Name: multi-enzyme screening using a high-throughput genetic enzyme screening system
Uploaded: 2016-08-08T13:00:00
Description: Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

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.

1. Preparing the Metagenomic Library with pNP-GESS
<ol>
	<li>Construct a metagenomic library in E. coli with a fosmid vector using a fosmid library production kit according to the manufacturer&#39;s protocol 5.</li>
	<li>Aliquot 100 &#181;l of the library for storage at &#8722;70 &#176;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.</li>
	<li>Thaw 100 &#18...

<ol>
	<li>Eggeling, L., Bott, M., Marienhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+screening+methods-biosensors.">Novel screening methods-biosensors.</a> Curr. Opin. Biotech. 35, 30-36 (2015).</li><li>Uchiyama, T., Abe, T., Ikemura, T., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-induced+gene-expression+screening+of+environmental+metagenome+libraries+for+isolation+of+catabolic+genes.">Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes.</a> Nat. Biotechnol. 23 (1), 88-93 (2005).</li><li>Van Sint Fiet, S., van Beilen, J. B., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+biocatalysts+for+chemical+synthesis.">Selection of biocatalysts for chemical synthesis.</a> Proc. Natl. Acad. Sci. U. S. A. 103 (6), 1693-1698 (2006).</li><li>Binder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+approach+to+identify+genomic+variants+of+bacterial+metabolite+producers+at+the+single-cell+level.">A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.</a> Genome Biol. 13 (5), R40 (2012).</li><li>Choi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+generalized+and+High-throughput+Enzyme+Screening+System+Based+on+Artificial+Genetic+Circuits.">Toward a generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits.</a> ACS Synthetic Biology. 3 (3), 163-171 (2014).</li><li>Lee, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+psychrophilic+alkaline+phosphatase+from+the+metagenome+of+tidal+flat+sediments.">A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments.</a> BMC Biotechnology. 15 (1), (2015).</li><li>Warnecke, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+and+functional+analysis+of+hindgut+microbiota+of+a+wood-feeding+higher+termite.">Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.</a> Nature. 450 (7169), 560-565 (2007).</li><li>Kim, U. J., Shizuya, H., de Jong, P. J., Birren, B., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+propagation+of+cosmid+sized+human+DNA+inserts+in+an+F+factor+based+vector.">Stable propagation of cosmid sized human DNA inserts in an F factor based vector.</a> Nucleic Acids Research. 20 (5), 1083-1085 (1992).</li><li>Jemli, S., Ayadi-Zouari, D., Hlima, H. B., Bejar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalysts:+application+and+engineering+for+industrial+purposes.">Biocatalysts: application and engineering for industrial purposes.</a> Crc. Cr. Rev. Biotechn. 36 (2), 246-258 (2014).</li><li>Lorenz, P., Eck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomics+and+industrial+applications.">Metagenomics and industrial applications.</a> Nat. Rev. Microbiol. 3 (6), 510-516 (2005).</li><li>Uchiyama, T., Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-induced+gene+expression,+a+product+responsive+reporter+assay+used+to+screen+metagenomic+libraries+for+enzyme+encoding+genes.">Product-induced gene expression, a product responsive reporter assay used to screen metagenomic libraries for enzyme encoding genes.</a> Applied and environmental microbiology. 76 (21), 7029-7035 (2010).</li><li>Mohn, W. W., Garmendia, J., Galvao, T. C., De Lorenzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+bio-transformations+with+&#224;+la+carte+genetic+traps:+translating+dehydrochlorination+of+lindane+(gamma-hexachlorocyclohexane)+into+lacZ-based+phenotypes.">Surveying bio-transformations with &#224; la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes.</a> Environmental microbiology. 8 (3), 546-555 (2006).</li><li>Tang, S. Y., Cirino, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+a+mevalonate-responsive+regulatory+protein.">Design and application of a mevalonate-responsive regulatory protein.</a> Angew Chem. Int. Ed. 50 (5), 1084-1086 (2011).</li><li>Jha, R. K., Kern, T. L., Fox, D. T., Strauss, C. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+an+Acinetobacter+regulon+for+biosensing+and+high-throughput+enzyme+screening+in+E.+coli+via+flow+cytometry.">Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry.</a> Nucleic Acids Res. 42 (12), 8150-8160 (2014).</li></ol>

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...

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 library5,6. 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.

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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">CopyControl</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCFOS110</td>
			<td style="width:736px;">Fosmid library production kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">CopyControl Induction Solution</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCIS125</td>
			<td>Fosmid copy induction solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">EPI300</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">EC300110</td>
			<td>Electrocompetent cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">pCC1FOS</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCFOS110</td>
			<td>Fosmid vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Gene Pulser Mxcell</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:88px;"></td>
			<td>Electroporation cuvette and electroporate system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">FACSAria III</td>
			<td style="width:109px;">Becton Dickinson</td>
			<td style="width:88px;"></td>
			<td>Flow Cytometry (FACS machine)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">AZ100M</td>
			<td style="width:109px;">Nikon</td>
			<td style="width:88px;"></td>
			<td>Microscope&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UltraSlim&#160;</td>
			<td style="width:109px;">Maestrogen</td>
			<td style="width:88px;"></td>
			<td>LED illuminator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">50-mL conical tube</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">14-mL round-bottom tube&#160;</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">5-mL round-bottom tube</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl phosphate</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N7653</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl &#946;-D-cellobioside</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N5759</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl butylate</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N9876&#160;</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Luria- Bertani (LB)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244620</td>
			<td>Tryptone 10g/L, Yeast extract 5g/L, Sodium Chloride 10g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Super Optimal broth (SOB)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244310</td>
			<td>Tryptone 20g/L, Yeast extract 5g/L, Sodium Chloride 0.5g/L, Magnesium Sulfate 2.4g/L, Potassium Chloride 186mg/L</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Super Optimal broth with Catabolite repression (SOC)</td>
			<td style="width:88px;"></td>
			<td>SOB, 0.4 % glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Yeast Extract Tryptone (2xYT)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244020</td>
			<td>Pancreatic digest of Casein 16g/L, Yeast extract 10g/L, Yeast extract 5g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Cell storage media</td>
			<td style="width:109px;"></td>
			<td style="width:88px;"></td>
			<td>2xYT broth, 15 % Glycerol, 2 % Glucose</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:319px;">pGESS(E135K)</td>
			<td style="width:109px;"></td>
			<td style="width:88px;"></td>
			<td style="width:736px;">A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator. 
			See the reference 5 in the manuscript for more details.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Chloramphenicol</td>
			<td style="width:109px;">Sigma</td>
			<td style="width:88px;">C0378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ampicillin</td>
			<td style="width:109px;">Sigma</td>
			<td style="width:88px;">A9518</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">BD FACSDiva</td>
			<td style="width:109px;">Becton Dickinson</td>
			<td style="width:88px;"></td>
			<td>Flow Cytometry Software Version 7.0</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:319px;">PBS</td>
			<td style="width:109px;">Gibco</td>
			<td style="width:88px;">70011-044</td>
			<td>0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4</td>
		</tr>
	</tbody>
</table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

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-&#946;-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.

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...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

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

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-&#946;-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 ...

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