Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris

Authors


S. Moukha, Laboratoire de Biotechnologie des Champignons Filamenteux, INRA, CESB-ESIL, Faculté des Sciences de Luminy, Parc Scientifique et Technologique, CP 925, 13288 Marseille Cedex 90, France. Fax: + 33 4 91828601, Tel.: + 33 4 91828607, E-mail: moukha@esil.univ-mrs.fr

Abstract

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiaeα-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.

Abbreviation
ABTS

2,2-azino-bis-(3-ethylthiazoline-6-sulfonate)

Laccases (ρ-diphenol:O2 oxidoreductase; EC 1.10.3.2) are multicopper containing enzymes, widely distributed in higher plants and fungi. They catalyse the oxidation of ρ-diphenols with the concurrent reduction of dioxygen to water [1]. Metal ions, such as Fe2+, and many nonphenolic aromatic compounds, such as ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate]), are oxidized by laccases [2], and this has generated considerable interest as an approach to the enzymatic bleaching of Kraft pulp and delignification. Lignin is a structurally complex aromatic biopolymer which is recalcitrant to degradation. It has been demonstrated that laccases are not only able to degrade lignin but are also able to polymerize phenolic compounds [3]. These features make laccases very interesting tools for industrial applications such as those of the pulp paper and agrochemical industries. However, recent studies suggest that in white-rot fungi the combination of laccase with either lignin peroxidase and/or manganese peroxidase are responsible for the lignin degradation. In the strain Pycnoporus cinnabarinus I-937 (ATCC 200478) neither lignin peroxidase nor manganese peroxidase were detected in lignin degradation conditions [4]. The blue multicopper oxidases, like laccases, are in the ascorbate oxidase and mammalian plasma ceruloplasmin family, which have been subjected to extensive biochemical and structural characterization [5]. Laccases from white-rot fungi are secreted glycoproteins with two disulphide bridges and four coppers distributed in one mononuclear (T1) and one trinuclear (T2/T3) domain. The T1 copper domain constitutes the primary electron acceptor from the reducing substrate. From this copper, electrons are transferred to the two-electron acceptor type-3 copper pair center [6,7]. The trinuclear center, which is the dioxygen-binding site, accepts these electrons with the concomitant reduction of molecular oxygen. This three-step process allows the oxidation of phenolic compounds.

White-rot fungi are good producers of laccases. These enzymes are considered to be part of their ligninolytic system [8]. Corresponding genes have been cloned and recently one laccase gene from P. cinnabarinus I-937 with 71% identity to the laccase gene from strain ATCC200478 [9] has been isolated (unpublished data, GenBank accession number AF170093). Ligninolytic enzymes are generally difficult to overexpress in heterologous organisms in an active form. However, the expression of active recombinant laccases has been reported in the filamentous fungus Aspergillus oryzae[10], and the yeasts Saccharomyces cerevisiae[11] and Pichia pastoris[12].

In this paper, we describe the isolation and characterization of a cDNA corresponding to the lac 1 gene isolated from P. cinnabarinus I-937 and its expression in the methylotrophic yeast P. pastoris. An important feature in the suitability of P. pastoris as host system for heterologous expression lies in the use of the highly efficient alcohol oxidase 1 gene (Aox 1) promoter. Like S. cerevisiae the methylotrophic yeasts combine ease of genetic manipulation with characteristics favourable for the fermentation process. In addition, it is claimed that yeasts can secrete heterologous proteins efficiently. Passage through the secretory pathway leads to important eukaryotic post-translational events such as glycosylation.

Heterologous expression of laccases in yeast would be of special interest in generating large amounts of enzyme. Although P. cinnabarinus is able to secrete significant amounts of laccase, the heterologous expression of laccase would be of special interest in that it would allow site-specific mutagenesis with the aim of producing improved laccase with a more neutral optimal pH or with different physicochemical abilities in terms of redox potential.

Experimental procedures

Strains, culture and media

The Escherichia coli strain JM109 (F′{tra D36 pro AB+lac Iq lac ZΔM15}end A1 rec A1 hsd R17(rk,mk+) sup E44 thi-1 gyr A96 rel A1Δ(lac-pro AB)) (Promega) was used in all DNA manipulations. E. coli was grown in Luria–Bertani medium (1% tryptone, 0.5% NaCl, 0.5% yeast extract; 1.6% agar in plates).

The P. pastoris stain used for heterologous expression was X33 (Invitrogen). P. pastoris was grown in yeast extract peptone dextrose medium (1% yeast extract, 2% peptone, 2% dextrose, 1 m sorbitol, 2% agar on plates) or in buffered minimal methanol medium [0.1 m potassium phosphate pH 6.0, 1.34% w/v yeast nitrogen base without amino acids, 4 × 10−5% biotin, 1.0% (v/v) methanol added daily] for heterologous expression in liquid medium.

Chemicals

Restriction enzymes, Pfu DNA polymerase and Taq DNA polymerase were purchased from Life Technologies. [α-32P]dCTP was purchased from Amersham Pharmacia Biotech.

Preparation of mRNA from P. cinnabarinus I-937

Fungus was grown in 250-mL baffled flasks containing 50 mL medium suitable for laccase production at 30 °C in a shaking incubator (120 r.p.m.). After 3 days, ferulic acid was added (300–500 mg·mL−1) to induce laccase expression. After 7 days mycelia were harvested and RNA was prepared as described previously [13]. Isolation of mRNA was performed using a poly(dT) column (Promega) according to the manufacturer's instructions.

Isolation of laccase cDNA by RT/PCR

The first strand cDNA was synthesized from mRNA by RT/PCR using Expand™ reverse transcriptase (Roche) and an adaptator-poly(dT) primer (5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3′) according to [14]; this created a heteroduplex mRNA/cDNA. This PCR product was used for vector construction using specific primers.

Vector construction

Subcloning of the laccase cDNA without laccase signal peptide. A 1.50-kb fragment corresponding to the laccase cDNA without the fragment encoding the signal peptide was synthesized by RACE/PCR, using the heteroduplex mRNA/cDNA as template, the upstream oligonucleotide linker adaptator (5′-CCGCTGCAGCCATAGGGCCTG) and the downstream oligonucleotide linker adaptator (3′-AACGCGGCCGCTCAGAGGTCG) generating a PstI and a NotI restriction site, respectively (in italics). Nucleotides in bold are complementary to the laccase cDNA. Pfu polymerase (Life Technologies) was used and PCR conditions were a first denaturing step at 94 °C for 5 min, followed by five pre-cycles of 94 °C for 1 min, 55 °C for 45 s and 72 °C for 5 min, and 35 cycles of 94 °C for 45 s, 55 °C for 45 s and 72 °C for 5 min in a GeneAmp 2400 thermocycler (PerkinElmer). A final extension at 72 °C for 90 min using Taq polymerase (Life Technologies) was performed to incorporate one adenine to both 5′ ends of the amplified product.

The PCR product was ligated overnight at 4 °C into the T vector pGEM-T Easy (Promega) using T4 DNA ligase (Promega). The ligation product was used to transform competent E. coli JM109. Cultures of JM109 were grown in Luria–Bertani plates supplemented with ampicillin (100 µg·mL−1) and X-Gal (50 µg·mL−1). Cultures were induced by the addition of 0.5 mm isopropyl thio-β-d-galactoside and recombinant white colonies were selected. Plasmids from recombinant colonies were prepared by alkaline lysis minipreparation. The presence of the described PCR product was verified by restriction enzyme digestion, agarose gel electrophoresis and sequencing.

Cloning in the yeast shuttle expression vector. The vector used for heterologous expression of the laccase cDNA without its own signal peptide in P. pastoris (pPICZαB) was purchased from Invitrogen. The vector pPICZαB was digested with Pst I and Not I, and the resulting 3.6-kb fragment was isolated by agarose gel electrophoresis and recovered using the Gel Extract Kit (Qiagen). The recombinant pGEM-T vector containing the laccase cDNA was digested with Pst I and Not I and the 1.50-kb fragment obtained was purified as described above. This digested fragment was ligated with the 3.6-kb Pst I–Not I fragment from pPICZαB using T4 DNA ligase overnight at 4 °C.

E. coli strain JM109 was transformed with the ligation mixture according to [15] and transformants were selected in low salt Luria–Bertani medium plates containing 25 µg·mL−1 Zeocin™ (Invitrogen). Plasmids were extracted from randomly choosen transformant colonies and the final construct pPICZαB/Lac1(Fig. 1A) was verified by restriction enzyme analysis and was checked by sequencing (Génome Express, Grenoble, France). The construct was linearized with SacI to target the integration of the expression cassette at the P. pastoris Aox 1 locus.

Figure 1.

Plasmids constructed for the expression of laccase in P. pastoris. The lac1 cDNA was cloned into either pPICZαB (A) downsteam of the native S. cerevisiaeα-factor secretion signal, or into pPICZB (B) together with its own signal sequence. The pPICZαB/Lac1 and pPICZB/Lac1 plasmids are 4.8 kbp and 4.6 kbp, respectively. 5′ Aox and Aox TT, promoter and transcription termination region of the alcohol oxidase 1 gene from P. pastoris, respectively; PTEF1, promoter region of the transcription elongation factor 1 gene from S. cerevisiae; PEM7, a constitutive E. coli promoter; Sh ble, Zeocin™ resistance gene; CYC1, transcription termination region of the S. cerevisiae CYC1 gene; ColE1, E. coli replication origin.

Subcloning of laccase cDNA with its own signal peptide. The laccase cDNA construct including its own signal peptide was synthesized from the heteroduplex mRNA/cDNA using the upstream oligonucleotide linker adaptator (5′-TCTTCGAAATCATGTCGAGGTTCCAGTC) and the downstream oligonucleotide linker adaptator (3′-AACGCGGCCGCTCAGAGGTCG) creating a SfuI and a NotI restriction site (in italic). Nucleotides in bold are complementary to the laccase cDNA. PCR was carried out under the same conditions as those used in the previous construction, and PCR products were cloned in pGEM-T Easy as described. Selection and verification of recombinant colonies and plasmid preparations were carried out as described above.

Cloning in the yeast shuttle expression vector. The vector pPICZB (Invitrogen), without the native S. cerevisiaeα-factor secretion signal, was used for cloning the entire laccase cDNA including its secretion signal sequence. The vector was digested with SfuI and Not I. The resulting 3.3-kb fragment was purified and recovered from agarose gel electrophoresis by using the Gel Extraction Kit (Qiagen).

From the recombinant pGEM-T vector containing the entire laccase cDNA, a 1.56-kb fragment was obtained by digestion with SfuI and Not I. The 1.56-kb fragment was isolated by agarose gel electrophoresis and recovered using the Gel Extract Kit (Qiagen). The 3.3-kb fragment from pPICZB was ligated using T4 DNA ligase with the 1.56-kb SfuI/Not I fragment overnight at 4 °C.

E. coli strain JM109 was transformed with the ligation mixture according to [15] and transformants were selected in low salt Luria–Bertani plates containing 25 µg·mL−1 Zeocin™ (Invitrogen). Preparation of recombinant plasmid was carried out as described above and the resulting plasmid pPICZB/Lac1 (Fig. 1B) was verified by restriction enzyme analysis and sequencing (Génome Express). As in the case of pPICZαB/Lac1, this construct was linearized with SacI to target the integration of the expression cassette at the Aox 1 locus of P. pastoris.

Yeast transformation, cultivation and laccase production

Yeast was transformed with 2–5 µg of pPICZ/Lac1, pPICZαB/Lac1 and pPICZαB and pPICZ parental vectors using the EasyComp™ transformation kit (Invitrogen). Transformants were selected for Zeocin™ resistance onto yeast extract peptone dextrose medium supplemented with 100 µg·mL−1 Zeocin™. Transformants were streaked onto minimal methanol histidine medium and minimal dextrose histidine medium plates supplemented with Zeocin™ to screen for fast methanol utilization (Mut+) and slow methanol utilization (Muts) phenotypes. To check for secreted laccase activity in liquid culture, a single transformant containing the laccase cDNA in genomic P. pastoris DNA (verified by PCR using the primers described) was inoculated in a 500-mL baffled flask containing 50 mL yeast extract peptone dextrose medium and incubated at 30 °C in a shaking incubator (250 r.p.m.). When the turbidity of the culture reached an optical density at 600 nm of ≈ 3.0 the cells were harvested by centrifugation at 2000 g for 5 min at room temperature. The cell pellet was washed extensively and re-suspended to an approximate D600 of 1.0 in buffered minimal methanol media. The culture was monitored for 10 days. The daily addition of methanol 1% (v/v) maintained the induction conditions of the Aox 1 promoter. Furthermore, pH was adjusted to 6.3 daily with potassium phosphate buffer 100 mm, pH 6.3.

Assay of laccase activity

Culture aliquots (1 mL) were collected daily and cells were removed by centrifugation (10 000 g for 15 min). Laccase activity in the culture supernatant was determined by monitoring the oxidation of 0.5 mm ABTS at 420 nm, in the presence of 50 mm sodium tartrate pH 4.0 at 25 °C. Activity is indicated in arbitrary units.

The agar plate assay, which allowed the selection of transformants secreting laccase was carried out on minimal methanol histidine plates supplemented with 0.2 mm ABTS. The plates were incubated for 3 days at 30 °C and checked for the developement of a green colour.

Characterization of the purified laccase

SDS/PAGE analysis was performed according to Laemmli [16] and laccase expression was confimed by immunoblotting with specific antibody. To determine the N-terminal sequence of both recombinant laccase proteins, the two proteins were submitted to Edman degradation. Analysis was carried out on an Applied Biosystem 470A by Angela Guevara (CNRS, Marseille, France). Phenylthiohydantoin amino acids were separated by reverse phase HPLC.

Northern blot analysis

Total RNA (15 µg) of from P. cinnabarinus I-937 was denatured at 65 °C in a loading mixture containing formamide and formaldehyde and loaded on to a 1% Tris/acetate/EDTA agarose gel containing 6% formaldehyde [17]. After electrophoresis, RNA was blotted onto Hybond N+ membranes (Amersham-Pharmacia Biotech) and UV cross-linked for 1 min (0.6 J·cm−2·min−1). A 531-bp radiolabelled probe was prepared by PCR from the heteroduplex mRNA/cDNA using the upstream (5′-CACAGGCAATAAGGGCGATC) and the downstream (3′-TGCTGAATGTATGGTTCGGATC) oligonucleotides in the presence of [α-32P]dCTP. PCR was carried out as already described, except that the final Taq polymerase step was omitted. Blotted membranes were hybridized overnight at 65 °C using a buffer containing 0.5 m sodium phosphate pH 7.2, 0.01 m EDTA, 7% (w/v) SDS, 2% (w/v) blocking reagent (Roche Molecular Biochemicals). After hybridization, the membranes were washed twice in 0.2 × NaCl/Cit (NaCl/Cit 20 × is 0.3 m sodium citrate buffer pH 7.0, 3 m NaCl) containing 1% SDS (w/v) at 65 °C. Blots were exposed to X-ray film (Biomax Mr, Eastman Kodak Company) for 5 days at −80 °C.

Results

Isolation of the laccase cDNA and characterization of the deduced protein

In previous work, a 3378-bp DNA fragment from P. cinnabarinus I-937 containing the laccase gene (lac1) was cloned (unpublished data, GenBank accession number AF170093). The coding region of the lac1 gene, represented by a fragment of 2131 bp from the ATG start codon to the TGA stop codon, was predicted to be interrupted by 10 introns. The location of the putative introns were inferred on the basis of alignment of the 2131 bp with other known laccase cDNA sequences and by identifying the 5′ and 3′ GT and AG dinucleotides of the consensus splicing sites. In this study, polyadenylated mRNA was prepared from laccase-producing mycelia of P. cinnabarinus I-937 and used for cDNA synthesis. The total cDNA fragment was amplified by PCR with the primers described above, using the heteroduplex mRNA/cDNA generated by RT/PCR as a template. Primers were defined in accordance with the predicted cDNA sequence from the 2131-bp coding region sequence of the lac1 gene cloned previously. The primers led to at least one band reasonably close to the expected size. After addition of an adenine to the 5′ ends the 1.57-kb PCR product was gel purified and subcloned in to the pGEM-T Easy vector. The complete sequence analysis of the cloned cDNA product was determined by the primer walking method. The cDNA sequence was deposited in the GenBank database under accession number AF152170. In this study, introns positions predicted in the lac1 gene were directly confirmed by alignment of the cDNA sequence (Fig. 2) with the 2131 bp genomic sequence using the conventional DNA Dot-Matrix comparison method (Fig. 3). The cDNA coding sequence was homologous to the 11 predicted exons building up the 2131 bp DNA genomic sequence, thus demonstrating the exact location of all the predicted introns. In addition, our results shows that the genomic nucleotide sequence cloned previously did correspond to an expressed gene. The intron lengths (56, 52, 52, 54, 62, 57, 62, 59, 55, 55 bases) are typical of fungal introns (49–85 bases). The cDNA open reading frame encodes a putative pre-protein of 518 amino acids. The predicted 21 amino-acid signal sequence was identified on the basis of the occurrence of the peptidase recognition site consensus, Ala-X-Ala [18]. An alanine residue was found in the putative signal peptide in position −3 with respect to the first amino acid of the mature protein. In addition, hydrophobic amino acids are found in the central region of this putative signal sequence. The mature protein would therefore consist of 496 amino acids with a calculated molecular mass of 53 901 Da and with an isoelectric point of 4.45.

Figure 2.

Nucleotide sequence and deducedamino-acid sequence of the Lac1 openreading frame. Numbering starts at the ATG starting codon of Lac1. The predicted signal peptide is underlined and the five putative N-glycosylation sites are boxed.

Figure 3.

DNA dot-matrix comparison. Comparison of the nucleotide sequence of Lac1 gene (vertical axis) vs. the corresponding cDNA (horizontal axis) from Pycnoporus cinnabarinus I-937.

Amino-acid comparison of laccase isoforms from different species

A multiple sequence alignment showed that the amino acid involved in copper centre (Type-2/Type-3 and Type-1) and disulfide bridges are quite well conserved among different species (data not shown). Based on a comparison with the amino-acid sequence of the laccase from Coprinus cinereus, the structure of which has been solved recently [7], Cys488, His430 and His493 are predicted to be involved in the trigonal coordination of the copper present in the Type-1 copper site. In this domain the distal amino acid Leu498, which is thought to play a crucial role [19,20], is replaced by a phenylalanine in the sequence of laccase from P. cinnabarinus I-937. The trinuclear Type-2/Type-3 copper site is normally coordinated with eight histidines in a highly conserved pattern of four His-X-His repeats. The Type3 Cu atom is coordinated to six of the conserved histidines with the remaining two histidines coordinating the Type2 Cu (His85, His87, His130, His132, His433, His435, His487, His489).

Construction of the vectors for the expression of laccase in P. pastoris

To study the expression of recombinant laccase in P. pastoris, two distinct expression plasmids were used. Plasmid pPICZB/Lac1 is composed of the inducible promoter Aox 1, the native signal sequence of lac1 upstream of the open reading frame for the mature laccase and a termination transcription signal. The second plasmid pPICZαB/Lac1 differs from the first in that it contains the native S. cerevisiaeα-factor secretion signal upstream of the sequence of mature laccase (Fig. 1). For each construct, approximately 50–100 transformants were obtained after direct selection of recombinants on agar containing Zeocin™. Integrants were checked for the presence of the expression cassette in their genome by PCR using primers specific for the lac1 cDNA (data not shown).

Expression of laccase in P. pastoris

Plate assay.P. pastoris recombinants carrying laccase cDNA, were tested for laccase expression by growing on minimal methanol plates supplemented with ABTS. Recombinants expressing laccase were identified by the appearance of a green zone around colonies detectable after 3 days of incubation at 30 °C (data not shown).

Coloured zones on assay plates were not observed in the case of control transformants lacking the laccase cDNA. All transformants that oxidized ABTS and that developed a green zone, were Mut+ (methanol utilization fast).

Laccase expression in baffled flasks. For both pPICZαB/Lac1 and pPICZ/Lac1 expression vectors, the laccase activity was found in the supernatant of the culture medium, indicating that laccase was secreted from yeast cells. Activity was detected neither in cells nor in the control culture (data not shown). For pPICZαB/Lac1 construction, the laccase activity gradually reached 20 units, and the cell culture density reached a D600 of 21 after 10 days of incubation (Fig. 4A). In addition, Fig. 4A shows that laccase production follows cellular growth. On the other hand, for the pPICZ/Lac1 expression vector, laccase activity reached a maximum of 12 units and the cellular density reached a D600 value of 17 after 8 days. The stationary phase was reached after 5 days (Fig. 4B) of growth instead of 9 days as observed for the previous construction (Fig. 4A). Both active laccase forms migrate as a band of ≈ 110 kDa on denaturing SDS/PAGE (Fig. 5, lanes 2 and 3). The Western blot analysis using specific antibodies confirms that the 110 kDa protein band is indeed a laccase (Fig. 5, lanes 4 and 5).

Figure 4.

Comparison of laccase production using either the native or the S. cerevisiaeα-factor secretion signal in P. pastoris X33. Activity (▪), D600 (▴) and pH (●) are plotted as a function of time for pPICZαB/Lac1 (A) and pPICZB/Lac1 (B).

Figure 5.

SDS/PAGE analysis of proteins secreted by P. pastoris. Five µL 100 × concentrated culture medium of P. pastoris expressing laccases were loaded on to a SDS/10% polyacrylamide gel for SDS/PAGE analysis (lane 2, pPICZαB/Lac1; lane 3, pPICZB/Lac1) or Western blot (lane 4, pPICZαB/Lac1; lane 5, pPICZB/Lac1).

Northern blot analysis

Northern blot analysis was performed to check whether the P. pastoris transformants accumulated the mRNA encoding laccase. RNA was blotted and probed using a 531-bp fragment of the cDNA laccase labelled with [α-32P]dCTP. The Northern blot shows that for each expression vector used, laccase cDNA was transcribed as a single mRNA species with a size close to 1600 nucleotides (data not shown). The transcript accumulation levels of the two recombinant strains are comparable and low as demonstrated by a long exposure time (data not shown). With either recombinant yeast grown on minimal medium without methanol, or with control yeast lacking the laccase cDNA no transcript was detected (data not shown).

Discussion

In this paper, we describe the molecular cloning and sequencing of the laccase cDNA from the white-rot fungus P. cinnabarinus I-937 and its expression in P. pastoris. Although several fungal laccase gene sequences are available, only the crystal structure of C. cinereus has been solved and published recently [7]. Unfortunately, this laccase structure was determined from a crystal of an inactive deglycosylated recombinant enzyme [7]. Therefore, the position of the amino acid involved in the copper site may have to be reconsidered in the future. The amino-acid sequence comparison revealed that Leu498 of C. cinereus laccase is replaced by a phenylalanine in the laccase from P. cinnabarinus I-937. A similar discrepancy occurs in the ascorbate oxidase, where a leucine is substituted by a methionine [5,21]. This modification does not appear to affect the activity, as these residues are located 3.5 Å away from the copper ion and so are not considered to be involved in direct coordination.

Whereas mature laccase purified from the culture medium of P. cinnabarinus migrates as an 81-kDa band on SDS/PAGE, the recombinant protein has an apparent molecular mass of 110 kDa. The identity of the 110-kDa band has been confirmed by Western blotting with anti-laccase antibodies. Laccases are known to be secreted glycoproteins [22], and five putative glycosylation sites (Asn-X-Thr/Ser) are observed in the predicted amino-acid sequence of the P. cinnabarinus laccase. The difference in molecular mass between the native and the recombinant laccase may be ascribed to the presence of additional oligosaccharide residues in the latter. Even if hyperglycosylation has already been described in S. cerevisiae[23], proteins secreted by P. pastoris are known to be subjected to a less extensive mannosylation. Nevertheless, recent studies showed differences in molecular mass between proteins secreted by the natural host and the corresponding recombinant proteins expressed in P. pastoris, thus strongly indicating polyglycosylation [24,25]. The hyperglycosylation of P. cinnabarinus recombinant laccase does not abolish activity, as demonstrated by the detection of a significant laccase activity in the culture medium.

The P. cinnabarinus laccase was secreted in the culture medium of P. pastoris and constructs with the mature laccase signal sequence and with the native S. cerevisiaeα-factor secretion signal gave approximately the same level of laccase activity. These two signal peptides have the same efficiency in triggering laccase production as detected by immunoblotting. Moreover, no intracellular laccase activity was detected at any time during growth in the presence of methanol. Both signal peptides are correctly processed as indicated by the N-terminal sequencing of the two recombinant laccase proteins.

Although the expression level is satisfactory (8 mg·L−1) with the two expression vectors used, we cannot exclude eventual proteolytic degradation events in the culture medium [26]. With P. pastoris the pH of the culture was adjusted each day because an acidic pH was detrimental for the production of laccase (data not shown). In the yeast Yarrowia lipolytica the pH regulates the expression of the acid (AXP) and alkaline (AEP) extracellular proteases [27–29]. It would be interesting to determine whether such regulation of proteases take place in also P. pastoris. The use of a proteinase A mutant (pep4) strain (SMD1168) has already been reported to be beneficial for the production of secreted recombinant proteins [30].

The P. pastoris expression system offers considerable improvements of some of the disadvantages encountered with the S. cerevisiae-based production system, namely mitotic instability of recombinants strains, a great extent of undesirable overglycosylation and difficulties in adapting production schemes to an industrial scale [31]. The use of a controlled fermentor instead of shake flasks improved protein production yield in P. pastoris[32,33]. This is probably due to limited gas and nutrients transfer.

In conclusion, even if the laccase expression level is high enough to allow structure–function studies to be carried out, laccase expression could be improved further by using the pep4 P. pastoris strain in fermentors. The availability of large amounts of purified recombinant laccase will allow its biochemical characterization including the elucidation of the role played by glycosylation in enzyme activity. Work is in progress to purify laccase from P. pastoris culture medium to obtain crystals of an active enzyme suitable for X-ray analysis.

Acknowledgements

L.O. is grateful to the Conseil Régional Provence-Alpes-Côte d’Azur (France), the Institut National de la Recherche Agronomique (INRA France) and the Cellulose du Rhône (France) for a Ph.D. scholarship. The authors thank A. Guevara (CNRS, Marseille, France) for protein analysis.

Footnotes

  1. Enzymes: Laccase (p-diphenol:O2 oxidoreductase; EC 1.10.3.2).

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