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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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 protein unaltered16. Libraries with several mutational loads can be readily prepared and recombined in vivo, along with the linearized plasmid, to generate a full autonomously replicating vector. It is critical that overlapping sequences flank each stretch to allow the fragments of the full gene to be reassembled through in vivo recombination, avoiding extra PCR reactions and in vitro ligation steps. In this protocol, the frequency of crossover events between PCR fragments can be increased by reducing the size of the overlapping regions, although this may compromise the transformation efficiency. Regardless of the DNA polymerases used for mutagenic PCR, the mutational loads can be adjusted by previously constructing and analyzing small mutant library landscapes (Figure 5A). If the GeneMorph II Kit is used, it is still advisable to follow this approach since in vivo DNA recombination can notably modify the mutational loads estimated by the manufacturer. In general terms, mutant landscapes in which 35 - 50% of the total clones screened have less than 10% of the parental activity are suitable for directed evolution campaigns, although this number varies in function of the target protein and its activity. Typically, the analysis of mutant libraries landscapes are further verified by DNA sequencing of a random sample of mutants. In the current example, the Taq DNA polymerase was used due to its high error rate, which is linked to the lack of 3&#180;&#8594;5&#180;proof-reading exonuclease activity. The mutational loads in Taq libraries were modified by the addition of different concentrations of MnCl2, but the use of unbalanced dNTPs and/or the reduction in gene template concentrations are also suitable options. Inherent limitations of MORPHING come from the number of segments to be recombined. According to our experience, up to four protein blocks (five crossover events counting the recombination areas with the linearized vector) can be spliced with good transformation yields (~105 clones per transformation reaction). This method can be easily modified to performed multiple site-saturation mutagenesis (e.g., using NDT degenerated primers or creating degeneracy for 22 unique codons) to explore several positions simultaneously while reducing significantly the screening efforts21,22.
The direct &#34;blind&#34; screening protocol for AAO is extremely sensitive and reliable (based on the direct detection of H2O2 regardless of the substrate used by the enzyme), representing a complementary assay to other well established indirect protocols to detect peroxides (mostly coupling peroxidases with colorimetric substrates). Indeed, the FOX assay has been routinely employed to measure H2O2 in biological fluids, and it can now be easily translated into protocols to evolve AAO and any other H2O2 producing enzymes (e.g., glucose oxidases, cellobiose dehydrogenases, glyoxal oxidases, methanol oxidases), particularly for activity on non-natural substrates where responses are otherwise hard to detect.
S. cerevisiae is the most adequate host for directed evolution of eukaryotic genes since it offers high transformation efficiencies (up to 1 x 106 transformants/&#181;g DNA), it performs complex post-translational processing and modifications (including N- and C-terminal processing, and glycosylation) and it exports foreign proteins into the culture broth via a secretory pathway. In addition, well-established molecular biology tools are available to work with this yeast, including uni- or bi-directional episomal (non-integrative) shuttle vectors under the control of promoters of different strengths. Last but not least, its high frequency of homologous DNA recombination has allowed a range of methods to be developed to obtain DNA diversity that are currently being used to evolve single proteins, as well as more complex enzyme pathways8, 12, 13, 23. The in vivo gap repair and the proof-reading device of this yeast can be also employed to create chimeras when recombining different genes (with approx. 60% of DNA sequence identity), as well as to shuffle best offspring/mutations from a directed evolution campaign, or to bring together in vitro and in vivo recombination methods in one round of evolution, thereby enriching mutant libraries in terms of foldability and function.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>CAS N&#186; 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>C-0378</td>
			<td>CAS N&#186; 56-75-7 M.W. 323.13</td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>CAS N&#186; 59-23-4 M.W. 180.16</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>CAS N&#186; 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&#186; 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&#186; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>2. PCR Reactions</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&#186; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3. Plasmid linearization</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>Gel Red</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>For staining DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4. FOX assays</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&#186; 7783-85-9 M.W. 392.14</td>
		</tr>
		<tr>
			<td>Anysil Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>W209902</td>
			<td>CAS N&#186; 105-13-5 M.W. 138.16</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td>CAS N&#186; 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&#186; 1611-35-4 M.W. 716.62</td>
		</tr>
		<tr>
			<td>Agarose gel stuff</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Norgen</td>
			<td>28035</td>
			<td>CAS N&#186; 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&#186; 39346-81-1</td>
		</tr>
	
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5. Kits and cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae strain BJ5465</td>
			<td>LGC Promochem, Spain</td>
			<td>ATTC 208289</td>
			<td>Protease deficient strain with genotype: MAT&#945; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>6. Plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655101</td>
			<td>Clear, non-sterile, Polystyrene (for activity measurements)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655161</td>
			<td>Clear, sterile, Polystyrene (for microfermentations)</td>
		</tr>
		<tr>
			<td>96-well plate lid</td>
			<td>Greioner 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 specific overlapping regions to favor the splicing between fragments and their cloning into the linearized vector. In the current example, for each PCR reaction, all the fragments had overhangs of approximately 50 bp to promote in vivo splicing in yeast. The number of recombination events is dependent on the number of segments to be assembled and cloned with the linearized vector (i.e., two crossover events took place between the three PCR segments -the two mutagenic segments flanking the non-mutagenized segment- plus two additional crossovers with the linearized vector; Figure 1). According to our experience, overlapping sequences longer than 50 bp decrease the likelihood of internal recombination while they do not improve transformation efficiency.
Mutational loads were adjusted by sampling mutant libraries with different landscapes, calculating the number of clones with &#60;10% of the parental enzyme activity, and further checking them by sequencing a random sample of active and non-active variants (Figure 5A). For the determination of the coefficient of variance S. cerevisiae cells were transformed with the parental AAO and plated on SC-drop out plates. Individual colonies were picked and inoculated in a 96 well-plate and the activity of the clones was evaluated from fresh preparations. Mutagenic sample 2 (Taq/MnCl2 0.05 mM) was chosen as the departure point for library construction and screening.
As the biological activity of AAO increases the H2O2 concentration in the reaction medium, we searched for a sensitive and accurate assay to quantify minor changes in H2O2. FOX is a chemical method based on the Fenton reaction20, whereby oxidation by H2O2 drives the reaction of Fe3+ with xylenol orange to form a blue-purple complex (o-cresolsulfone-phthalein 3&#39;,3&#39;&#39;-bis(methylimino)diacetate &#949;560 = 1.5 x 104 M-1cm-1). The ferrous oxidation step was amplified by adding sorbitol to enhance the sensitivity of the assay, increasing the propagation of radicals with an apparent &#949;560 = 2.25 x 105 M-1cm-1 (Figure 4).
The detection limit of this assay (in the &#181;M range) was calculated by the Blank determination method in a 96-well plate with standards in triplicate (0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 &#181;M H2O2) and using several supernatants from S. cerevisiae lacking the URA3- plasmid (Figure 5B). The assay was linear in the presence of sorbitol (up to 8 &#181;M of H2O2), and although linearity was more persistent in the absence of this sugar (at least up to 30 &#181;M of H2O2) the response was weaker (e.g., at 6 &#181;M of H2O2, a 4-fold enhancement was obtained in the presence of sorbitol -deep purple- from an absorbance of 0.24 in its absence -dark orange- (Figure 5B)). The relationship between Abs and the AAO concentration was evaluated with increasing amounts of enzyme (from yeast supernatants) and a linear response was observed; R2&#160;= 0.997 (Figure 5C).
It is notable that the FOX signal was stable for several hours without any apparent interference by the different elements in the culture broth. The estimated sensitivity of FOX was ~0.4 &#181;M of H2O2 produced by the AAO in the supernatant in the presence of sorbitol, and ~2 &#181;M in its absence.
A mutant library of 2,000 clones was constructed and screened with this assay. Several AAO mutants were identified with notably improved secretion and activity against p-methoxybenzyl alcohol (Figure 5D)19.
<img alt="Figure 1" src="/files/ftp_upload/53761/53761fig1.jpg" /> 
Figure 1.&#160;MORPHING Protocol for AAO Evolution. Two different regions of AAO were targeted for random mutagenesis and recombination: M1 (blue, 590 bp) that includes the signal peptide (SP); M2 (yellow, 528 bp). The HF region (grey, 844 bp) was amplified with high fidelity polymerases. Mutagenic regions were mapped in the crystal structure of AAO (PDB ID: 3FIM). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53761/53761fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53761/53761fig2.jpg" /> 
Figure 2. Preparation of PCR Products and the Linearized Vector. (A) Analytical agarose gel (1% w:v) containing a molecular weight marker (1 kb ladder) in lanes 1 and 7; the BamHI and XhoI linearized vector, lane 2; PCR segment M1, lane 3; PCR segment M2, lane 4; PCR segment HF, lane 5; the in vivo reassembled vector linearized with NheI (containing the full AAO gene with regions M-I, HF and M-II), lane 6. (B) Vector linearization, lanes 1 and 6 molecular standards, 1 Kb ladder; plasmid miniprep, lane 2; plasmid linearized with NheI, lane 3; plasmid linearized with BamHI and XhoI, lane 4; linearized plasmid obtained by gel extraction and clean-up after digestion, lane 5. (C) Protocol for plasmid purification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53761/53761fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53761/53761fig3.jpg" /> 
Figure 3. High-throughput Screening Protocol. Overview of the process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53761/53761fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53761/53761fig4.jpg" /> 
Figure 4. The FOX Method. White-rot fungi attack the cell wall of wood through a Fenton reaction that produces hydroxyl radical OH&#8226;. The FOX method couples this reaction to xylenol orange (XO), and the absorbance of the XO-Fe3+ complex is measured at 560 nm. Ferrous oxidation is amplified by the addition of sorbitol to the reagent mixture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53761/53761fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53761/53761fig5.jpg" /> 
Figure 5. Mutagenic Landscapes for MORPHING Libraries Using Different Error Prone PCR Conditions and Validation of the Screening Assay. (A) MORPHING landscapes. Solid horizontal line shows the activity of the parental type in the assay while the dashed lines indicate the coefficient of variance of the assay. The percentages indicate the number of clones with less than 10% of the parental enzyme activity. Activities are plotted in descending order. (B) The FOX detection limit was evaluated with increasing concentrations of H2O2 in the presence (black circles) and absence (white squares) of sorbitol. (C) Linear correlation between AAO concentration (transformant supernatants) and Abs560nm. Each point corresponds to the average of 8 experiments and includes the standard deviation. (D) Mutant library landscape. The selected variants (shadowed square) were rescreened as reported elsewhere19. Solid line shows the activity of AAO parental type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53761/53761fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Biology

10.8K 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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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 ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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.