Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast
© García-Ruiz et al; licensee BioMed Central Ltd. 2010
Received: 3 December 2009
Accepted: 18 March 2010
Published: 18 March 2010
In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.
Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.
The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.
During the last couple of decades, thermostability has been considered by many as a key feature in terms of protein robustness, evolvability and catalytic function [1–4]. From a practical point of view, the engineering of thermo-tolerant biocatalysts is highly desirable since transformations at high temperatures intrinsically supply a box-set of key biotechnological advantages (higher entropies -better reaction yields-, solubilisation of hydrophobic compounds or low levels of microbial side-contamination, among others). Besides, thermostable enzymes are typically tolerant to many other harsh conditions often required in industry, such as the presence of organic co-solvents, extreme pHs, high salt concentrations, high pressures, etc [5, 6]. Few exceptions aside [7, 8], the discovery of stabilizing mutations is not always straightforwardly accomplished without significant drops in turnover rates . Most of these mutations, which establish new interactions by salt bridges, hydrogen bonds, hydrophobic contacts or even disulfide bridges, are placed either at the protein surface or in internal cores pursuing the tightly packing of the tertiary protein structure in order to prevent unfolding and denaturation under extreme environments . On the contrary, improvements in activity are generally accomplished by introducing beneficial but destabilizing mutations in hot regions for catalysis (substrate binding sites, channels of access to the active pockets) although sometimes distant mutations can also vary the catalytic function by altering the dynamics and geometry in the protein scaffold . There are several examples in literature about the stabilization of enzymes by directed evolution or rational design but unfortunately, main constraints still remain from the lack of appropriate methods to recreate diversity in conjunction with reliable screening strategies, especially if one wants to surpass the existing tradeoff between activity and thermostability for many single residue substitutions [10–20].
Among the enzymes forming the ligninolytic system of white-rot fungi (i.e. involved in lignin biodegradation), high redox potential laccases HRPL (EC 126.96.36.199) and peroxidases, including versatile peroxidases (VP; EC 188.8.131.52) are outstanding biocatalysts finding potential applications in paper pulp bleaching and functionalization, bioremediation, organic synthesis, food and textile industries, nanobiodevice construction and more [21–23]. Indeed, HRPL can oxidize dozens of different compounds releasing water as the only by-product and in the presence of redox mediators (diffusible electron carriers from natural or synthetic sources) their substrates specificities are further expanded [24, 25]. On the other hand, VP (with redox potential above +1000 mV) shares the catalytic features of lignin and manganese peroxidase in terms of substrate specificity, together with the ability to oxidize phenols and dyes characteristic of low redox-potential peroxidases. Indeed, the presence of different catalytic sites in a small and compact protein structure (around 300 amino acids) makes VP an ideal platform for laboratory evolution strategies [23, 26, 27].
Here, we have employed these two enzymatic systems as departure points to improve their protein thermostability by directed evolution. VP and HRPL were functionally expressed in yeast and mutant libraries were constructed combining several methodologies of in vitro evolution to guarantee the library complexity, favoring the selection of optimal crossover events or the discovery of beneficial mutations. Highly functional/soluble expressed mutants were stressed under high temperatures and explored for activity and stability. The analysis of the data from screening (ratio residual activity/initial activity in combination with the T50 values) enabled us to discover stabilizing mutations in both systems.
Materials and methods
HRPL from basidiomycete PM1  (PM1-7H2 mutant) and VP from Pleurotus eryngii (10C3, 6B1, 13E4, 6E7 and 11F3 mutants of the allelic variant VPL2, GenBank AF007222) were used as parent types for library construction. Both systems are from previous engineering work by several rounds of directed evolution in S. cerevisiae including the replacement of their original native signal sequences by the alpha factor prepro-leader, ( and unpublished material). ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), bovine haemoglobine, Taq polymerase and the S. cerevisiae transformation kit were purchased from Sigma-Aldrich (Madrid, Spain). The E. coli XL2-blue competent cells and the Genemorph Random mutagenesis kit were from Stratagene (La Jolla, CA, USA). The protease deficient S. cerevisiae strain BJ5465 was from LGCPromochem (Barcelona, Spain). The uracil independent and ampicillin resistance shuttle vector pJRoC30 was obtained from the California Institute of Technology (CALTECH, USA), while the zymoprep yeast plasmid miniprep kit, zymoclean gel DNA recovery kit, and the DNA clean and concentrator TM-5 kit were all from Zymo Research (Orange, CA). NucleoSpin Plasmid kit was purchased from Macherey-Nagel (Germany) and the restriction enzymes BamH I and Xho I were from New England Biolabs (Hertfordshire, UK). All chemicals were of reagent-grade purity.
1. Culture media
Minimal medium contained 100 mL 6.7% sterile yeast nitrogen base, 100 mL 19.2 g/L sterile yeast synthetic drop-out medium supplement without uracil, 100 mL sterile 20% raffinose, 700 mL sdd H2O and 1 mL 25 g/L chloramphenicol. YP medium contained 10 g yeast extract, 20 g peptone and dd H2O to 650 mL. Expression medium contained 720 mL YP, 67 ml 1 M KH2PO4 pH 6.0 buffer, 111 mL 20% galactose, 1 ml 25 g/L chloramphenicol and dd H2O to 1000 mL. For HRPL the expression medium was supplemented with 2 mM CuSO4 and 25 g/L ethanol. For VP the expression medium was supplemented with 100 mg/L bovine haemoglobine. YPD solution contained 10 g yeast extract, 20 g peptone, 100 mL 20% sterile glucose, 1 ml 25 g/L chloramphenicol and dd H2O to 1000 mL. SC drop-out plates contained 100 mL 6.7% sterile yeast nitrogen base, 100 mL 19.2 g/L sterile yeast synthetic drop-out medium supplement without uracil, 20 g bacto agar, 100 mL 20% sterile glucose, 1 mL 25 g/L chloramphenicol and dd H2O to 1000 ml.
2. Library construction for laboratory evolution
Unless otherwise specified, PCR fragments were cleaned, concentrated and loaded onto a low melting point preparative agarose gel and purified using the Zymoclean gel DNA recovery kit (Zymo Research). PCR products were cloned under the control of the Gal 10 promoter of the expression shuttle vector pJRoC30, replacing the corresponding parental gene in pJRoC30. To remove the parental gene, the pJRoC30 plasmid was linearized (with Xho I and BamH I for HRPL- and VP-libraries). Linearized vector was concentrated and purified as described above for the PCR fragments.
Mutagenic StEP (Staggered Extension Process) followed by in vivo DNA shuffling and IvAM (In vivo A ssembly of M utant libraries with different mutational spectra) were used to create the VP and HRPL libraries, respectively, as described below.
VP Library: mutagenic StEP + in vivo DNA shuffling
10C3, 6B1, 13E4, 6E7 and 11F3 VP-mutants were used as parental types. StEP was performed as reported elsewhere  with some modifications. In order to favor random mutagenesis during StEP, Taq DNA-polymerase was employed for the PCR reaction along with low concentration of templates to promote the introduction of point mutations during the amplification. The primers used were: RMLN-sense (5'-CCTCTAATACTTTAACGTCAAGG-3') and RMLC-antisense (5'-GGGAGGGCGTGAATGTAAGC-3'). For the in vivo ligation, overhangs of 40 bp and 66 bp that were homologous to linearized vector were designed. PCR reactions were performed in a final volume of 50 μL containing 90 nM RMLN, 90 nM RMLC, 0.3 mM dNTPs, 3% dimethylsulfoxide (DMSO), 0.05 U/μL of Taq polymerase (Sigma), 1.5 mM MgCl2 and 0.1 ng/μL of 10C3, 6B1, 13E4, 6E7 and 11F3 DNA-template mixture. StEP was carried out using a gradient thermocycler (Mycycler, Biorad, USA). The thermal cycling parameters were as follows: 95°C for 5 min (1 cycle), 94°C for 30 s and 55°C for 20 s (90 cycles). Purified PCR products were further recombined by in vivo DNA-shufflling . PCR mutated/recombined products were mixed equimolarly (160 ng of each product) and transformed along with linearized vector (ratio PCR product:vector, 4:1) into competent cells using the yeast transformation kit (Sigma). A mutant library of ~2000 clones was explored.
HRPL Library: IvAM
IvAM (~1300 clones) was performed as reported elsewhere  with some modifications. HRPL PM1-7H2 mutant was used as parent type. Mutagenic PCR was carried out using the following thermal cycling parameters: 95°C for 2 min (1 cycle), 94°C for 0.45 min, 53°C for 0.45 min, 74°C for 3 min (28 cycles), 74°C for 10 min (1 cycle). For the Taq library the concentrations of each ingredient in 50 μL final volume were as follows: 90 nM RMLN; 90 nM RMLC; 0.1 ng/μL HRPL template; 0.3 mM dNTPs (0.075 mM each); 3% DMSO; 1.5 mM MgCl2; 0.01 mM MnCl2 and 0.05 U/μL Taq polymerase. For the Mutazyme library the concentrations of each reagent in 50 μL final volume were as follows: 370 nM RMLN; 370 nM RMLC, 40 ng/μL HRPL template; 0.8 mM dNTPs; 3% DMSO; and 0.05 U/μL Mutazyme DNA polymerase. Taq/MnCl2 and Mutazyme libraries were equimolarly mixed and transformed along with linearized vector (ratio equimolar library:vector, 8:1) into competent S. cerevisiae cells as described above.
3. High-throughput thermostability assay
Individual clones were picked and cultured in 96-well plates (Sero-well, Staffordshire, UK) containing 50 μL of minimal medium per well. In each plate, column number 6 was inoculated with standard (parental HRPL or VP), and one well (H1-control) was either not inoculated for HRPL libraries or inoculated with untransformed S. cerevisiae cells for VP-libraries. Plates were sealed to prevent evaporation and incubated at 30°C, 225 RPM and 80% relative humidity in a humidity shaker (Minitron-INFORS, Biogen, Spain). After 48 h, 160 μL of expression medium were added to each well, and the plates were incubated for 24 h. The plates (master plates) were centrifuged (Eppendorf 5810R centrifuge, Germany) for 5 min at 3000 × g at 4°C and 20 μL of supernatant was transferred from the master plate with the help of a robot (Liquid Handler Quadra 96-320, Tomtec, Hamden, CT, USA) onto the replica plate. Subsequently, 180 μL of stability buffer (10 mM sodium tartrate buffer pH 5.1 for VP-library and 10 mM Britton and Robinson buffer pH 6.0 for HRPL-library) were added to each replica and briefly stirred. Replica plate was duplicated with the help of the robot by transferring 50 μL of mixture to a thermocycler plate (Multiply PCR plate without skirt, neutral, Sarstedt, Germany) and 20 μL to the initial activity plate. Thermocycler plates were sealed with thermoresistant film (Deltalab, Spain) and incubated at the corresponding temperature using a thermocycler (MyCycler, Biorad, USA). Incubation took place for 10 min (so that the assessed activity was reduced 2/3 of the initial activity). Afterwards, thermocycler plates were placed on ice for 10 min and further incubated for 5 min at room temperature. 20 μL of supernatants were transferred from both thermocycler and initial activity plates to new plates to estimate the initial activities and residual activities values by adding ABTS containing specific buffers. For VP-libraries 180 μL of 100 mM sodium tartrate buffer pH 3.5 containing 2 mM ABTS and 0.1 mM H2O2 were added to each plate. For HRPL-libraries 180 μL of 100 mM sodium acetate buffer pH 5.0 containing 3 mM ABTS were added. Plates were stirred briefly and the absorption at 418 nm (εABTS•+ = 36,000 M-1 cm-1) was recorded in the plate reader (SPECTRAMax Plus 384, Molecular Devices, Sunnyvale, CA). The plates were incubated at room temperature until a green color developed, and the absorption was measured again. The same experiment was performed for both the initial activity plate and residual activity plate. Relative activities were calculated from the difference between the absorption after incubation and that of the initial measurement normalized against the parental type in the corresponding plate. Thermostability values came from the ratio between residual activities and initial activities values. To rule out false positives, two consecutive rescreenings were carried out according to the protocol previously reported  with some modifications. A third rescreening was incorporated to calculate the T50 of selected mutants.
aliquots of 5 μL of the best clones were removed from master plates to inoculate 50 μL of minimal media in new 96-well plates. Columns 1 and 12 (rows A and H) were not used to prevent the appearance of false positives. After 24 h of incubation at 30°C and 225 RPM, 5 μL were transferred to the adjacent wells and further incubated for 24 h. Finally, 160 μL of expression medium were added and plates were incubated for 24 h. Accordingly, every single mutant was grown in 4 wells. Parent types were subjected to the same procedure (lane D, wells 7-11). Plates were assessed using the same protocol of the screening described above but including not only an endpoint assay but also a kinetic assay. In the ABTS kinetic assay, linear absorption increases over a wide range of enzyme concentration (1-20 mU/mL) allowing the estimation of initial rates.
an aliquot from the wells with the best clones of first rescreening was inoculated in 3 mL of YPD and incubated at 30°C and 225 RPM for 24 h. Plasmids from these cultures were extracted (Zymoprep yeast plasmid miniprep kit, Zymo Research). As the product of the zymoprep was very impure and the concentration of extracted DNA was very low, the shuttle vectors were transformed into super-competent E. coli cells (XL2-Blue, Stratagene) and plated onto LB-amp plates. Single colonies were picked and used to inoculate 5 mL LB-amp media and were grown overnight at 37°C and 225 RPM. Plasmids were then extracted (NucleoSpin® Plasmid kit, Macherey-Nagel, Germany). S. cerevisiae was transformed with plasmids from the best mutants and also with parent type. Five colonies of every single mutant were picked and rescreened as described above (using both end-point and kinetic assays).
Third rescreening (T50 determination)
fresh transformants of selected mutants and parent types were cultivated (10 mL) in 100 mL flask for VP and HRPL production. Supernatants were subjected to a thermostability assay to accurate estimate their T50 using 96/384 well gradient thermocyclers (Mycycler, Biorad, US). Appropriate dilutions of supernatants were prepared with the help of the robot in such a way that aliquots of 20 μL give rise to a linear response in kinetic mode. 50 μL (from both selected mutants and parent types) were used for each point in the gradient scale. A temperature gradient profile ranging from 30 to 90°C was established. After 10 min of incubation, samples were removed and chilled out on ice for 10 min. Afterthat, samples of 20 μL were removed and incubated at room temperature for 5 min. Finally, samples were subjected to the same ABTS-based colorimetric assay described above for the screening. Thermostabilities values were deduced from the ratio between the residual activities incubated at different temperature points and the initial activity at room temperature.
4. Determination of thermostabilities in VP and HRPL parent types
Thermostabilities of 7H2-HRPL and 10C3-VP mutants were assessed mimicking the growth conditions established for the screening assay as described above. Two 96 well-plates containing 50 μL minimal media were inoculated with 7H2 and 10C3 respectively and cultivated until reaching functional expression following the conditions used for the assay. Afterwards, supernantants of 7H2 and 10C3 were pooled and employed to estimate their respective thermostabilities with the gradient thermocycler. The gradient of temperature was set at the following points (in°C): 30.0, 31.7, 34.8, 39.3, 45.3, 49.9, 53.0, 55.0, 56.8, 59.9, 64.3, 70.3, 75.0, 78.1 and 80 for the VP mutant and 35.0, 36.7, 39.8, 44.2, 50.2, 54.9, 58.0, 60.0, 61.1, 63.0, 65.6, 69.2, 72.1, 73.9, 75.0, 76.2, 78.0, 80.7, 84.3, 87.1, 89.0 and 90.0 for the HRPL mutant. The protocol followed the general rules described for the third re-screening.
5. DNA sequencing
Plasmid-containing variant HRPL and VP genes were sequenced by using a BigDye Terminator v 3.1 Cycle Sequencing Kit. Primers were designed with Fast-PCR software (University of Helsinki, Finland). Primers used for VP variants were: RMLN; 3R-direct (5'-GTTCCATCATCGCGTTCG-3'); 5F-reverse (5'-GGATTCCTTTCTTCTTGG-3') and RMLC. For HRPL primers were: RMLN; PM1FS (5'-ACGACTTCCAGGTCCCTGACCAAGC-3'); PM1RS (5'-TCAATGTCCGCGTTCGCAGGGA-3') and RMLC.
6. Protein modelling
We carried out a search in the Protein Data Bank for proteins with known structural homology to laccase PM1. The most similar protein to PM1 was a laccase from Trametes trogii (crystal structure solved with a resolution of 1.58 Å), showing 97% sequence identity (PDB id: 2hrgA) . A model from the Swiss-Model protein automated modelling server was generated http://swissmodel.expasy.org/ and analyzed with DeepView/Swiss-Pdb Viewer.
Results and discussion
1. Library construction
2. High-throughput screening assay
3. Library analysis
HRPL selected mutants generated by IvAM
Amino acid Substitution
Secondary structure motif
Distance to the T1 Site (Å)
Distance to the T2/T3 (Å)
G TA238C TA
GC A38GT A
AT T98AC T
G CG1354A CG
TC G1718TT G
In summary, S. cerevisiae is a valuable cell factory for the directed evolution of ligninolytic enzymes for thermostability and taking together, the VP and the HRPL evolved variants share several common features. First, the thermostability improvements obtained for both VP and HRPL systems are especially significant near the enzyme inactivation temperatures: the best VP (24E10) showed ~30% of its maximal activity at 65°C (3-fold more than the initial VP) and the best laccase (16B10) up to 40% of its maximal activity at 72°C (over 10-fold more than the corresponding parent type). Second, an apparently inherent tradeoff between activity and stability appeared in both enzymes for different amino acid substitutions. Although not physically incompatible, in general protein scaffolds activity and thermostability tend to act as communicating vessels and the laboratory design of any of them usually come at the cost of its counterpart. For protein engineers, to find single mutations which improve both properties simultaneously is extremely difficult. In nature, stability is under selection just in the case that it is required for biochemical function, hence mutations which join activity and stability are rare taking into account the genetic drift and that a selective pressure towards both features at the same time is not frequently exerted. It has been reported that in principle is easier to evolve thermostability while keeping activity than vice versa, although recent research indicates that evolving activity while maintaining stability can be accomplished as well [1, 39]. We have demonstrated that the generation of complex crossover events along with the introduction of new mutations facilitates the improvement in the stability of ligninolytic oxidoreductases buffering the drops on their activities. In the evolutionary scenario, the recombination methods described in this work for the generation of diversity along with the screening assay engineered for this specific task can be valuable tools not only to tailor thermostable ligninolytic oxidoreductases but also other enzymatic systems.
2,2'-azino-bis (3 ethylbenzothiazoline 6 sulfonic acid)
High Redox Potential Laccases
In vivo Assembly of Mutant libraries with different mutational spectra
Residual Activity/Initial Activity
Staggered Extension Process
Temperature at which the enzyme loses 50% of its activity following incubation for 10 minutes
Authors truly thank Prof. Ramón Santamaria from Salamanca University for providing PM1 laccase gene. This material is based upon work funded by National Projects CCG08-CSIC/PPQ-3706 and CSIC 200880I033; and EU Projects NMP4-SL-2009-229255 and NMP2-CT-2006-026456. D.M. thanks the CSIC for a JAE contract.
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