Reagents and enzymes.
The pGEM-T Easy vector containing native PcL cDNA (lac1 from P. cinnabarinus I-937, GenBank accession no. AF170093) was provided by E. Record (INRA, Marseille, France). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), Taq polymerase, and an S. cerevisiae transformation kit were all purchased from Sigma-Aldrich (Madrid, Spain). Escherichia coli XL2-Blue competent cells and Genemorph I and II random mutagenesis kits were obtained from Stratagene (La Jolla, CA). Protease-deficient S. cerevisiae strain BJ5465 was bought from LGCPromochem (Barcelona, Spain). The uracil-independent and ampicillin resistance shuttle vector pJRoC30 was kindly donated by Novozymes, and the pGAPZα vector containing the α-factor preproleader was from Invitrogen. A Zymoprep yeast plasmid miniprep kit, Zymoclean gel DNA recovery kit, and DNA clean and concentrator TM-5 kit were all obtained from Zymo Research (Orange, CA). A NucleoSpin plasmid kit was purchased from Macherey-Nagel (Germany), and the restriction enzymes BamHI and XhoI were from New England BioLabs (Hertfordshire, United Kingdom). All chemicals were of reagent-grade purity.
Minimal medium contained 100 ml 6.7% sterile yeast nitrogen base, 100 ml 19.2 g/liter sterile yeast synthetic dropout medium supplement without uracil, 100 ml sterile 20% raffinose, 700 ml sterile double-distilled H2O (ddH2O), and 1 ml 25 g/liter chloramphenicol. Yeast extract-peptone (YP) medium contained 10 g yeast extract, 20 g peptone, and ddH2O to 650 ml. Expression medium contained 720 ml YP, 67 ml 1 M KH2PO4, pH 6.0, buffer, 111 ml 20% galactose, 2 mM CuSO4, 25 g/liter ethanol, 1 ml 25 g/liter chloramphenicol, and ddH2O to 1,000 ml. The yeast extract-peptone-dextrose (YPD) solution contained 10 g yeast extract, 20 g peptone, 100 ml 20% sterile glucose, 1 ml 25 g/liter chloramphenicol, and ddH2O to 1,000 ml. Synthetic complete (SC) dropout plates contained 100 ml 6.7% sterile yeast nitrogen base, 100 ml 19.2 g/liter sterile yeast synthetic dropout medium supplement without uracil, 20 g Bacto agar, 100 ml 20% sterile glucose, 1 ml 25 g/liter chloramphenicol, and ddH2O to 1,000 ml.
Construction of α-PcL.
The pGEM-T Easy vector containing the PcL cDNA was used as a template to amplify PcL using the primers NEcoRI sense (5′-CGGAATTCGCCATAGGGCCTGTGGCGG-3′) and CNotI antisense (5′-AAGGAAAAAAGCGGCCGCTCAGAGGTCGCTGGGGTCAAGTGC-3′), which included targets for EcoRI and NotI (underlined), respectively, and the optimized stop codon for Pichia pastoris. The PcL fragment generated lacked its natural signal peptide, which was replaced by the signal leader of the α-factor preproleader, resulting in detectable laccase secretion. The pGAPZα vector (Invitrogen) containing the α-factor preproleader was linearized with EcoRI and NotI. The amplified PcL fragment was digested with EcoRI and NotI and cloned into the linearized pGAPZα, giving rise to pGAPZα-PcL. This pGAPZα-PcL was used to amplify the fusion gene α-PcL using the following primers: NpJBglII sense (5′-GAAGATCTATGAGATTTCCTTCAATTTTTACTGC-3′) and CNotI antisense (5′-AAGGAAAAAAGCGGCCGCTCAGAGGTCGCTGGGGTCAAGTGC-3′), which included the BglII site (compatible with BamHI) and the NotI site (underlined), respectively. The resulting fragment was digested with BglII and NotI. The episomal shuttle vector pJRoC30 was digested with BamHI and XhoI, and α-PcL was ligated with the vector to produce the pJRoC30-α-PcL construct. PCRs were performed in a final volume of 50 μl containing 400 nM each primer, 25 ng of template, deoxynucleoside triphosphates (dNTPs; 0.25 mM each), 4 mM MgCl2, 5 μl Taq polymerase buffer, and 2.5 units of Taq polymerase. The PCR cycles followed were 94°C for 5 min, 55°C for 5 min, and 72°C for 5 min (1 cycle); 95°C for 0.35 min, 50°C for 2 min, and 72°C for 4 min (25 cycles); and 72°C for 10 min (1 cycle).
For each generation, PCR fragments were cleaned and concentrated, loaded onto a low-melting-point preparative agarose gel, and purified using the Zymoclean gel DNA recovery kit (Zymo Research). The PCR products were cloned under the control of the gal1 promoter of the expression shuttle vector pJRoC30, which was linearized with XhoI and BamHI. The linearized vector was concentrated and purified as described above for the PCR fragments.
(i) First generation: mutagenic PCR.
Two libraries (1,000 mutants each) with different mutation rates were generated by mutagenic PCR with Mutazyme II DNA polymerase, using α-PcL as template. The first mutagenic library was constructed with a mutation rate of between 0 and 4.5 mutations per 1,000 bp, and the second was constructed with a rate of between 4.5 and 9 mutations per 1,000 bp. Error-prone PCR was carried out in a gradient thermocycler (Mycycler; Bio-Rad) in a final volume of 50 μl containing 185 nM each primer, 4.65 μg and 2 μg of template for low- and medium-mutation-rate libraries, respectively, dNTPs (0.2 mM each), 3% dimethyl sulfoxide (DMSO), and 2.5 units of Mutazyme II DNA polymerase. PCRs were performed as follows: 95°C for 2 min (1 cycle); 94°C for 0.45 min, 53°C for 0.45 min, and 74°C for 3 min (28 cycles); and 74°C for 10 min (1 cycle). The primers used for amplification were RMLN sense (5′-CCTCTATACTTTAACGTCAAGG-3′ which binds to bp 160 to 180 of pJRoC30-αPcL) and RMLC antisense (5′-GGGAGGGCGTGAATGTAAGC-3′, which binds to bp 2031 to 2050 of pJRoC30-αPcL). To promote in vivo ligation, overhangs of 40 and 66 bp homologous to the linear vector were designed. The PCR products (400 ng) were mixed with the linearized vector (100 ng) and transformed into competent cells using a yeast transformation kit (Sigma). Transformed cells were plated on SC dropout plates and incubated for 3 days at 30°C. Colonies containing the whole autonomously replicating vector were selected and screened. This protocol was applied for each round of evolution.
(ii) Second generation: mutagenic PCR and in vivo DNA shuffling.
The best mutants from the first generation (1D12, 3D3, 5F7, and 3G10) were subjected to Taq-MnCl2 amplification, and they were recombined by in vivo DNA shuffling (~2,000 clones). PCR amplification mixtures were prepared in a final volume of 50 μl containing 90 nM each primer (RMLN and RMLC), 4.6 ng of the mutant template, 0.3 mM dNTPs (0.075 mM each), 3% DMSO, 1.5 mM MgCl2, 0.01 mM MnCl2, and 2.5 units of Taq polymerase. The PCRs and the primers used were the same as those used in the previous generation. Mutated PCR products were mixed in equimolar amounts and transformed into S. cerevisiae with the linearized vector (ratio of PCR products/vector = 4:1).
(iii) Third generation: mutagenic PCR and in vivo DNA shuffling.
The best mutants from the 2nd generation (10A7, 19C8, 20C7, 1F10, 1C9, and 2D8) were submitted to Taq-MnCl2 amplification and recombined by in vivo DNA shuffling (2,000 clones), as described for the second generation.
(iv) Fourth generation: mutational exchange by IVOE.
Mutational exchange was carried out using the 7A9 mutant from the 3rd cycle and evolved PM1L template (41
) as templates. Mutations were introduced by site-directed mutagenesis/recombination by in
xtension (IVOE) (3
). Mutations R[α2]S, A[α9]D, A240P, and P394H were introduced between both laccase scaffolds by mutational exchange. PCR mixtures were prepared in a final volume of 50 μl containing 0.25 μM each primer, 100 ng mutant template (7A9 or evolved PM1L), 1 mM dNTPs (0.25 mM each), 3% DMSO, and 2.5 units of Pfu Ultra
DNA polymerase. The PCRs were performed as follows: 95°C for 2 min (1 cycle); 94°C for 0.45 min, 55°C for 0.45 min, and 74°C for 2 min (28 cycles); and 74°C for 10 min (1 cycle).
(a) P394H mutant.
The primers for PCR 1 were RMLN and 3CP484HREV (5′-GCAAGTGGAAGGGGTGGTGGAAGCCGGGGGCGGCGGAGG-3′, which binds to bp 1639 to 1678 of pJRoC30-αPM1 and where the underlining indicates the targeted codon for mutagenesis). The primers for PCR 2 were 3CP484HFOR (5′-CCTCCGCCGCCCCCGGCTTCCACCACCCCTTCCACTTGC-3′, which binds to bp 1639 to 1678 of pJRoC30-αPM1) and RMLC.
(b) A[α9]D mutant.
The primers for PCR 1 were RMLN-2 (5′-GGTAATTAATCAGCGAAGC-3′, which binds to bp 5 to 24 of pJRoC30-αPM1) and 1C-REVDI (5′-GAGGATGCTGCGAATAAATCATCAGTAAAAATTGAAGG-3′, which binds to bp 219 to 257 of pJRoC30-αPM1). The primers for PCR 2 were 1C-FORDI (5′-CCTTCAATTTTTACTGATGATTTATTCGCAGCATCCTC-3′, which binds to bp 219 to 257 of pJRoC30-αPM1) and RMLC.
(c) Site-directed recombination library R[α2]S, A240P.
the primers for PCR 1 were RMLN and 3SA240P antisense (5′-GCACGAAGGAGTAGCGCTGCGCAGGAAAAATCTGGATTGAATC-3′, which binds to bp 195 to 235 of PjRoC30-αPcL). The primers for PCR 2 were 2SPREAL sense (5′-GGATCCATAAGATCTATGAGTTTTCCTTCAATTTTTACTGC-3′, which binds to bp 1182 to 1224 of PjRoC30-αPcL) and RMLC antisense. Approximately 400 clones were explored.
(v) Fifth generation: mutagenic PCR.
The 7A9 mutant was subjected to mutagenic PCR with Mutazyme II DNA polymerase (~1,200 clones), using the same conditions described for the construction of the medium-mutation-rate library of the 1st generation.
(vi) Sixth generation: in vivo DNA shuffling and backcrossing recombination and mutagenic PCR.
Two different libraries were prepared in the 6th generation. Library 1 was built by in vivo DNA shuffling of the 7 best mutants from the 5th generation (1H3, 6A10, 12B4, 3B7, 9E2, 7F11, and 8B9) and with the 5D3 mutant from the 3rd generation for backcrossing. The mutants were amplified by PCR in a final volume of 50 μl containing 0.25 mM each primer (RMLN and RMLC), 100 ng of each mutant template, 1 mM dNTPs (0.25 mM each), 3% DMSO, and 2.5 units of Pfu Ultra DNA polymerase. PCRs were performed as follows: 95°C for 2 min (1 cycle); 94°C for 0.45 min, 55°C for 0.45 min, and 74°C for 2 min (28 cycles); and 74°C for 10 min (1 cycle). The amplified products were then cotransformed (100 ng of each of the 8 mutants) with the linearized plasmid (200 ng) using the yeast transformation kit (Sigma). Library 2 was built by error-prone PCR using Taq-MnCl2 and the 1H3 mutant as the parental type. The PCR products (400 ng) were mixed with the linearized vector in a 4:1 ratio and transformed into competent cells using the yeast transformation kit (Sigma).
Engineering α*-PcL and α-3PO fusion genes.
Two fusion genes were constructed by using IVOE (3
). (i) The α*-PcL fusion gene comprised the evolved α-factor preproleader (harboring mutations A[α9]D, F[α48]S, S[α58]G, G[α62]R, and E[α86]G) plus the native PcL. (ii) The α-3PO fusion gene comprised the native (nonevolved) α-factor preproleader plus the ultimate evolved mature PcL (3PO laccase) harboring C117C, N208S, L279L, R280H, N331D, D341N, P394H, A410A, and L457L.
(i) α*-PcL fusion.
The primers for PCR 1 were RMLN and alpha-rev (5′GCATTGGTAAGGGTCAGGTCC3′, which binds to bp 5′-550 to 521-3′ of pJRoC30-α*-3PO). The primers for PCR 2 were PcL-dir (5′ AATTCGCCATAGGGCCTGTGG 3′, which binds to bp 5′-477 to 499-3′ of pJRoC30-α-PcL) and RMLC. The products from PCR 1 and PCR 2 have overhangs with homologous regions of 44 bp between each other and of 40 bp and 66 bp with the linearized vector for in vivo cloning.
(ii) α-3PO fusion.
The primers for PCR 1 were RMLN and alpha-rev (5′GCATTGGTAAGGGTCAGGTCC3′, which binds to bp 5′-550 to 521-3′ of pJRoC30-α-PcL). The primers for PCR 2 were PcL-dir (5′ AATTCGCCATAGGGCCTGTGG 3′, which binds to bp 5′-501 to 522-3′ of pJRoC30-α*-3PO) and RMLC. The products from PCR 1 and PCR 2 have overhangs with homologous regions of 44 bp between each other and of 40 bp and 66 bp with the linearized vector for in vivo cloning.
For both fusions, the linearized plasmid (100 ng) was mixed with products from PCR 1 and PCR 2 (400 ng each) and transformed into competent S. cerevisiae cells. Individual clones were picked and cultured in 96-well plates (GreinerBio-One, Germany) containing 50 μl of minimal medium per well and subjected to the screening procedure described below. Positive clones were rescreened (see below), the in vivo-repaired plasmid was recovered, and the fusion genes were confirmed by DNA sequencing.
High-throughput (HTP) multiscreening. (i) General aspects.
In the first cycles of evolution, the low secretion levels required performing screenings in liquid format after 5 days of induction. From the 3rd cycle onwards, expression was strong enough to reduce protein induction to 24 h. It is worth noting that in the first rounds of evolution, screening in endpoint mode was required after incubating the supernatants for 24 h in the presence of the substrates. In the last cycles, the improvements were assessed in a few minutes, mainly due to the increases in expression/activity.
(ii) HTP assay.
Individual clones were selected and cultured in 96-well plates (Sero-Wel; Sterilin, Staffordshire, United Kingdom) containing 50 μl minimal medium per well. In each plate, column number 6 was inoculated with the parental type and one well (H1 control) was not inoculated. The 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 was added to each well, and the plates were incubated at 20°C for 5 days in the first two generations and for 24 h in subsequent generations. The plates (master plates) were centrifuged (Eppendorf 5810R centrifuge; Germany) for 5 min at 3,000 × g at 4°C, and 20 μl of the supernatant was transferred from the master plate onto three replica plates with the help of a robot (Liquid Handler Quadra 96-320; Tomtec, Hamden, CT). The first replica plate was filled with 180 μl of 100 mM sodium acetate buffer (pH 5.0) containing 3 mM ABTS. The second replica plate was filled with 180 μl of 100 mM sodium acetate buffer (pH 5.0) containing 3 mM DMP. The third replica plate was filled with 180 μl of 100 mM sodium acetate buffer (pH 5.0) containing 250 μM sinapic acid (SA). The plates were briefly stirred, and the absorption at 418 nm (εABTS·+ = 36,000 M−1 cm−1), 469 nm (εDMP = 27,500 M−1 cm−1), and 512 nm (εSA = 14,065 M−1 cm−1) was recorded in a plate reader (SPECTRAMax Plus 384; Molecular Devices, Sunnyvale, CA). The plates were incubated at room temperature in darkness until the color developed, and the absorption was then remeasured. Relative activities were calculated from the difference in absorption over time, and that of the initial measurement was normalized against the parental type in the corresponding plate. The coefficients of variance (CV) for the screening assays were adjusted throughout the evolution process (resulting in CVs below 11% from the third round of evolution onward).
(iii) First rescreening.
Aliquots (5 μl) of the best clones were removed from the master plates to inoculate 50 μl of minimal medium 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 of the growth medium was transferred to the adjacent well and the plates were incubated for a further 24 h. Finally, 160 μl of expression medium was added and the plates were incubated for 24 h at 20°C. Accordingly, each mutant was grown in 4 wells. Parental types were subjected to the same procedure (lane D, wells 7 to 11). Finally, the plates were assessed using the same screening protocols described above.
(iv) Second rescreening.
An aliquot from the wells with the best clones from the first rescreening was inoculated in 3 ml of YPD and incubated at 30°C for 24 h at 225 rpm. The plasmids from these cultures were extracted (Zymoprep yeast plasmid miniprep kit; Zymo Research), and as the product of the Zymoprep extraction was very impure and the concentration of extracted DNA was very low, the shuttle vectors were transformed into supercompetent E. coli cells (XL2-Blue; Stratagene) and plated onto LB-ampicillin (LB-amp) plates. Single colonies were selected, used to inoculate 5 ml LB-amp medium, and grown overnight at 37°C and 225 rpm, after which the plasmids were extracted (NucleoSpin plasmid kit; Macherey-Nagel, Germany). S. cerevisiae was transformed with plasmids from the best mutants and also with the parental type. Five colonies of each mutant were selected and screened as described above.
Production and purification of PcL variants. (i) Production of laccases in S. cerevisiae.
A single colony from the S. cerevisiae clone containing the parental or mutant laccase genes was selected from an SC dropout plate, inoculated in 3 ml of minimal medium, and incubated for 48 h at 30°C and 225 rpm (Micromagmix shaker; Ovan, Spain). An aliquot of the cells was removed and inoculated in a final volume of 50 ml of minimal medium in a 500-ml flask (optical density at 600 nm [OD600] = 0.25). The cells were incubated for two complete growth phases (6 to 8 h). Thereafter, 250 ml of expression medium was inoculated with the 50-ml preculture in a 1-liter flask (OD600 = 0.1). After incubation for 120 h at 20°C and 225 rpm (laccase activity was maximal; OD600 = 25 to 30), the cells were separated by centrifugation for 20 min at 3,000 × g (4°C) and the supernatant was double filtered (using both a glass membrane and a nitrocellulose membrane of 0.45-μm pore size).
(ii) Laccase purification.
The crude extract was first concentrated and dialyzed in 20 mM Tris-HCl buffer (pH 7.4) by tangential ultrafiltration through a 10-kDa-pore-size membrane (Minisette; Filtron) using a peristaltic pump (Masterflex easy load; Cole-Parmer). The concentrate was then precipitated with ammonium sulfate at 65%, and after centrifugation, the supernatant was dialyzed and concentrated by pressure ultrafiltration through a 10-kDa-pore-size membrane (Amicon; Millipore). The sample was filtered and loaded onto a weak anion-exchange column (HiTrap Q FF; Amersham Bioscience) preequilibrated with Tris-HCl buffer and coupled to an ÄKTA purifier system (GE Healthcare). The proteins were eluted with a linear gradient of from 0 to 1 M NaCl at a flow rate of 1 ml/min in two phases: from 0 to 50% over 75 min and from 50 to 100% over 15 min. Fractions with laccase activity were pooled, concentrated, dialyzed against Tris-HCl buffer, and loaded onto a high-resolution, strong-anion-exchange column (MonoQ HR 5/5; Amersham Bioscience) that was preequilibrated with Tris-HCl buffer. The proteins were eluted with a linear gradient of 0 to 1 M NaCl at a flow rate of 1 ml/min in two phases: from 0 to 25% in 25 min and from 25 to 100% in 1 min. Fractions with laccase activity were again pooled, dialyzed against Tris-HCl buffer, concentrated, and further purified by high-pressure liquid chromatography (HPLC) with a Superose 12 HR 10/30 molecular exclusion column (Amersham Bioscience) preequilibrated with 150 mM NaCl in Tris-HCl buffer at a flow rate of 0.5 ml/min. The fractions with laccase activity were pooled, dialyzed against Tris-HCl buffer, concentrated, and stored at −20°C. Throughout the purification protocol the fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 7.5% gels, in which the proteins were stained with Coomassie blue. All protein concentrations were determined using the Bio-Rad protein reagent and bovine serum albumin as a standard.
Overproduction of evolved mutants in Aspergillus niger.
The cDNA corresponding to the 7A9 mutant (3rd generation) was cloned in the expression vector pAN52-4. The sequence encoding the evolved α-factor preproleader of 7A9 for secretion in S. cerevisiae
was replaced by the 24-amino-acid glucoamylase preprosequence from Aspergillus niger
, under the Emericella nidulans gpd
promoter and trpC
terminator. Cotransformants were selected on agar plates of selective minimum medium without uridine containing 200 mM ABTS, which gave green colonies when laccase was expressed (51
). In order to screen the laccase production in liquid medium, 50 ml of culture medium containing 70 mM NaNO3
, 7 mM KCl, 200 mM Na2
, 2 mM MgSO4
, 10% (wt/vol) glucose, and trace elements and adjusted to pH 5.0 with a 1 M citric acid solution was inoculated with 1 × 106
spores/ml. The culture was monitored for 11 days at 30°C in a shaker incubator (200 rpm). The pH was adjusted to 5.0 daily with 1 M citric acid. Twenty positive clones were cultured in liquid for each construction. Results for laccase activity ranged from 150 to 2,400 units/liter. The best producer was selected for the purification of laccase. For protein purification, 450-ml cultures were prepared in 1-liter flasks under the same conditions and purified as reported elsewhere (51
Characterization of evolved laccase variants. (i) Determination of thermostability.
The thermostability of different laccase samples was estimated by assessing their T50
values, defined as the temperature at which the enzyme retains 50% of its activity after 10 min of incubation (18
), using 96/384-well gradient thermocyclers. Appropriate laccase dilutions were prepared in such a way that 20-μl aliquots produced a linear response in the kinetic mode. Subsequently, 50-μl samples (three independent incubations for each point in the gradient scale) were subjected to a temperature gradient ranging from 35 to 90°C. The temperature profile was established as follows (in °C): 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. After a 10-min incubation, the samples were chilled on ice for 10 min and incubated at room temperature for a further 5 min. Afterwards, 20-μl samples were added to 180-μl volumes of 100 mM acetate buffer (pH 5) containing 3 mM ABTS, and the activities were measured in triplicate in kinetic mode. The thermostability values were deduced from the ratio between the residual activities for incubations at the different temperature points and the initial activity at room temperature (23
(ii) Determination of optimum pH.
Appropriate laccase dilutions were prepared in such a way that 10-μl aliquots produced a linear response in the kinetic mode. Plates containing 10 μl of laccase samples and 180 μl of 100 mM Britton and Robinson buffer were prepared at pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. The assay commenced when 10 μl of 60 mM ABTS or DMP was added to each well to give a final substrate concentration of 3 mM. The activities were measured in triplicate in kinetic mode, and the relative activity (in percent) is based on the maximum activity for each variant in the assay.
(iii) Steady-state kinetic constants.
The kinetics parameters were estimated at two different pH values for each substrate. Reactions were carried out in triplicate in a final volume of 250 μl containing the corresponding substrate in 100 mM acetate buffer (pH 5.0) or 100 mM tartrate buffer (pH 3.0). Substrate oxidation was followed by measurement of the absorption at 418 nm for ABTS (ε418 = 36,000 M−1 cm−1), 469 nm for DMP (ε469 = 27,500 M−1 cm−1), and 312 nm for sinapic acid (ε312 = 17,600 M−1 cm−1) using the plate reader. To calculate the values of Km and kcat, the average Vmax was represented versus substrate concentration and fitted to a single rectangular hyperbola function in SigmaPlot (version 10.0) software, where parameter a was equal to kcat and parameter b was equal to Km.
Plasmids containing HRPL variants were sequenced using a BigDye Terminator (version 3.1) cycle sequencing kit. The following primers were designed with Fast-PCR software (University of Helsinki, Helsinki, Finland): RMLN, PcLF1 (5′-CGACGACACTGTCATTACGC-3′), PcLF2 (5′-CGAGGTCGACTTGCATCC-3′), PcLR3 antisense (5′-GCTTCGTAGGAGTAGTC-3′), PcLR4 antisense (5′-ACAAGAACGAGTGGTTTG-3′), and RMLC.
A structural model of the P. cinnabarinus
laccase crystal structure at a resolution of 1.75 Å (Protein Data Bank [PDB] accession no. 2XYB
) was kindly provided by K. Piontek (University of Munich). Mutations selected upon PcL evolution were analyzed by use of a DeepView/Swiss-Pdb viewer (Glaxo SmithKline) and PyMOL viewer (DeLano Scientific LLC). Using the Swiss-Model protein automated modeling server (http://swissmodel.expasy.org/
), evolved PM1L was modeled. The crystal structure of Trametes trogii
laccase (PDB accession no. 2HRG
; 1.58-Å resolution), which shares 97% sequence identity with PM1L (43
), served as a template.