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Journal of Biotechnology 209 (2015) 76–84

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

ecombinant expression of four oxidoreductases in Phanerochaetehrysosporium improves degradation of phenolic and non-phenolicubstrates

ancy Coconi-Linares a, Elizabeth Ortiz-Vázquez b, Francisco Fernández c,chim M. Loske c,d, Miguel A. Gómez-Lim a,∗

Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-León, 36500 Irapuato,uanajuato, MexicoInstituto Tecnológico de Mérida, Av. Tecnológico, 97118 Mérida, Yucatán, MexicoCentro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Boulevard Juriquilla 3001, 76230 Querétaro, MexicoDivisión de Ciencias de la Salud, Universidad del Valle de México, Villas del Mesón 1000, 76230 Querétaro, Mexico

r t i c l e i n f o

rticle history:eceived 23 April 2015eceived in revised form 5 June 2015ccepted 16 June 2015vailable online 22 June 2015

eywords:hanerochaete chrysosporiumungal oxidoreductasesaccase

a b s t r a c t

Phanerochaete chrysosporium belongs to a group of lignin-degrading fungi that secretes various oxidore-ductive enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP). Previously, wedemonstrated that the heterologous expression of a versatile peroxidase (VP) in P. chrysosporium recom-binant strains is possible. However, the production of laccases (Lac) in this fungus has not been completelydemonstrated and remains controversial. In order to investigate if the co-expression of Lac and VP in P.chrysosporium would improve the degradation of phenolic and non-phenolic substrates, we tested theconstitutive co-expression of the lacIIIb gene from Trametes versicolor and the vpl2 gene from Pleurotuseryngii, and also the endogenous genes mnp1 and lipH8 by shock wave mediated transformation. Theco-overexpression of peroxidases and laccases was improved up to five-fold as compared with wild type

henolic compounds degradationeterologous expressionhock waves

species. Transformant strains showed a broad spectrum in phenolic/non-phenolic biotransformation anda high percentage in synthetic dye decolorization in comparison with the parental strain. Our results showthat the four enzymes can be constitutively expressed in a single transformant of P. chrysosporium in min-imal medium. These data offer new possibilities for an easy and efficient co-expression of laccases andperoxidases in suitable basidiomycete species.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

The white-rot basidiomycetes have been extensively studiedver the last 30 years due to their high capacity for biodegrada-ion of environmental pollutants, bleaching treatments of papernd pulp, as well as for solubilization and modification of lignin foriofuel production (Camarero et al., 2014). This high efficiency haseen mainly attributed to the synthesis of extracellular oxidoreduc-ases, including manganese peroxidase (MnP), lignin peroxidaseLiP), versatile peroxidase (VP), and laccase (Lac) (Janusz et al.,

013). Previous reports have shown that the redundancy andiversity of these enzymes are important elements affecting therocesses of lignin degradation (Knezevic et al., 2013; Camarero

∗ Corresponding author. Fax: +52 462 624 58 46.E-mail address: mgomez@ira.cinvestav.mx (M.A. Gómez-Lim).

ttp://dx.doi.org/10.1016/j.jbiotec.2015.06.401168-1656/© 2015 Elsevier B.V. All rights reserved.

et al., 2014). Interestingly, these factors are also correlated withthe efficiency to oxidize a variety of aromatic and recalcitrant com-pounds (Salame et al., 2013; Barrasa et al., 2014; Benghazi et al.,2014). Owing to the versatility in their modes of action, it has beensuggested that multiple enzymatic activities would increase thebiotransformation of diverse compounds (Verma and Madamwar,2002; Elisashvili and Kachlishvili, 2009; Yang et al., 2011; Knopet al., 2014).

No natural microorganism has been found to exhibit all theoxidoreductases desired to degrade a wide group of recalcitrantcompounds. For that reason, a number of bacteria and fungi havebeen genetically modified to overproduce these enzymes (Bugget al., 2011). Recombinant organisms resulting from these engi-neering works may have great potential in achieving a direct

microbial configuration that incorporates the production of mul-tiple ligninolytic enzymes under the same cultivation conditionsin a single process. These studies have been traditionally focused

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N. Coconi-Linares et al. / Journa

n the expression of a single laccase or peroxidase limiting thedvantages of complementing activity of fungal oxidoreductasesJanusz et al., 2013; Rivera-Hoyos et al., 2013). Furthermore, over-xpression of multiple recombinant proteins in the same fungalell has been hampered due to the low efficiency of the geneticanipulation of filamentous fungi, as well as by an inadequate

election of the expression host (Kück and Hoff, 2010). The white-ot Phanerochaete chrysosporium is an attractive host organism forhis purpose, because it possesses two of the most important lignin-

odifying enzymes, MnP and LiP (Sakamoto et al., 2013). Othertudies have reported the expression of laccases in some strainsf this fungus, but this is still controversial because analysis ofhe genome has not confirmed the presence of these enzymesMartínez et al., 2009; Kersten and Cullen, 2014). On the other hand,e have successfully transformed P. chrysosporium using underwa-

er shock waves and expressed and purified a recombinant VPL2Coconi-Linares et al., 2014).

In an effort to increase the diversity of oxidoreductases in P.hrysosporium, we have attempted the constitutive co-expressionf lacIIIb gene from Trametes versicolor and vpl2 gene from Pleu-otus eryngii, as well as the overexpression of endogenous mnp1nd lipH8 genes. Although we previously reported the efficient co-ransformation of P. chrysosporium with the encoding genes hph,np1, lipH8 and vpl2 (Coconi-Linares et al., 2014), to the best of our

nowledge, this is the first time that the expression of five recom-inant enzymes is demonstrated in P. chrysosporium. By using thistrategy, we have recovered recombinant strains that exhibitedigh activity over a broad range of substrates and with a muchreater degradative potential in comparison to wild type strains.hese results confirm that it is possible to obtain novel strainsf basidiomycete species with biotechnological potential by co-xpression of laccases and peroxidases. Heterologous expression ofhese enzymes may allow optimal degradation of a large variety ofromatic and recalcitrant compounds derived from several indus-ries as pollutants. Finally, the strains generated may be employedo improve the degradation of lignin, a necessary step in biofuelroduction.

. Materials and methods

.1. Strains and culture condition

Escherichia coli strain DH5� (Life Technologies Inc., Waltham,A, USA) was used for all cloning experiments and grown

n Luria–Bertani medium. P. chrysosporium (ATCC 24275) wasmployed as a parental strain throughout this study and wasaintained as previously described (Coconi-Linares et al., 2014).

fter 2 days at 37 ◦C, agar plates containing actively growingycelium was used as inoculum for further studies. Minimaledium contained 0.1 mM CuS04, 1% glucose, 0.1% thiamine, 0.1%

race elements, and 1% nitrate salts at pH 5.5.

.2. Vector construction

The endogenous mnp1 and lipH8 genes were ampli-ed from P. chrysosporium genomic DNA using primersnpF (5′-ACTAGTATGGCCTTCAAGTCCCTCA-3′) and MnpR (5′-

CATGGTTATGCAGGGCCGTTGAAC-3′) for mnp1, and the primersipF (5′-ACTAGTATGGCCTTCAAGCAGCTCT-3′) and LipR (5′-CGGCCGCAAGCACCCGGAGGCGG-3′) for lipH8. The PCR productsere digested with SpeI and NcoI and inserted in the expression

lasmids, each with the glyceraldehyde-3-phosphate dehydroge-ase (gpd) promoter and anthranilate synthase (trpC) terminator

rom P. chrysosporium as described previously (Coconi-Linarest al., 2014). The resulting plasmids were designated as pPCMNP1

otechnology 209 (2015) 76–84 77

and pPCLIPH8. The vpl2 cDNA optimized sequence (GenBankaccession no. KM206768) was commercially synthesized by Gen-Script (Piscataway, NJ, USA) using the signal peptide of mnp1 forimproved expression in P. chrysosporium. The synthesized DNAfragment was cloned into the expression vector with restrictionsites SpeI and NotI to generate pPCVPL2. The lacIIIb cDNA sequence(GenBank accession no. AF414109) with its native signal peptidewas synthesized by GenScript and subsequently cloned betweenthe SpeI and NotI sites onto the fungal expression vector, resultingin pPCLACIIIb. The selectable hygromycin B (hph) marker gene wassynthesized (GenScript) and inserted into the expression vector togenerate pPCHPH.

2.3. Phanerochaete co-transformation

Conidia were obtained as reported previously (Coconi-Linareset al., 2014). Fungal co-transformation was carried out by under-water shock waves essentially as described by Coconi-Linares et al.(2014) using each of the laccase and peroxidases expression vectorsand pPCHPH, in a 12:1 ratio. The suspensions of P. chrysospo-rium intact conidia were placed inside 15 mm × 10 mm heat sealedpolyethylene bags and exposed to 150 shock waves, generated ata rate of 0.5 Hz and a voltage of 5.7 kV with a Piezolith 2501-basedshock wave source (Richard Wolf GmbH, Knittlingen, Germany).The shock wave generator consists of a mosaic pattern of severalhundred small piezoceramic elements arranged on the inner sur-face of a bowl-shaped aluminum backing. Application of a highvoltage discharge to the array results in an abrupt deformation ofeach crystal, generating a pressure pulse that propagates towardthe center (F) of the device. A shock wave is produced in the vicin-ity of F due to nonlinear effects. Water is used as a coupling media totransfer the acoustic energy into the polyethylene bag. The piezoce-ramic elements are insulated from the water by a flexible polymericlayer. A solid-state trigger switch driven by a pulse generator con-trols the discharge rate of the shock waves. The electric circuitmainly consists of a capacitor charging unit and a discharge controlsystem. Each polyethylene bag was fastened horizontally inside thewater tank of the shock wave source and centered at F using a spe-cially designed Lucite holder. The error in positioning the samplewas estimated to be ±0.5 mm. The shock waves had a peak positivepressure of approximately 38 MPa, a rise time of a few nanosec-onds and a full width at half maximum of 0.85 �s, followed by apressure through of approximately −16 MPa. The number of shockwaves and the energy applied to each sample were determinedbased on previous experiments. Transformants were selected fortheir ability to grow on a rich medium plate supplemented with100 �g/ml hygromycin (Invitrogen, Carlsbad, CA, USA).

2.4. Enzymatic assays and dye decolorization in agar plates

Hygromycin resistant co-transformants were tested for enzymeactivity assays and dye degradation in agar plates. The selectedsubstrates were 2,6-dimethoxyphenol (DMP) and guaiacol (GUA)as representatives of phenolic substrates, -N,N-dimethyl-p-phenylenediamine sulfate (DMPPDA) as azo dye, and RemazolBrilliant Blue R (RBBR) as an anthraquinone dye. All compoundsused for the substrate screen were purchased from Sigma–Aldrich(St. Louis, MO, USA). For enzyme activity assays in agar plates, min-imal medium plates were inoculated with one mycelial disc (5 mmin diameter) and incubated at 37 ◦C during 3 days. Subsequently,5 ml solution overlay of either 2 mM DMP, 2 mM guaiacol or 20 mMDMPPDA in 100 mM citrate-phosphate buffer (pH 4.0) was applied.

The overlay solutions were discarded after 30 min of incubation.Plates were incubated again at 37 ◦C and checked for developmentof colored halo around the colonies that indicates enzyme activ-ity. For degradation of anthraquinone dye, minimal medium plates

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8 N. Coconi-Linares et al. / Journa

ere supplemented with 0.05% RBBR and inoculated with one agariece of growing mycelium. Plates were incubated at 37 ◦C andhecked for decolorization activities.

.5. Enzyme production and preparation

Six parallel Erlenmeyer flasks, each containing 50 ml of minimaledium, were inoculated with one agar piece (10 mm in diameter)

overed with 2-days grown P. chrysosporium mycelium of transfor-ants and wild type strain. The cultures were maintained statically

t 37 ◦C in darkness and collected each 2 days after inoculation.ulture fluid was separated from the mycelia by filtering throughiracloth (Calbiochem, San Diego, CA, USA). The mycelia were

ried to constant mass at 110 ◦C. The supernatants of liquid cul-ures were vacuum filtered through 0.22 �m membranes (Milliporeorp., Billerica, MA, USA) and concentrated 30X with an Amicon 10Da cutoff membrane (Millipore) at 4 ◦C. The extracellular cultureuid was used in assays for MnP, LiP, VP and Lac activities. The totalrotein concentration of the samples was determined using theradford protocol (Bradford, 1976) with the Sigma protein reagentccording to the manufacturer’s instructions using bovine serumlbumin as the standard.

.6. Determination of laccase and peroxidases activities

Assays for laccase activity were evaluated by monitoringhe oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-ulfonic acid) (ABTS) at 420 nm (� = 36,000 M−1 cm−1), DMPt 469 nm (� = 27,500 M−1 cm−1) and guaiacol at 465 nm� = 12,100 M−1 cm−1) (Jolivalt et al., 2005). The assay mixtureontained either 1 mM ABTS, 1 mM DMP or 2 mM guaiacol and0 �l of concentrated extracellular proteins in 100 mM citrate-hosphate buffer (pH 3.0). Assays were performed in the presencef 10 U/ml of catalase (Sigma) to ensure the absence of H2O2 inhe protein extracts. Manganese peroxidase activity was mea-ured at room temperature by the H2O2-dependent formationf Mn(III)–malonate complex at 270 nm (� = 11,590 M−1 cm−1)Wariishi et al., 1992). Reactions contained 0.5 mM MnSO4 and0 �l concentrated extracellular proteins in 50 mM malonateuffer (pH 4.5). The reaction was started by the addition of 0.1 mM2O2. Lignin peroxidase activity was determined by measuring thexidation of veratryl alcohol (VA) to veratryl aldehyde at 310 nm� = 9300 M−1 cm−1) (Tien and Kirk, 1988). The reaction mixtureontained 2 mM VA, 50 �l concentrated extracellular proteins,.4 mM H2O2 in 50 mM tartaric acid buffer pH 3.0. Versatileeroxidase activity was measured by monitoring the oxidationf 1 mM DMP at 469 nm (� = 27,500 M−1 cm−1) (Pérez-Boadat al., 2002). The assay mixture contained 1 mM DMP and 50 �loncentrated extracellular proteins in 100 mM Na-tartrate bufferpH 3). The reaction was started by the addition of 0.1 mM H2O2.n UV/visible spectrophotometer (Pharmacia Biotech, Piscataway,J, USA) was used in all the measurements. The amount of enzyme

hat transforms 1 �mol of substrate per minute was defined as 1 Uf enzyme activity.

.7. Southern blot analysis

The five transformants showing the highest enzymectivity were analyzed by Southern blot analysis. Briefly,enomic DNA was isolated and digested with BstXI. Theigested DNA was electrophoresed and transferred ontoybond N+ membranes (GE Healthcare, Buckinghamshire,

K). The primers LacF (5′-GCCGACCTAACCATCACCAA-3′) andacR (5′-TGAACGTGTAGTTGGGGTCG-3′) for lacIIIb, and therimers VpF (5′-GCCTCACCTTCCACGACGCC-3′) and VpR (5′-ATCGTAGCGGCGAAGCGGT-3′) for vpl2, were used to generate a

otechnology 209 (2015) 76–84

probe labeled with biotin using the Biotin DecaLabel DNA LabelingKit (Thermo Scientific, Vilnius, Lithuania). Hybridization and detec-tion procedures were performed according to the manufacturer’sinstructions using Biotin Chromogenic Detection Kit (ThermoScientific).

2.8. Laccase purification

The transformant with the highest Lac activity was selected forenzyme purification and kinetic characterization. The culture fil-trate was dialyzed against 20 mM citrate-phosphate buffer (pH 5.0)using cellulose dialysis tubing membrane (Sigma). The dialysatewas applied to a DEAE-Sepharose Fast Flow column (GE Healthcare,Bio-Sciences, Uppsala, SE) equilibrated in the same buffer. The elu-tion of protein was performed with a linear NaCl gradient from 0to 0.75 M. Fractions that showed Lac activity were pooled and con-centrated with an Amicon 10 kDa cutoff membrane (Millipore). Thesample was loaded onto Sephadex 75 (Sigma) equilibrated with50 mM citrate-phosphate buffer (pH 3.5). Fractions showing Lacactivity were pooled and concentrated. Finally, purification of Lacwas confirmed by 10% SDS-PAGE according to Laemmli (1970) withCoomassie brilliant blue R250 (Sigma) staining.

2.9. Characterization of the recombinant LACIIIb

The determinations of Km and kcat of the recombinant LacIIIbwere measured from a Lineweaver–Burk plot using the substratesABTS and DMP as described (Bertrand et al., 2002). The effect ofpH on recombinant laccase activity towards ABTS was evaluated atroom temperature using 100 mM citrate-phosphate buffer adjustedat pH range 2.0–9.0 under laccase activity conditions describedabove. The effect of temperature on the enzymes was determined inthe same conditions at pH 3 in the temperature range of 20–80 ◦C.

2.10. Dye decolorization assay

Erlenmeyer flasks containing 50 ml liquid minimal mediumwere inoculated with one agar plug (10 mm diameter) of grow-ing mycelia of transformants and wild type strain. The cultureswere incubated stationary at 37 ◦C for 8 days. Culture medium withmycelia was filtrated on miracloth membranes and fresh biomasswas weighted. The mycelia were re-inoculated to a new flaskscontaining 0.25% Coomassie Brilliant Blue G250 (Sigma) as triph-enylmethane dye, 5% (v/v) acetic acid and 20% (v/v) methanol ina total volume of 40 ml and incubated at 37 ◦C for 24 days. Non-inoculated flask with Coomassie dye was used as blank. Aliquotsof 100 �l were taken each 4 days and examined for decoloriza-tion. Percentage of decolorization was performed by monitoringthe decrease in absorbance at 595 nm and calculated as reportedpreviously (Bao et al., 2013).

3. Results

3.1. Cloning of oxidoreductases recombinant genes in expressionvectors

P. chrysosporium genomic DNA was used to amplify the full-length genomic of mnp1 and lipH8 genes. Single bands of 1527 bp formnp1 and 1570 bp for lipH8 were obtained and sequenced (data notshown). The amplified genes were cloned under control of the con-stitutive gpd promoter and trpC terminator. The resulting plasmidswere designated as pPCMNP1 and pPCLIPH8.

Codons of vpl2 gene were optimized to P. chrysosporium codonusage and the full-length gene had an expected size of 1090 bp.For expression of lacIIIb in Phanerochaete, we used the unmodi-fied DNA sequence and its native signal peptide with a total size of

l of Biotechnology 209 (2015) 76–84 79

1lp

3c

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Fig. 1. Screening of oxidoreductase activity in P. chrysosporium strains on agarplates. General oxidoreductase activity of the transformants (TF) and wild type(WT) was determined on minimal media in the presence of substrates RBRR, DMP,DMPPDA and GUA. The expression of oxidoreductases in each transformant was

N. Coconi-Linares et al. / Journa

570 bp. The synthetic genes (vpl2, lacIIIb, hph) were successfullyigated downstream of the respective promoter region to generatePCVPL2, pLACIIIB and pPCHPH.

.2. Co-transformation of five recombinant genes in the P.hrysosporium strain

P. chrysosporium was successfully co-transformed with a mix-ure of the four expression vectors and the hygromycin resistanceelection marker. After co-transformation, several single sporesolates were obtained. Individual isolates were selected forheir ability to grow on rich medium plates in the presence ofygromycin. Forty-five isolated transformants were resistant toygromycin, while untransformed P. chrysosporium did not exhibitesistance (data not shown). Because co-transformation of five dif-erent plasmids was used in this work, ten transformants wereelected randomly for subsequent studies. The stability of theransformation was confirmed by subculturing colonies onto freshelective liquid medium (up to 5 times) to verify that the phenotypeas maintained.

.3. Dye decolorization and screening for oxidoreductase activityn agar plates

In an attempt to analyze the substrate spectrum of oxidation ofhe recombinant enzymes, comprising four synthetic compounds,ransformants were subjected to a general screen on agar plates.fter 3 days of growth at 37 ◦C, transformants and wild type wereompared for their ability to decolorize the synthetic RBBR dye. Ashown in Fig. 1, 7 out of the 10 transformants decolorized the RBBRye faster than the wild type strain. The appearance of decolorizedrea was evident in the plates after only 5 days. When the generalxidoreductase activities of the transformants were analyzed withMP as substrate, oxidation of this compound produces an orange-rown zone (de Jong et al., 1994). This area was observed in theurrounding region of TF1, TF2, TF3, TF4, TF5, TF8, and TF9 mutantsFig. 1). In another analysis using DMPPDA, a toxic aromatic amine,ntense purple halos were formed during oxidation of this azo dyeWong and Yuen, 1998). These colored zones were observed aroundF1, TF2, TF3, TF4, TF5, TF7, TF8, and TF9. In contrast, non-coloredalos appeared in the wild type (Fig. 1). Also, significant differencesere also observed when GUA was used in plate assays. GUA natu-

ally has a yellowish color but the complex turns yellowish brownhen there is oxidation by activity of laccases or peroxidases. In

his assay, 7 out of 10 transformants exhibited activity in the plateest, but only 6 (TF1, TF2, TF3, TF4, TF8, and TF9) showed a clear-ng brown halo around the transformant mycelium, while coloredones were not observed in the parental strain (Fig. 1).

.4. Evaluation of the enzyme activity of transformant strains

Enzymatic activity in liquid cultures of the transformants andild type strain was also evaluated. The selected mutants presented

accase activity in the culture medium by oxidation of ABTS, DMPnd GUA, confirming that recombinant laccase was being secretedy P. chrysosporium transformants. The experimental results wereisualized in three-dimensional (3D) graphics showing the cor-elation between the enzymatic activities of the three substratesFig. 2A). Interestingly, the 10 transformants exhibited an increasen activity on three compounds than the parental strain. In particu-ar, transformants TF1, TF2, TF3, TF8 and TF9 exhibited significantlyigher laccase activity (approximately 4-fold) than wild type strain.

oreover, the peroxidase activities of the transformants were

creened independently by the oxidation of MnSO4, VA and DMP.he 3D graphics obtained from the multi-screening demonstratedhat most of the 10 transformants showed slight improvements in

evaluated by a change in color.

activity respect of the wild type (Fig. 2B). Also, we could detect sig-nificant differences in peroxidase activity (at least six-fold) amongfour different transformants (Fig. 2B).

In general, we observed a direct correlation of oxidoreductaseactivity on agar plates in these assays, which allowed us to select

80 N. Coconi-Linares et al. / Journal of Biotechnology 209 (2015) 76–84

Fig. 2. 3D graphics from the multi-screening with phenolic and non-phenolic compounds of transformants obtained of P. chrysosporium. (A) Laccase activities of transformantsa B) Peroxidase activities in recombinant strains of P. chrysosporium. DMP, MnSO4 and VAw ansformants (TF, blue points); wild type strain (WT, red point). (For interpretation of ther this article.)

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Table 1Comparison of kinetic parameters of native and recombinant lacIIIb on twosubstrates.

Laccases ABTS 2,6-DMP

Km (�M) kcat (s−1) Km (�M) kcat (s−1)

Native lacIIIba 50 107 275 54Recombinant lacIIIb 52 ± 1.9 71 ± 3.08 311 ± 14.2 24 ± 1.1

a Data taken from Jolivalt et al. (2005).

nd wild type analyzed with three independent substrates (ABTS, GUA and DMP). (ere employed to analyze the enzymatic activity of transformants and wild type. Tr

eferences to color in this figure legend, the reader is referred to the web version of

ransformants TF1, TF2, TF3, TF8 and TF9 as those with the highestnzyme activity.

.5. Analysis of the integration of recombinant genes in theenome of Phanerochaete

Five transformants were grown in liquid minimal media in threeubsequent cultures and the DNA extracted was used to verify thetability of the gene insertion by Southern blot analysis, whichevealed that the co-transformation resulted in insertion of one orwo copies of the lacIIIb gene (Fig. 3). Transformants TF1, TF2 andF8 had the same blot pattern, while that TF3 and TF9 showed twoopies with almost equal intensity. Moreover, analysis of genomicNA of transformants revealed the presence of only one copy of theloned vpl2 gene in TF1, TF2, TF3 and TF8, with the exception of TF9,hich exhibited two copies. In addition, lacIIIb and vpl2 genes were

ncorporated into the P. chrysosporium genome of the five trans-ormants evaluated under the regulation of the gpd promoter. Onhe other hand, genomic DNA extracted from a wild type colony of. chrysosporium did not show hybridization with lacIIIb and vpl2robes (Fig. 3).

.6. Purification of recombinant laccase from P. chrysosporium

Purification of recombinant laccase was carried out fromtationary cultures of the TF1 transformant. Laccase was puri-ed in two steps, involving anion-exchange and size exclusionhromatography. Fig. 4A shows the last step used for laccase purifi-ation. Two protein peaks were obtained during size exclusionhromatography but only one exhibited Lac activity. SDS-PAGEnalysis revealed that the purified rLacIIIb migrated as a single bandith a molecular mass of around 60 kDa (Fig. 4B).

.7. Characterization of recombinant laccase

Two substrates were oxidized by rLacIIIb and the kinetic

arameters of the enzyme were calculated for each of them, asummarized in Table 1. The Michaelis constant (Km) showed thatLacIIIb (52 �M) had higher affinity towards ABTS than 2,6-DMP.he catalytic constants (Kcat) of ABTS and 2,6-DMP were 71 s−1 and

24 s−1, respectively, thus indicating that the recombinant enzymeexhibited increased catalytic efficiency on ABTS compared with2,6-DMP. A comparison between recombinant and native laccasesshowed similar values (Table 1).

The effect of the temperature and pH on rLacIIIb activity wasdetermined for the transformants TF1, TF2, TF3, TF8 and TF9. Themaximum relative laccase activity of the evaluated transformantswas observed at a pH range from 3.0 to 4.0, and a temperaturebetween 30 ◦C and 40 ◦C (Fig. 5). All transformants presented thelowest relative activity at >50 ◦C.

3.8. Coomassie dye decolorization by co-transformants

The ability of the co-transformants to decolorize synthetic dyeswas tested with Coomassie G-250, which belongs to the triph-enylmethane dyes. A change in the color of the supernatants thatcontained the transformants was observed over 24 days after trans-fer of the mycelia to the dye. Coomassie dye was decolorized by thewild type and co-transformants to different extents (Fig. 6). Amongthese five transformants, TF1 and TF9 showed the highest decol-orization efficiency in comparison with the parental strain, with74.5, 83.9 and 46.9% decolorization, respectively. The results of thisanalysis suggested that the transformants produced oxidoreduc-tases extracellularly at higher levels than the wild type, indicatingthat these recombinant fungi have potential for triphenylmethane

dye decolorization.

N. Coconi-Linares et al. / Journal of Biotechnology 209 (2015) 76–84 81

Fig. 3. Southern blot analysis for lacIIIb and vpl2 genes in the wild type P. chrysosporium and the five transformants with higher laccase and peroxidase activity.

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ig. 4. Purification of recombinant laccase (rLACIIIb) from P. chrysosporium. (A) Profiline) and Lac activity (continuous line) are shown. (B) SDS-PAGE of recombinant lacclane MW); purified rLACIIIb (lane 1, arrow).

. Discussion

In this research, we present evidence that P. chrysosporium co-ransformants can improve the oxidation of different syntheticompounds in a similar way as native fungal oxidoreductases.lthough the oxidation of phenolic compounds (DMP and GUA),on-phenolic substrates (DMPPDA, ABTS, and VA) and syntheticyes (RBBR and Coomassie) by fungal oxidoreductases has beenidely studied, to the best of our knowledge, this is the first report

escribing the constitutive expression of three peroxidases and oneaccase and their improved efficiency in a basidiomycete species.

The ligninolytic system of P. chrysosporium is one of the mosttudied, but efforts to improve the strain have been hampered

y the absence of efficient methods for introduction of multipleenetic modifications in a single transformation step. In this regard,enetic engineering of P. chrysosporium has been severely limitedy the low efficiency of current genetic transformation methods

ig. 5. Effect of pH and temperature on the enzymatic activity of rLACIIIb. Laccase activitiide) using ABTS as substrate; relative activity of purified rLACIIIb is shown as a discontin

esponding to size exclusion chromatography: absorbance at 280 nm (discontinuoushe molecular mass of rLACIIIb was estimated to be around 60 kDa. Protein standard

using protoplasts or Agrobacterium (Kersten and Cullen, 2007). Asit has been previously reported (Bradshaw, 2006) the use of proto-plast may promote the formation of heterokaryons and genomicinstability, this phenomenon does not occur with shock waves(Magana-Ortíz et al., 2013). Moreover, Agrobacterium-mediatedtransformation has been tested for P. chrysosporium only employ-ing mycelial pellets but some strains of Agrobacterium are unable toinfect certain cell types (Sharma and Kuhad, 2010). Owing to the useof intact conidia in shock waves transformation method the viabil-ity of P. chrysosporium spores is not dramatically reduced as in othermethods that degrade the fungal cell wall. By employing shockwaves, the stability of multiple gene insertion is improved and itallows obtaining a high number of transformants with the desired

phenotype (Magana-Ortíz et al., 2013; Coconi-Linares et al., 2014).The data presented in this article show that with this method,co-expression of five genes is possible and, to the best of our

es of transformants were tested at different pHs (left side) and temperatures (rightuous line.

82 N. Coconi-Linares et al. / Journal of Bi

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ig. 6. Time course for Coomassie G-250 dye decolorization of recombinant andild type strains during 24 days of incubation.

nowledge, similar results have not been reported previously inlamentous fungi.

The results presented here demonstrated that genetic trans-ormation using underwater shock waves can produce valuabletrains of P. chrysosporium with enhanced activity of key fungalxidoreductases. This high activity was demonstrated using severalhenolic and non-phenolic substrates (Fig. 1). Seven transformantsf P. chrysosporium (TF1, TF2, TF3, TF4, TF5, TF8 and TF9) were ableo degrade a wide group of synthetic compounds in extracellularorm. The fact that the recombinant strains can directly transformeveral substrates is consistent with the properties of inserted fun-al oxidoreductases, while in another transformants (TF6, TF7 andF10) a variation of enzymatic profiles was observed. For exam-le, laccase activity was detected in TF6, but the degradation ofromatic compounds such as RBRR, DMP and DMPPDA was notbserved. This could be caused by the expression of lacIIIb gene, ashown in the enzymatic assays, but not by the expression of per-xidase recombinant genes. The co-transformation protocol maybenly allowed the integration of hph-resistance and lacIIIb genes; theenes that encoded the peroxidases were not integrated probablyue to the degradation of DNA by the shock waves (Campos-Guillént al., 2012). Comparable phenomena could be involved in enzy-atic profile of TF7 and TF10. Differences in enzymatic activity

y Phanerochaete recombinant strains were also observed by Hirait al. (2013).

Furthermore, substrate oxidation by recombinant strains wasxtended to phenols such as DMP and GUA. In previous reports,t was demonstrated that oxidation of GUA is much better corre-ated with decolorization of multiple synthetic dyes than standardcreening substrates (Wong et al., 2013). It is known that theechanisms of oxidation of phenolic compounds by oxidore-

uctases involve generation of phenoxyl radical followed byo-oligomerization similar to the oxidation mechanism of azo dyesWong et al., 2013). The different properties of the oxidoreduc-ases expressed indicate that the combined action of these enzymesupported the high degradation activity of Phanerochaete transfor-ants under various conditions and substrates.

The recombinant strains with relatively high enzymatic activityn plates and liquid cultures were selected for Southern blot anal-sis. This scrutiny showed the insertion of vpl2 and lacIIIb genesf high molecular weight in transformants, confirming that theecombinant genes were integrated in the chromosomal DNA ofhe transformants. Notably, the behavior of multiple transgenesnserted by co-transformation in P. chrysosporium was similar tohat for a single gene (Ma et al., 2003; Magana-Ortíz et al., 2013).

correlation in the number of insertions and relatively high enzy-atic activity was observed in the case of TF3 and TF9. Despite

hat correlation between copy number and yield is a controversialssue (Ma et al., 2003; Sharma et al., 2009), we hypothesize thathe insertion of multiple copies of recombinant gene enhanced thexpression of lacIIIb and vpl2 in strains TF3 and TF9. Furthermore,

otechnology 209 (2015) 76–84

using the gpd promoter to drive the expression of the five recombi-nant genes in the same fungal cell did not appear to cause silencingproblems or be unstable as was evidenced in this work.

The recombinant laccase was purified in only two steps; thisallowed us to study their physico-chemical properties for compar-ison with native laccase. The Km for rLacIIIb at 2,6-DMP slightlyvaried with respect to the native enzyme. It is tempting to speculatewhether the reason for this difference could be the experimentalconditions, which may have been not quite so similar in both cases,or that the host P. chrysosporium made an alternative processing ofthe recombinant enzyme. Further research is needed to determinethe causes of these differences in the recombinant strain. In addi-tion, the main characteristics of rLacIIIb (molecular mass, kineticparameters, optimal temperature and pH) were similar to those ofthe T. versicolor laccase (Jolivalt et al., 2005). Hence, P. chrysosporiumseems to be a good system for fungal oxidoreductase expression.

Triarylmethane dyes such as Coomassie represent the mostcommon synthetic dyes, however, concentrations of these dyes atless than 1 ppm cause potential toxicity to animals and humans(Yang et al., 2011). Hence, removal of this kind of dyes from wastew-ater effluents is a major environmental concern. Interestingly,fungal oxidoreductases are widely used in synthetic dye degra-dation. In this work, the use of Coomassie G-250 confirmed thepotential of the recombinant strains to decolorize this recalcitrantaromatic compound. Montiel-González et al. (2009) demonstratedthat the heterologous expression of LiP or MnP from P. chrysospo-rium in the zygomycete Amylomyces rouxii, which only produceslaccases, increased pentachlorophenol removal by this fungus. Ourresults confirm the positive effect of the heterologous expression ofoxidoreductases in the degradation of synthetic dyes. Additionally,a positive correlation was found between higher LACIIIb and VPL2activity and decolorization of Coomassie G-250 in recombinantstrains. These results are in accordance with Liu et al. (2004). Wedetected an improvement in percentage of decolorization between2 and 3 fold in comparison with the wild type strain. Similar resultswere obtained when the versatile peroxidase or laccase were usedfor the degradation of recalcitrant dyes (Pérez-Boada et al., 2005;Bao et al., 2013; Zheng et al., 2014; Benghazi et al., 2014). Ingeneral, a synergistic action of the enzymes present in the crudeextract could be possibly responsible for the higher oxidation rateobserved.

The constitutive co-expression of LiP and MnP in a single recom-binant strain has never been achieved in any strain, although someattempts have been reported in Aspergillus species (Conesa et al.,2000). Our results demonstrated that the enhanced homologousexpression of two enzymes can improve volumetric activity up to3.7 fold (66.8 U/ml) and 4.2 fold (85.9 U/ml) in the case of MnP andLiP, respectively, when compared with the wild type strain. In con-trast, previous reports showed that the homologous expression ofrMnP and rLiP independently under a constitutive promoter onlyreached the levels of the wild type strain (Mayfield et al., 1994;Gelpke et al., 1999). Our results have demonstrated that trans-formation of P. chrysosporium with rVP allowed the production ofVPL2 in functional form and this suggests that the use of basid-iomycetes can offer a better processing of recombinant peroxidasesin comparison with other platforms (Coconi-Linares et al., 2014).Unexpectedly, the volumetric activity was up to 4 fold (89.2 U/ml)higher in comparison to previously work published by our group,but additional work is required to elucidate the mechanisms ofthese improvements.

Although laccase is very common in white-rot fungi, it has beenconfirmed that the genome of P. chrysosporium does not contain

typical laccase-encoding genes (Kersten and Cullen, 2007; Kerstenand Cullen, 2014). Several studies have shown than under highconcentrations of nitrogen and copper, and the use of celluloseas a carbon source, laccase activity is detected in P. chrysosporium

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ultures (Srinivasan et al., 1995; Dittmer et al., 1997; Rodríguezt al., 1997; Gnanamani et al., 2006). Remarkably, we detectedow levels of endogenous laccase activity in the P. chrysosporium

ild type strain during growth on minimal medium and stationaryulture conditions. In contrast, the recombinant strains obtainedhowed higher volumetric laccase activity, up to 8 fold (9 U/ml) inomparison with the wild type strain, demonstrating the successf this strategy. The production of active fungal oxidoreductasesequires a post-translational modification, which might impact onheir activity, protein folding, secretion efficiency, pH and temper-ture stability and interactions with other proteins (Martínez et al.,009). The similar characteristics of the peroxidases and laccasesmong basidiomycetes indicate that P. chrysosporium may be moreuitable than other filamentous fungi for expressing recombinantxidoreductases.

. Conclusions

We have demonstrated co-expression of four oxidoreductases in basidiomycete (P. chrysosporium) in functional form. This had noteen achieved before. Co-expression of the four oxidoreductasesignificantly improved the degradation of recalcitrant substrates.

cknowledgements

The authors would like to thank Paula Bernardino and Guillermoázquez for technical assistance.

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