Plot button on the global worksheet. Set a sorting gate R2 on the plot using the Polygon Gate button on the tool bar so that the gate is located between +5% and -5% cells from the center of the distribution (total 10% cells around the peak of the bell-shaped curve).
10. Place a collection tube containing 1.2 ml LB containing 50 µg/ml ampicillin and 12.5 µg/ml chloramphenicol at the outlet of the FACS instrument and sort out 10⁶ cells satisfying both the R1 and R2 gates.
11. Remove the collection tube, cap, and gently vortex.

3. Metagenomic Enzyme Screening

1. Incubate the sorted cells (from Step 2.11) at 37 °C with shaking at 200 rpm until the OD₆₀₀ reaches 0.5.
2. Add 1 μl copy induction solution to amplify the intracellular fosmid copy number. Incubate the cells for an additional 3 hr at 37 °C with shaking at 250 rpm.
3. To prepare cells for sorting, add 0.5 ml of the cultured cells and an appropriate substrate (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, or phenyl phosphate) into a 14-ml round-bottom tube at a final concentration of 100 µM.
   1. For a control sample, add 0.5 ml of the cultured cells from Step 3.2 into a 14-ml round-bottom tube. Add the same volume of PBS as the substrate volume in Step 3.3.
4. Incubate the two samples at 37 °C with shaking at 200 rpm for 3 hr.
5. Meanwhile, prepare the FACS machine with the same configurations as Step 2.3.
6. Add 5 µl of the cells (for sorting) and control cells to two 5 ml round-bottom tubes containing 1 ml PBS, respectively.
7. Place the tube containing control cells onto the loading port of the FACS and click on Load button on Acquisition Dashboard of the FACS software. Adjust the event Rate to 1,000 - 3,000 events/sec by controlling Flow Rate buttons on the dashboard.
8. Create log scaled FSC-A vs. log scaled SSC-A scatter plot by clicking on Dot Plot button on the global worksheet of the software and adjust the scatter gate R1 to encompass the singlet events (bacterial population) using the Polygon Gate button on the tool bar.
9. Create log scaled FSC-A vs. log scaled FITC-A scatter plot by clicking on the Dot Plot button on the worksheet and set a sorting gate R2 on the plot using the Polygon Gate button on the tool bar so that less than 0.1 % of negative cells are detected within the R2 gate.
10. Replace the control sample tube with the sorting sample tube, and adjust the event rate to 1,000 - 3,000 events/sec by controlling the Flow Rate buttons on the dashboard.
11. Place a collection tube containing 0.5 ml LB at the outlet of the FACS instrument and sort out 10⁴ cells satisfying both the R1 and R2 gates.
    Note: The sorting criterion can range from top 0.1% to 5% of FITC-A in the R2 gate. In this protocol, top 1% cells were collected as positives.
12. Remove the collection tube, cap, and gently vortex.
13. Spread the 0.5 ml collected samples on two agar plates (90 mm Petri dishes) containing LB, 50 µg/ml ampicillin, 12.5 µg/ml chloramphenicol and incubate the plate at 37 °C overnight.
14. If necessary, perform additional rounds of sorting for FITC-A enrichment by repeating Steps 3.1-3.14.

4. Hit Selection and Enzyme Activity Confirmation

1. Flow cytometry analysis
   1. From the incubated plate at Step 3.14, select a colony showing no fluorescence using a colony picker under the observation of 488 nm wavelength of a LED illuminator.
   2. Inoculate the selected colony in a 14-ml round-bottomed tube containing 2 ml LB containing 50 µg/ml ampicillin and 12.5 µg/ml chloramphenicol at 37 °C with shaking at 200 rpm until its OD₆₀₀ reaches 0.5.
   3. Add 2 μl copy induction solution to amplify the intracellular fosmid copy number. Incubate the cells for an additional 3 hr at 37 °C with shaking at 250 rpm.
   4. Prepare a 14 ml round-bottom tube and add 1 ml of the cultured cells (Step 4.1.3). Add an appropriate substrate (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, or phenyl phosphate) at a final concentration of 100 µM.
      1. For a control, add the 1 ml cultured cells from Step 4.1.3 into a 14 ml round-bottom tube and add the same volume of PBS as the substrate volume in Step 4.1.4.
   5. Incubate the two samples at 37 °C with shaking at 200 rpm for 3 hr.
   6. Meanwhile, prepare the FACS machine with the same configurations as Step 2.3.
   7. Add 5 µl of the control and substrate treated cells to 5 ml round-bottom tubes containing 1 ml PBS, respectively.
   8. Place the tube containing control cells on the loading port of the FACS device and click on Load button on Acquisition Dashboard of the FACS software. Adjust the event rate to 1,000 - 3,000 events/sec using Flow Rate buttons on the dashboard.
   9. Click on Acquisition button to measure FITC-A.
   10. Replace the control sample tube with the substrate treated sample tube. Adjust the event rate to 1,000 - 3,000 events/sec using Flow Rate buttons on the dashboard.
   11. Click on Acquisition button to measure FITC-A.
   12. Compare the fluorescence of the two groups of cells by plotting a histogram of cell count vs. the log scaled FITC-A.
2. Other assays to confirm enzyme activity
   1. For further identification of the selected enzyme candidates, extract fosmid DNA using standard extraction procedures from the commercial extraction kits and analyze the nucleotide sequence, or test the in vitro enzyme activity^6,7.

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

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 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