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Biology

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Published: August 8th, 2016

DOI:

10.3791/54059

1Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Biosystems and Bioengineering Program, University of Science and Technology, Daejeon, South Korea

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

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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 .......

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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.......

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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.......

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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.

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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

  1. Eggeling, L., Bott, M., Marienhagen, J. Novel screening methods-biosensors. Curr. Opin. Biotech. 35, 30-36 (2015).
  2. Uchiyama, T., Abe, T., Ikemura, T., Watanabe, K. Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat. Biotechnol. 23 (1), 88-93 (2005).
  3. Van Sint Fiet, S., van Beilen, J. B., Witholt, B. Selection of biocatalysts for chemical synthesis. Proc. Natl. Acad. Sci. U. S. A. 103 (6), 1693-1698 (2006).
  4. Binder, S., et al. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Genome Biol. 13 (5), R40 (2012).
  5. Choi, S., et al. Toward a generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits. ACS Synthetic Biology. 3 (3), 163-171 (2014).
  6. Lee, D. H., et al. A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments. BMC Biotechnology. 15 (1), (2015).
  7. Warnecke, F., et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature. 450 (7169), 560-565 (2007).
  8. Kim, U. J., Shizuya, H., de Jong, P. J., Birren, B., Simon, M. I. Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Research. 20 (5), 1083-1085 (1992).
  9. Jemli, S., Ayadi-Zouari, D., Hlima, H. B., Bejar, S. Biocatalysts: application and engineering for industrial purposes. Crc. Cr. Rev. Biotechn. 36 (2), 246-258 (2014).
  10. Lorenz, P., Eck, J. Metagenomics and industrial applications. Nat. Rev. Microbiol. 3 (6), 510-516 (2005).
  11. Uchiyama, T., Miyazaki, K. Product-induced gene expression, a product responsive reporter assay used to screen metagenomic libraries for enzyme encoding genes. Applied and environmental microbiology. 76 (21), 7029-7035 (2010).
  12. Mohn, W. W., Garmendia, J., Galvao, T. C., De Lorenzo, V. Surveying bio-transformations with à la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes. Environmental microbiology. 8 (3), 546-555 (2006).
  13. Tang, S. Y., Cirino, P. C. Design and application of a mevalonate-responsive regulatory protein. Angew Chem. Int. Ed. 50 (5), 1084-1086 (2011).
  14. Jha, R. K., Kern, T. L., Fox, D. T., Strauss, C. E. M. Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry. Nucleic Acids Res. 42 (12), 8150-8160 (2014).

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