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

Summary

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

Abstract

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.

Introduction

Directed molecular evolution is a robust, fast and reliable method to design enzymes^{1, 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 goals^3-5. Among the hosts used in directed evolution, the brewer's yeast Saccharomyces cerevisiae offers a repertoire of solutions for the functional expression of complex eukaryotic proteins that are not otherwise available in prokaryotic counterparts^6,7.

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 pathways^9-12. 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.

Protocol

1. Mutant Library Construction

1. Choose the regions to be subjected to MORPHING with the help of computational algorithms based on the available crystal structure or homology models¹⁸.
   1. Here, target two regions of AAO from Pleurotus eryngii for random mutagenesis and recombination (Met[α1]-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.
2. Amplify the targeted areas by mutagenic PCR. Create overlapping areas between segments (~50 bp each) by superimposing PCR reactions of the defined regions.
   1. Prepare mutagenic PCR of targeted segments in a final volume of 50 µl containing DNA template (0.92 ng/µl), 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/µl Taq DNA polymerase. Primers sequences are detailed in Figure 1.
   2. Use the following PCR program: 95 °C for 2 min (1 cycle); 95 °C for 45 sec, 50 °C for 45 sec, 74 °C for 45 sec (28 cycles); and 74 °C for 10 min (1 cycle).
3. Amplify the non-mutagenic regions with ultra-high fidelity polymerase and include the corresponding areas overlapping the mutagenic segments and/or linearized vector overhangs.
   1. Prepare reaction mixtures in a final volume of 50 µl containing: DNA template (0.2 ng/µl), 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/µl iproof DNA polymerase. Primers sequences are detailed in Figure 1.
   2. Use the following PCR program: 98 °C for 30 sec (1 cycle); 98 °C for 10 sec, 55 °C for 25 sec, 72 °C for 45 sec (28 cycles); and 72 °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.
   3. Purify all the PCR fragments (mutagenic and non-mutagenic) with a commercial gel extraction kit according to manufacturer's protocol.
4. Linearize the vector such that flanking regions of approximately 50 bp are created that are homologous to the 5´- and 3´-ends of the target gene.
   1. Prepare a linearization reaction mixture containing 2 µg DNA, 7.5 U BamHI, 7.5 U XhoI, 20 µg BSA and 2 µl of Buffer BamHI 10x in a final volume of 20 µl.
   2. Incubate the reaction mixture at 37 °C for 2 hr and 40 min. Afterwards, proceed with inactivation at 80 °C for 20 min.
5. Purify the linearized vector by agarose gel extraction to avoid contamination with the residual circular plasmid (Figure 2).
   1. 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 µl) of the reaction mix in the adjacent well as a reporter.
   2. Run DNA electrophoresis (5 V/cm between electrodes, 4 ᵒC) and separate the agarose gel corresponding to the mega-well and store it at 4 ᵒC in 1x TAE.
   3. 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.
   4. 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.
   5. Extract the linearized vector from agarose and purify it with a commercial gel extraction kit according to manufacturer'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.
6. 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).
   1. Measure the absorbance of the PCR fragments and linearized vector at 260 nm and 280 nm to determine their concentration and purity.
7. Transform yeast competent cells with the DNA mixture using a commercial yeast transformation kit (see Table for supplies) according to manufacturer's instructions.
   1. 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., Δkre2 that is only capable of attaching smaller mannose oligomers) could be a suitable option.
8. Plate the transformed cells on SC drop-out plates and incubate them at 30 °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).

2. High-Throughput Screening Assay (Figure 3)

1. Fill an appropriate number of sterile 96-well plates (23 plates to analyze a library of 2,000 clones) with 50 µl minimal medium per well with the help of a pipetting robot.
2. Pick individual colonies from the SC-drop out plates and transfer them to the 96-well plates.
   1. 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.
3. Cover the plates with their lids and wrap them in Parafilm. Incubate plates for 48 hr at 30 °C, 225 rpm and 80% relative humidity in a humid shaker.
4. Remove the Parafilm, add 160 µl 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.
5. Centrifuge the plates (master plates) at 2,800 x g for 10 min at 4 °C.
6. Transfer 20 µl 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 α 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.
7. Add 20 µl 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.
8. With the pipetting robot, add 160 µl of the FOX reagent to each replica plate and stir briefly with the mixer (final concentration of FOX mixture in the well: 100 µM xylenol orange, 250 µM Fe(NH₄)₂(SO₄)₂ and 25 mM H₂SO₄).
   1. 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).
9. Read the plates (end-point mode, t₀) at 560 nm on a plate reader.
10. Incubate the plates at RT until the color develops and measure the absorption again (t₁).
    1. 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 (Δt₁ - t₀).
11. 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.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
1. Culture media
Ampicillin sodium salt	Sigma-Aldrich	A0166	CAS Nº 69-52-3 M.W. 371.39
Bacto Agar	Difco	214010
Cloramphenicol	Sigma-Aldrich	C0378	CAS Nº 56-75-7 M.W. 323.13
D-(+)-Galactose	Sigma-Aldrich	G0750	CAS Nº 59-23-4 M.W. 180.16
D-(+)-Glucose	Sigma-Aldrich	G5767	CAS Nº 50-99-7 M.W. 180.16
D-(+)-Raffinose pentahydrate	Sigma-Aldrich	83400	CAS Nº 17629-30-0 M.W. 594.51
Peptone	Difco	211677
Potassium phosphate monobasic	Sigma-Aldrich	P0662	CAS Nº 7778-77-0 M.W. 136.09
Uracil	Sigma Aldrich	U1128
Yeast Extract	Difco	212750
Yeast Nitrogen Base without Amino Acids	Difco	291940
Yeast Synthetic Drop-out Medium Supplements without uracil	Sigma-Aldrich	Y1501
Name	Company	Catalog Number	Comments
2. PCR Reactions
dNTP Mix	Agilent genomics	200415-51	25 mM each
iProof High-Fidelity DNA polymerase	Bio-rad	172-5301
Manganese(II) chloride tetrahydrate	Sigma-Aldrich	M8054	CAS Nº 13446-34-9 M.W. 197.91
Taq DNA Polymerase	Sigma-Aldrich	D4545	For error prone PCR
Name	Company	Catalog Number	Comments
3. Plasmid linearization
BamHI restriction enzyme	New England Biolabs	R0136S
Bovine Serum Albumin	New England Biolabs	B9001S
XhoI restriction enzyme	New England Biolabs	R0146S
Not I restriction enzyme	New England Biolabs	R0189S
Gel Red	Biotium	41003	For staining DNA
Name	Company	Catalog Number	Comments
4. FOX assays
Ammonium iron(II) sulfate hexahydrate	Sigma-Aldrich	F3754	CAS Nº 7783-85-9 M.W. 392.14
Anysil Alcohol	Sigma Aldrich	W209902	CAS Nº 105-13-5 M.W. 138.16
D-Sorbitol	Sigma-Aldrich	S1876	CAS Nº 50-70-4 M.W. 182.17
Hydrogen peroxide 30%	Merck Millipore	1072090250	FOX standard curve
Xylenol Orange disodium salt	Sigma-Aldrich	52097	CAS Nº 1611-35-4 M.W. 716.62
Name	Company	Catalog Number	Comments
5. Agarose gel stuff
Agarose	Norgen	28035	CAS Nº 9012-36-6
Gel Red	Biotium	41003	DNA analysis dye
GeneRuler 1kb Ladder	Thermo Scientific	SM0311	DNA M.W. standard
Loading Dye 6x	Thermo Scientific	R0611
Low-melting temperature agarose	Bio-rad	161-3112	CAS Nº 39346-81-1
Name	Company	Catalog Number	Comments
6. Kits and cells
S. cerevisiae strain BJ5465	LGC Promochem	ATTC 208289	Protease deficient strain with genotype: MATα ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL
E. coli XL2-Blue competent cells	Agilent genomics	200150	For plasmid purification and amplification
NucleoSpin Gel and PCR Clean-up Kit	Macherey-Nagel	740,609,250	DNA gel extraction
NucleoSpin Plasmid Kit	Macherey-Nagel	740,588,250	Column miniprep Kit
Yeast Transformation Kit	Sigma-Aldrich	YEAST1-1KT	Included DNA carrier (Salmon testes)
Zymoprep yeast plasmid miniprep I	Zymo research	D2001	Plasmid extraction from yeast
Name	Company	Catalog Number	Comments
7. Plates
96-well plates	Greiner Bio-One	655101	Clear, non-sterile, polystyrene (for activity measurements)
96-well plates	Greiner Bio-One	655161	Clear, sterile, polystyrene (for microfermentations)
96-well plate lid	Greiner Bio-One	656171	Clear, sterile, polystyrene (for microfermentations)