Published: August 8th, 2016
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).
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.
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
1. Preparing the Metagenomic Library with pNP-GESS
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.......
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.......
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.....
|Fosmid library production kit
|CopyControl Induction Solution
|Fosmid copy induction solution
|Gene Pulser Mxcell
|Electroporation cuvette and electroporate system
|Flow Cytometry (FACS machine)
|50-mL conical tube
|14-mL round-bottom tube
|5-mL round-bottom tube
|Luria- Bertani (LB)
|Tryptone 10g/L, Yeast extract 5g/L, Sodium Chloride 10g/L
|Super Optimal broth (SOB)
|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)
|Pancreatic digest of Casein 16g/L, Yeast extract 10g/L, Yeast extract 5g/L
|Cell storage media
|2xYT broth, 15 % Glycerol, 2 % Glucose
|A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.
See the reference 5 in the manuscript for more details.
|Flow Cytometry Software Version 7.0
|0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4
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