We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.
Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that 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.
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'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.
1. Mutant Library Construction
2. High-Throughput Screening Assay (Figure 3)
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 <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',3''-bis(methylimino)diacetate ε560 = 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 ε560 = 2.25 x 105 M-1cm-1 (Figure 4).
The detection limit of this assay (in the µM 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 µM 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 µM of H2O2), and although linearity was more persistent in the absence of this sugar (at least up to 30 µM of H2O2) the response was weaker (e.g., at 6 µM 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 = 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 µM of H2O2 produced by the AAO in the supernatant in the presence of sorbitol, and ~2 µM 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.
Figure 1. 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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3. High-throughput Screening Protocol. Overview of the process. Please click here to view a larger version of this figure.
Figure 4. The FOX Method. White-rot fungi attack the cell wall of wood through a Fenton reaction that produces hydroxyl radical OH•. 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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´→5´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 "blind" 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/µg 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.
The authors have nothing to disclose.
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. Culture media | |||
Ampicillin sodium salt | Sigma-Aldrich | A0166 | CAS Nº 69-52-3 M.W. 371.39 |
Bacto Agar | Difco | 214010 | |
Cloramphenicol | Sigma-Aldrich | C-0378 | 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 | |
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 |
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 |
5. Kits and cells | |||
S. cerevisiae strain BJ5465 | LGC Promochem, Spain | 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 |
6. Plates | |||
96-well plates | Greioner Bio-One | 655101 | Clear, non-sterile, Polystyrene (for activity measurements) |
96-well plates | Greioner Bio-One | 655161 | Clear, sterile, Polystyrene (for microfermentations) |
96-well plate lid | Greioner Bio-One | 656171 | Clear, sterile, Polystyrene (for microfermentations) |