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>

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<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>
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<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>
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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 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...

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

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

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 ...

directed-evolution-method-saccharomyces-cerevisiae-mutant-library

Research

JoVE Journal

Biology

10.9K Views.  Institute of Catalysis, CSIC. We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae. 

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

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

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

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.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Genetics

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

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

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

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

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

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

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

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

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

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

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.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

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

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Optimizing Protein Functions Through Yeast Surface Display Techniques

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

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 enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

Bioengineering

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

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.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

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

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