Name: Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening (Video) | JoVE Journal
Uploaded: 2016-04-01T13:00:00.000+00:00
Duration: 10 min 50 s
Description: Learn directed evolution using computational algorithms to select target regions for mutagenesis in Saccharomyces cerevisiae. This method constructs and screens mutant libraries to enhance fungal aryl-alcohol oxidase activity.

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5 minutes

This work was supported by the European Commission project Indox-FP7-KBBE-2013-7-613549; a Cost-Action CM1303-Systems Biocatalysis; and the National Projects Dewry [BIO201343407-R] and Cambios [RTC-2014-1777-3].

1. Mutant Library Construction
<ol>
	<li>Choose the regions to be subjected to MORPHING with the help of computational algorithms based on the available crystal structure or homology models18.
	<ol>
		<li>Here, target two regions of AAO from Pleurotus eryngii for random mutagenesis and recombination (Met[&#945;1]-Val109, Phe392-Gln566), while amplifying the remainder of the gene (844 bp) by high-fidelity PCR (Figure 1). 
		Note: Several segments can be studied by MORPHING in an independent or combined manner16.</li>
	</ol>
	</li>
	<li>Amplify the targeted areas by mutagenic PCR. Create overlapping areas between segments (~50 bp each) by superimposing PCR reactions of the defined regions.
	<ol>
		<li>Prepare mutagenic PCR of targeted segments in a final volume of 50 &#181;l containing DNA template (0.92 ng/&#181;l), 90 nM oligo sense (RMLN for segment M-I and AAO-BP for segment M-II), 90 nM antisense primer (AAO-92C for segment M-I and RMLC for segment M-II) , 0.3 mM dNTPs (0.075 mM each), 3% (v/v) dimethylsulfoxide (DMSO), 1.5 mM MgCl2, 0.05 mM MnCl2 and 0.05 U/&#181;l Taq DNA polymerase. Primers sequences are detailed in Figure 1.</li>
		<li>Use the following PCR program: 95 &#176;C for 2 min (1 cycle); 95 &#176;C for 45 sec, 50 &#176;C for 45 sec, 74 &#176;C for 45 sec (28 cycles); and 74 &#176;C for 10 min (1 cycle).</li>
	</ol>
	</li>
	<li>Amplify the non-mutagenic regions with ultra-high fidelity polymerase and include the corresponding areas overlapping the mutagenic segments and/or linearized vector overhangs.
	<ol>
		<li>Prepare reaction mixtures in a final volume of 50 &#181;l containing: DNA template (0.2 ng/&#181;l), 250 nM oligo sense HFF, 250 nM oligo antisense HFR, 0.8 mM dNTPs (0.2 mM each), 3% (v/v) dimethylsulfoxide (DMSO) and 0.02 U/&#181;l iproof DNA polymerase. Primers sequences are detailed in Figure 1.</li>
		<li>Use the following PCR program: 98 &#176;C for 30 sec (1 cycle); 98 &#176;C for 10 sec, 55 &#176;C for 25 sec, 72 &#176;C for 45 sec (28 cycles); and 72 &#176;C for 10 min (1 cycle). 
		Note: With conditions described in 1.2 and 1.3 overlaps of 43 bp (plasmid-M1 region); 46 bp (M1 region-HF region); 47 bp (HF region-M2 region) and 61 bp (M2 region- plasmid) are designed (Figure 1) to favor in vivo splicing in yeast.</li>
		<li>Purify all the PCR fragments (mutagenic and non-mutagenic) with a commercial gel extraction kit according to manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Linearize the vector such that flanking regions of approximately 50 bp are created that are homologous to the 5&#180;- and 3&#180;-ends of the target gene.
	<ol>
		<li>Prepare a linearization reaction mixture containing 2 &#181;g DNA, 7.5 U BamHI, 7.5 U XhoI, 20 &#181;g BSA and 2 &#181;l of Buffer BamHI 10x in a final volume of 20 &#181;l.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 2 hr and 40 min. Afterwards, proceed with inactivation at 80 &#176;C for 20 min.</li>
	</ol>
	</li>
	<li>Purify the linearized vector by agarose gel extraction to avoid contamination with the residual circular plasmid (Figure 2).
	<ol>
		<li>Load the digestion reaction mix into the mega-well of a semi-preparative low melting point agarose gel (0.75%, w:v) as well as an aliquot (5 &#181;l) of the reaction mix in the adjacent well as a reporter.</li>
		<li>Run DNA electrophoresis (5 V/cm between electrodes, 4 &#7506;C) and separate the agarose gel corresponding to the mega-well and store it at 4 &#7506;C in 1x TAE.</li>
		<li>Stain the lane with the molecular weight ladder and the reporter. Visualize the bands under UV light. Nick the position where the linearized vector places. 
		Note: As the quality of the purified linearized vector is a critical factor for successful recombination and assembly in yeast, avoid gel staining for semi-preparative DNA electrophoresis. The use of dyes and UV exposure for gel extraction may affect the stability of the DNA vector, compromising the in vivo recombination efficiency. As alternative to toxic EtBr dyes, Gel Red and SYBR dyes are commonly used for gel staining.</li>
		<li>In the absence of UV light, identify the linearized vector in the mega-well fragment using the guidance of the nicks in the stained reporter lane so that it can be isolated.</li>
		<li>Extract the linearized vector from agarose and purify it with a commercial gel extraction kit according to manufacturer&#39;s protocol. 
		Note: Use high-copy episomal shuttle vectors with antibiotic and auxotrophy markers: In this example we employed the uracil independent and ampicillin resistance pJRoC30 vector, under the control of the yeast GAL1 promoter.</li>
	</ol>
	</li>
	<li>Prepare an equimolar mixture of the PCR fragments and mix it with the linearized vector at a 2:1 ratio, with no less than 100 ng of linearized plasmid (test different ratios of equimolar library/open vector to achieve good transformation yields).
	<ol>
		<li>Measure the absorbance of the PCR fragments and linearized vector at 260 nm and 280 nm to determine their concentration and purity.</li>
	</ol>
	</li>
	<li>Transform yeast competent cells with the DNA mixture using a commercial yeast transformation kit (see Table for supplies) according to manufacturer&#39;s instructions.
	<ol>
		<li>Here, use a protease deficient and URA3- dependent S. cerevisiae strain, BJ5465. Transform the cells with the parental circularized vector as an internal standard during screening (see below). Additionally, check the background by transforming the linearized vector in the absence of PCR fragments. 
		Note: In case of detecting initial low secretion levels, use S. cerevisiae protease deficient strains like BJ5465 to foster the accumulation of active protein in culture supernatants. If the target enzyme undergoes hyperglycosylation, the use of glycosylation-deficient strains (e.g., &#916;kre2 that is only capable of attaching smaller mannose oligomers) could be a suitable option.</li>
	</ol>
	</li>
	<li>Plate the transformed cells on SC drop-out plates and incubate them at 30 &#176;C for three days. Plate (on SC drop-out plates supplemented with uracil) URA3-S. cerevisiae cells lacking the plasmid as a negative control for screening (see below).</li>
</ol>
2. High-Throughput Screening Assay (Figure 3)
<ol>
	<li>Fill an appropriate number of sterile 96-well plates (23 plates to analyze a library of 2,000 clones) with 50 &#181;l minimal medium per well with the help of a pipetting robot.</li>
	<li>Pick individual colonies from the SC-drop out plates and transfer them to the 96-well plates.
	<ol>
		<li>In each plate, inoculate column number 6 with the parental type as an internal standard and well H1 with URA3-S. cerevisiae cells (in SC medium supplemented with uracil) with no plasmid as a negative control. 
		Note: Well H1 is filled specifically with drop-out media supplemented with uracil. A blank well containing media without cells can be also prepared as an additional sterility control.</li>
	</ol>
	</li>
	<li>Cover the plates with their lids and wrap them in Parafilm. Incubate plates for 48 hr at 30 &#176;C, 225 rpm and 80% relative humidity in a humid shaker.</li>
	<li>Remove the Parafilm, add 160 &#181;l of expression medium to each well with the help of the pipetting robot, reseal the plates and incubate them for a further 24 hr. 
	Note: Minimal medium and expression medium are prepared as reported elsewhere19. Secretion levels may vary depending on the gene under study and accordingly, the incubation times must be optimized in each case to synchronize the cell growth in all the wells.</li>
	<li>Centrifuge the plates (master plates) at 2,800 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer 20 &#181;l of the supernatant from the wells in the master plate to the replica plate using a liquid handling robotic multistation. 
	Note: To favor enzyme secretion it is advisable to replace the native signal peptide of the target protein by signal peptides commonly used for heterologous expression in yeast (e.g., the &#945; factor prepro-leader, the leader of the K1 Killer toxin from S. cerevisiae, or even chimeric versions of both peptides13). Alternatively, the native signal peptide can be exclusively evolved for secretion in yeast.</li>
	<li>Add 20 &#181;l of 2 mM p-methoxybenzylalcohol in 100 mM sodium phosphate buffer pH 6.0 with the help of the pipetting robot. Stir the plates briefly with a 96-well plate mixer and incubate them for 30 min at RT.</li>
	<li>With the pipetting robot, add 160 &#181;l of the FOX reagent to each replica plate and stir briefly with the mixer (final concentration of FOX mixture in the well: 100 &#181;M xylenol orange, 250 &#181;M Fe(NH4)2(SO4)2 and 25 mM H2SO4).
	<ol>
		<li>Add several additives to the reagent to enhance sensitivity, such as organic co-solvents (DMSO, ethanol, methanol) or sorbitol17. Here, amplify the response by adding sorbitol to a final concentration of 100 mM (Figure 4).</li>
	</ol>
	</li>
	<li>Read the plates (end-point mode, t0) at 560 nm on a plate reader.</li>
	<li>Incubate the plates at RT until the color develops and measure the absorption again (t1).
	<ol>
		<li>Calculate the relative activity from the difference between the Abs value after incubation and that of the initial measurement normalized to the parental type for each plate (&#916;t1&#160;- t0).</li>
	</ol>
	</li>
	<li>Subject the best mutant hits to two consecutive re-screenings to rule out false positives. 
	Note: Typically, re-screenings include plasmid isolation from yeast, amplification and purification in Escherichia coli, followed by transformation of fresh yeast cells with the plasmid19. Each selected clone is re-screened in pentaplicate.</li>
</ol>

<ol><li>Jäckel, C., Hilvert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biocatalysts+by+evolution%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">Biocatalysts by evolution.</a> Curr. Opin. Biotechnol. 21 (6), 753-759 (2010).</li>
<li>Bornscheuer, U. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+the+third+wave+of+biocatalysis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Engineering the third wave of biocatalysis.</a> Nature. 485 (7397), 185-194 (2012).</li>
<li>Renata, H., Wang, Z. W., Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+enzyme+universe%3A+accessing+non-natural+reactions+by+mechanism-guided+directed+evolution%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.</a> Angew. Chem. Int. Ed. 54 (11), 3351-3367 (2015).</li>
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<li>Gonzalez-Perez, D., Garcia-Ruiz, E., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Saccharomyces+cerevisiae+in+directed+evolution%3A+an+efficient+tool+to+improve+enzymes%5BTitle%5D)+AND+%22Bioeng+Bugs%22%5BJournal%5D)">Saccharomyces cerevisiae in directed evolution: an efficient tool to improve enzymes.</a> Bioeng Bugs. 3, 172-177 (2012).</li>
<li>Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutagenesis+protocols+in+Saccharomyces+cerevisiae+by+In+Vivo+Overlap+Extension%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Mutagenesis protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension.</a> Methods Mol. Biol. 634, 3-14 (2010).</li>
<li>Bulter, T., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparing+libraries+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Methods.+Mol.+Biol%22%5BJournal%5D)">Preparing libraries in Saccharomyces cerevisiae.</a> Methods. Mol. Biol. 231, 17-22 (2003).</li>
<li>Ostrov, N., Wingler, L. M., Cornish, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+assembly+and+combinatorial+libraries+in+S.+cerevisiae+via+reiterative+recombination%5BTitle%5D)+AND+%22Methods.+Mol.+Biol%22%5BJournal%5D)">Gene assembly and combinatorial libraries in S. cerevisiae via reiterative recombination.</a> Methods. Mol. Biol. 978, 187-203 (2013).</li>
<li>Shao, Z., Zhao, H., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+assembler%2C+an+in+vivo+genetic+method+for+rapid+construction+of+biochemical+pathways%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways.</a> Nucleic Acids Res. 37 (2), e16(2009).</li>
<li>Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+the+ligninolytic+enzyme+consortium%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Engineering the ligninolytic enzyme consortium.</a> Trends Biotechnol. 33 (3), 155-162 (2015).</li>
<li>Garcia-Ruiz, E. Directed evolution of ligninolytic oxidoreductases: from functional expression to stabilization and beyond. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGarcia-Ruiz%2C+E+%22Cascade+Biocatalysis%3A+integrating+stereoselective+and+environmentally+friendly+reactions%22">Cascade Biocatalysis: integrating stereoselective and environmentally friendly reactions</a>. , Wiley-VCH. 1-22 (2014).</li>
<li>Hernandez-Ortega, A., Ferreira, P., Martinez, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fungal+aryl-alcohol+oxidase%3A+a+peroxide-producing+flavoenzyme+involved+in+lignin+degradation%5BTitle%5D)+AND+%22Appl.+Microbiol.+Biotechnol%22%5BJournal%5D)">Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation.</a> Appl. Microbiol. Biotechnol. 93 (4), 1395-1410 (2012).</li>
<li>Gonzalez-Perez, D., Molina-Espeja, P., Garcia-Ruiz, E., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutagenic+organized+recombination+process+by+homologous+in+vivo+grouping+%28MORPHING%29+for+directed+enzyme+evolution%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) for directed enzyme evolution.</a> PLoS One. 9, e90919(2014).</li>
<li>Rhee, S. G., Chang, T., Jeong, W., Kang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+Detection+and+Measurement+of+Hydrogen+Peroxide+Inside+and+Outside+of+Cells%5BTitle%5D)+AND+%22Mol.+Cells%22%5BJournal%5D)">Methods for Detection and Measurement of Hydrogen Peroxide Inside and Outside of Cells.</a> Mol. Cells. 29 (6), 539-549 (2010).</li>
<li>Sebestova, E., Bendl, J., Brezovsky, J., Damborsky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+tools+for+designing+smart+libraries%5BTitle%5D)+AND+%22Methods.+Mol.+Biol%22%5BJournal%5D)">Computational tools for designing smart libraries.</a> Methods. Mol. Biol. 1179, 291-314 (2014).</li>
<li>Viña-Gonzalez, J., Gonzalez-Perez, D., Ferreira, P., Martinez, A. T., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focused+directed+evolution+of+aryl-alcohol+oxidase+in+yeast+using+chimeric+signal+peptides%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Focused directed evolution of aryl-alcohol oxidase in yeast using chimeric signal peptides.</a> Appl. Environ. Microbiol. , (2015).</li>
<li>Gay, C., Collins, J., Gebicki, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydroperoxide+Assay+with+the+Ferric-Xylenol+orange+Complex%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Hydroperoxide Assay with the Ferric-Xylenol orange Complex.</a> Anal. Biochem. 273 (2), 149-155 (1999).</li>
<li>Reetz, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biocatalysis+in+organic+chemistry+and+biotechnology%3A+Past%2C+present%2C+and+future%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Biocatalysis in organic chemistry and biotechnology: Past, present, and future.</a> J. Am. Chem. Soc. 135 (34), 12480-12496 (2013).</li>
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</ol>

The authors have nothing to disclose.

In this article, we have summarized most of the tips and tricks employed in our laboratory to engineer enzymes by directed evolution in S. cerevisiae (using AAO as an example) so that they can be adapted for use with many other eukaryotic enzyme systems by simply following the common approach described here.
In terms of library creation, MORPHING is a fast one-pot method to introduce and recombine random mutations in small protein stretches while leaving the remaining regions of the p...

Directed molecular evolution is a robust, fast and reliable method to design enzymes1, 2. Through iterative rounds of random mutation, recombination and screening, improved versions of enzymes can be generated that act on new substrates, in novel reactions, in non-natural environments, or even to assist the cell to achieve new metabolic goals3-5. Among the hosts used in directed evolution, the brewer&#39;s yeast Saccharomyces cerevisiae offers a repertoire of solutions for the functional expression of complex eukaryotic proteins that are not otherwise available in prokaryotic counterparts6,7.
Used exhaustively in cell biology studies, this small eukaryotic model has many advantages in terms of post-translational modifications, ease of manipulation and transformation efficiency, all of which are important traits to engineer enzymes by directed evolution8. Moreover, the high frequency of homologous DNA recombination in S. cerevisiae coupled to its efficient proof-reading apparatus opens a wide array of possibilities for library creation and gene assembly in vivo, fostering the evolution of different systems from single enzymes to complex artificial pathways9-12. Our laboratory has spent the past decade designing tools and strategies for the molecular evolution of different ligninases in yeast (oxidoreductases involved in the degradation of lignin during natural wood decay)13-14. In this communication, we present a detailed protocol to prepare and screen mutant libraries in S. cerevisiae for a model flavooxidase, -aryl-alcohol oxidase (AAO15)-, that can be easily translated to many other enzymes. The protocol involves a focused-directed evolution method (MORPHING: Mutagenic Organized Recombination Process by Homologous in vivo Grouping) assisted by the yeast cell apparatus16, and a very sensitive screening assay based on the Fenton reaction in order to detect AAO activity secreted into the culture broth17.

<table><tbody><tr><td>&lt;strong&gt;1. Culture media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A0166</td><td>CAS N&amp;ordm; 69-52-3 M.W. 371.39</td></tr><tr><td>Bacto Agar</td><td>Difco</td><td>214010</td><td></td></tr><tr><td>Cloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td>CAS N&amp;ordm; 56-75-7 M.W. 323.13</td></tr><tr><td>D-(+)-Galactose</td><td>Sigma-Aldrich</td><td>G0750</td><td>CAS N&amp;ordm; 59-23-4 M.W. 180.16</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>CAS N&amp;ordm; 50-99-7 M.W. 180.16</td></tr><tr><td>D-(+)-Raffinose pentahydrate</td><td>Sigma-Aldrich</td><td>83400</td><td>CAS N&amp;ordm; 17629-30-0 M.W. 594.51</td></tr><tr><td>Peptone</td><td>Difco</td><td>211677</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P0662</td><td>CAS N&amp;ordm; 7778-77-0 M.W. 136.09</td></tr><tr><td>Uracil</td><td>Sigma Aldrich</td><td>U1128</td><td></td></tr><tr><td>Yeast Extract</td><td>Difco</td><td>212750</td><td></td></tr><tr><td>Yeast Nitrogen Base without Amino Acids</td><td>Difco</td><td>291940</td><td></td></tr><tr><td>Yeast Synthetic Drop-out Medium Supplements without uracil</td><td>Sigma-Aldrich</td><td>Y1501</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. PCR Reactions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>dNTP Mix</td><td>Agilent genomics</td><td>200415-51</td><td>25 mM each</td></tr><tr><td>iProof High-Fidelity DNA polymerase</td><td>Bio-rad</td><td>172-5301</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M8054</td><td>CAS N&amp;ordm; 13446-34-9 M.W. 197.91</td></tr><tr><td>Taq DNA Polymerase</td><td>Sigma-Aldrich</td><td>D4545</td><td>For error prone PCR</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Plasmid linearization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BamHI restriction enzyme</td><td>New England Biolabs</td><td>R0136S</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>XhoI restriction enzyme</td><td>New England Biolabs</td><td>R0146S</td><td></td></tr><tr><td>Not I restriction enzyme</td><td>New England Biolabs</td><td>R0189S</td><td></td></tr><tr><td>Gel Red</td><td>Biotium</td><td>41003</td><td>For staining DNA</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;4. FOX assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonium iron(II) sulfate hexahydrate</td><td>Sigma-Aldrich</td><td>F3754</td><td>CAS N&amp;ordm; 7783-85-9 M.W. 392.14</td></tr><tr><td>Anysil Alcohol</td><td>Sigma Aldrich</td><td>W209902</td><td>CAS N&amp;ordm; 105-13-5 M.W. 138.16</td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td>CAS N&amp;ordm; 50-70-4 M.W. 182.17</td></tr><tr><td>Hydrogen peroxide 30%</td><td>Merck Millipore</td><td>1072090250</td><td>FOX standard curve</td></tr><tr><td>Xylenol Orange disodium salt</td><td>Sigma-Aldrich</td><td>52097</td><td>CAS N&amp;ordm; 1611-35-4 M.W. 716.62</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;5. Agarose gel stuff&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Norgen</td><td>28035</td><td>CAS N&amp;ordm; 9012-36-6</td></tr><tr><td>Gel Red</td><td>Biotium</td><td>41003</td><td>DNA analysis dye</td></tr><tr><td>GeneRuler 1kb Ladder</td><td>Thermo Scientific</td><td>SM0311</td><td>DNA M.W. standard</td></tr><tr><td>Loading Dye 6x</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>Low-melting temperature agarose</td><td>Bio-rad</td><td>161-3112</td><td>CAS N&amp;ordm; 39346-81-1</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;6. Kits and cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>S. cerevisiae strain BJ5465</td><td>LGC Promochem</td><td>ATTC 208289</td><td>Protease deficient strain with genotype: MAT&amp;alpha; ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL</td></tr><tr><td>E. coli XL2-Blue competent cells</td><td>Agilent genomics</td><td>200150</td><td>For plasmid purification and amplification</td></tr><tr><td>NucleoSpin Gel and PCR Clean-up Kit</td><td>Macherey-Nagel</td><td>740,609,250</td><td>DNA gel extraction</td></tr><tr><td>NucleoSpin Plasmid Kit</td><td>Macherey-Nagel</td><td>740,588,250</td><td>Column miniprep Kit</td></tr><tr><td>Yeast Transformation Kit</td><td>Sigma-Aldrich</td><td>YEAST1-1KT</td><td>Included DNA carrier (Salmon testes)</td></tr><tr><td>Zymoprep yeast plasmid miniprep I</td><td>Zymo research</td><td>D2001</td><td>Plasmid extraction from yeast</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;7. Plates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well plates</td><td>Greiner Bio-One</td><td>655101</td><td>Clear, non-sterile, polystyrene (for activity measurements)</td></tr><tr><td>96-well plates</td><td>Greiner Bio-One</td><td>655161</td><td>Clear, sterile, polystyrene (for microfermentations)</td></tr><tr><td>96-well plate lid</td><td>Greiner Bio-One</td><td>656171</td><td>Clear, sterile, polystyrene (for microfermentations)</td></tr></tbody></table>

directed evolution method in saccharomyces cerevisiae: mutant library creation and screening

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

AAO from P. eryngii is an extracellular flavooxidase that supplies fungal peroxidases with H2O2 to start attacking lignin. Two segments of AAO were subjected to focused-directed evolution by MORPHING in order to enhance its activity and its expression in S. cerevisiae 19. Irrespective of the foreign enzymes harbored by S. cerevisiae, the most critical issue when constructing mutant libraries in yeast concerns the engineering of s...

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

The overall goal of this protocol is to show how to construct and screen mutant libraries in Saccharomyces Cerevisiae for directed evolution experiments. This method can help answer key questions in laboratory creation use for focused directed evolution experiments such as how to assign overlapping areas for in the assembly and cloning. The main advantage of this technique is its simplicity and robustness since in just one transformation step it's something levels with good quality can be assigned.This protocol for creating and screening mutant libraries will be demonstrated for a fungal aryl-alcohol oxidase or AAO. The regions for focused directed evolution are first chosen with the help of computational algorithms based on the available crystal structure or homology models of the enzyme. Two regions of AAO will be targeted for random mutagenesis and recombination:the M1 region and the M2 region.Mutagenic PCR will be performed to amplify the targeted areas. To promote in vivo splicing in yeast, overlapping areas between segments of approximately 50 base pairs each will be created by superimposing PCR reactions of the defined regions. Prepare 50 microliter volume mutagenic PCRs, each containing 46 nanograms of DNA template 90 nanomolar each of sense and antisense primers 0.3 milimolar of DNTPs, 3%of DMSO, 1.5 milimolar of magnesium chloride, 05 milimolar of manganese chloride, and 05 units per microliter of tack DNA polymerase.Use the following PCR program. 95 degrees Celsius for two minutes, 95 degrees Celsius for 45 seconds, 50 degrees Celsius for 45 seconds, and 74 degrees Celsius for 45 seconds for 28 cycles, and 74 degrees Celsius for 10 minutes. The remainder of the AAO gene, the HF region, will be amplified by high fidelity PCR with the corresponding areas overlapping the mutagenic segments, and or linearized vector overhangs included.Prepare the high-fidelity PCR in a final volume of 50 microliters containing 10 nanograms of DNA template, 250 nanomolar each of sense and antisense primers, 0.8 milimolar of DNTPs, 3%of DMSO, and 02 units per microliter of iProof ultra high-fidelity DNA polymerase. Use the following PCR program. 98 degrees Celsius for 30 seconds, 98 degrees Celsius for 10 seconds, 55 degrees Celsius for 25 seconds, and 72 degrees Celsius for 45 seconds for 28 cycles, and 72 degrees Celsius for 10 minutes.All the PCR fragments are then purified with a commercial gel-extraction kit according to the manufacturer's protocol. The next step is to linearize the vector to create flanking regions of approximately 50 base pairs that are homologous to the five prime and three prime ends of the target gene. Prepare a 20 micoliter linearization reaction mixture containing 2 micrograms of DNA, 7.5 units of BamH-one, 7.5 units of XHO-one, 20 micrograms of BSA, and two microliters of 10x buffer BamH-one.Incubate the reaction mixture at 37 degrees Celsius for two hours and 40 minutes. After that, inactivate the reaction at 80 degrees Celsius for 20 minutes. To purify the linearized vector to avoid contamination with the residual circular plasmid, load the digestion reaction mix into the mega-well of a semi-preparative low-melting point Agarose gel.Load five microliters of the reaction mix in the adjacent well as a reporter. Run DNA electrophoresis at four degrees Celsius and five volts per centimeter between electrodes. Then separate the Agarose gel corresponding to the mega-well and store it at four degrees Celsius in one XTAE.Stain the lane with the molecular weight ladder and the reporter. Visualize the bands under UV light, and nick the position where the linearized vector places. In the absence of UV light and using the guidance of the nicks in the stained reporter lane, identify the linearized vector in the mega-well fragment and excise it.Extract the linearized vector from the Agarose and purify it with a commercial gel extraction kit according to the manufacturer's protocol. Subsequently, the purified linearized vector is mixed with the PCR fragments and this DNA mixture is used to transform competent yeast cells using a commercial kit according to the manufacturer's instructions. Plate the transformed cells on SC dropout plates and incubate them at 30 degrees Celsius for three days.To prepare for this assay, pick individual colonies from the SC dropout plates and transfer them to 96 well-plates containing 50 microliters of minimal medium per well. In each plate inoculate column number six with the parental type as an internal standard. As a negative control, inoculate well H-one filled with SC mediums supplemented with uracil with URA3 negative ESI visceral cells with no plasmid.Cover the plates with their lids, wrap them in para film, and incubate at 30 degrees Celsius in a humid shaker for 48 hours. After 48 hours, add 160 microliters of expression medium to each well and reseal the plates. Incubate for a further 24 hours.After centrifuging the plates use a liquid-handling robotic multi-station to transfer 20 microliters of the supernatant from the wells in each plate to a replica plate. Add 20 microliters of two milimolar P-methoxybenzyl alcohol and 100 milimolars sodium phosphate buffer ph 6.0 to each well. Stir the plates briefly with a 96-well plate mixer and incubate them for 30 minutes at room temperature.Next, add 160 microliters of the FOX reagant to each replica plate and stir briefly with the mixer. Read the plates at 560 nanometers on a plate reader. Incubate the plates at room temperature until the color develops.Measure the absorption again. The relative activity is calculated from the difference between the absorption value after incubation and that of the initial measurement normalized to the parental type for each plate. This representative gel image shows the linearized vector in lane two, the M1 PCR segment in lane three, the M2 PCR segment in lane four, the HF PCR segment in lane five, and the in vivo reassembled vector linearized with NHE one in lane six.This next gel shows the plasmid mini-prep of the reassembled vector in lane two, the plasmid linearized with NHE one in lane three, the plasmid linearized with BamH one and XHO one in lane four, and the linearized plasmid obtained by gel extraction and cleanup in lane five. Mutational loads were adjusted by sampling mutant libraries with different landscapes calculating the number of clones with less than 10%of the parental enzyme activity and further verification by sequencing a random sample of active and non-active variants. An evaluation of the detection limit of this assay showed that the assay was linear in the presence of sorbitol represented by black circles.In the absence of sorbitol represented by the white squares, linearity was more persistent but the response was weaker. A linear correlation was observed between AAO concentration and absorbance. A mutant library of 2, 000 clones was constructed and screened with this assay.The shadowed square indicates the AAO mutants with notably improved secretion and activity against P-methoxybenzyl alcohol. Once mastered, mutant libraries can be made in approximately four hours if it is performed properly. When attempting this procedure it is important to remember to assign proper overlapping areas for each PCR family and linearized vector for in vivo splicing and cloning in a single transformational step.Following a similar approach, other methods like in vivo DNA sampling, saturation mutagenesis library or DNA assembly can be performed in order to construct mutant libraries and, or metabolic pathways. After its development, this technique paved the way for researchers in the field of bioengineering to explore activities, stabilities, or even to invent infinite kinds of interaction. After watching this video, you should have a good understanding of how to harness this Saccharomyces Cerevisiae device for library construction and screening

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Research

JoVE Journal

Biology

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitive Genomic Screens of Barcoded Yeast Libraries

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Mating-based Overexpression Library Screening in Yeast

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and  selective  for  mutations that  often  occur at   a  single  genomic  locus.  The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein Engineering by Yeast Surface Display

Learn directed evolution using computational algorithms to select target regions for mutagenesis in Saccharomyces cerevisiae. This method constructs and screens mutant libraries to enhance fungal aryl-alcohol oxidase activity.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

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