TY - JOUR
AU - Bianchi, Michele M.
AB - Abstract Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production. heterologous protein, fermentation process, dye decolorization Introduction Kluyveromyces lactis has the rare capability among yeasts to grow on lactose. This peculiarity has favored the introduction of K. lactis in basic research as a model for lactose metabolism and in the industrial world as a source of lactase (β-galactosidase) for many decades. The ease of genetic research with this organism, together with the availability of molecular tools for genetic engineering, such as transformation protocols (Das & Hollenberg, 1982) and plasmids (de Louvencourt, 1983; Bianchi, 1987), greatly promoted the use of this organism in many fields of investigation. More recently, the genome of K. lactis has been completely sequenced (Dujon, 2004), annotated and made freely accessible (http://cbi.labri.fr/Genolevures). Another important aspect of K. lactis is the flexibility of its glucose metabolism. The best-known yeast Saccharomyces cerevisiae has a strong Crabtree effect, which does not allow respiratory metabolism in the presence of abundant glucose, which is the cheapest and most widely used carbon source in industrial fermentation. The fermentative metabolism has many negative procedural drawbacks, including low energy and biomass yield, and the presence of ethanol in the medium. For these reasons, yeasts with aerobic metabolism, such as Pichia pastoris, are preferred in industrial applications. Kluyveromyces lactis is considered a ‘Crabtree-negative’ yeast and can grow on glucose media by respiration or fermentation, depending on the oxygen supply (Kiers, 1998). Glucose metabolism regulation in K. lactis, together with the facts that this yeast is a GRAS organism, can use alternative low-cost carbon sources (lactose) and has a well-established background in industrial processes, makes this organism an attractive host for scalable processes such as synthesis of heterologous products. In K. lactis, glycolytic and fermentative genes are not redundant as in S. cerevisiae. This genetic structure makes it possible to select mutant strains unable to ferment glucose in the presence of mitochondrial drugs, such as antimycin A [resistance to antimycin on high glucose (Rag) phenotype; Goffrini, 1989; Wésolowski-Louvel, 1992b], thus facilitating the study of glucose transport and metabolism. The production of proteins and enzymes from microbial cells is greatly facilitated in the downstream process if the product is secreted. A comparative study revealed that K. lactis is more effective in secreting heterologous proteins than S. cerevisiae (van den Berg, 1990), and mutant strains with improved secretion properties have also been selected. A recent survey of examples of excreted heterologous proteins from K. lactis (van Ooyen, 2006) indicates that native signal peptides are efficiently recognized by the secretory apparatus, as well as fusions with K. lactis peptides (killer toxin) or S. cerevisiae peptides (α factor). Protein production is also affected by the dosage of the heterologous gene and its expression. An inducible system for gene dosage increase has been described for vectors based on the stable and multicopy plasmid pKD1 (Morlino, 1999). In terms of regulation of the expression of the heterologous gene, commonly used inducible promoters are the PHO5 promoter of S. cerevisiae and the LAC4 promoter of K. lactis. The strong and regulated promoter of the pyruvate decarboxylase gene (KlPDC1) also proved to be effective for this purpose, especially when induced by blocking autoregulation (Salani & Bianchi, 2006) or by hypoxia (Camattari, 2007). Laccases (benzenediol : oxygen oxidoreductase, EC 1.10.3.2) are very flexible enzymes able to oxidize an extensive list of aromatic compounds containing hydroxyl or amino groups, including pesticides, polycyclic aromatic hydrocarbons and dyes. These properties make laccases good candidates for applications in the pulp and paper industry, textile industry, biosensor development and bioremediation of polluted water and soil (Baldrian, 2006). Recent studies have shown that fungal laccases can decolorize and detoxify industrial dyes in vitro (Palmieri, 2005; Zille, 2005) and that the substrate specificity of the enzyme can be broadened in the presence of redox mediators (Claus, 2002; Colao, 2006). Organisms naturally producing laccases usually produce a mixture of isoenzymes. For the analysis of individual isoforms, in particular of minor forms, large amounts of purified proteins are required. For this reason, production of a specific isoform of laccase is advantageously obtained by heterologous expression even if the product yield is lower than in the original organism. Heterologous expression of laccase genes has been studied both in yeast species such as S. cerevisiae (Cassland & Jönsson, 1999; Bulter, 2003; Kiiskinen & Saloheimo, 2004), P. pastoris (Jönsson, 1997; Otterbein, 2000; Liu, 2003) and K. lactis (Piscitelli, 2005; Camattari, 2007; Faraco, 2008) and in filamentous fungi, Trichoderma reseei (Kiiskinen, 2004) and Aspergillus (Larrondo, 2003; Sigoillot, 2005). Laccases are notoriously difficult to express in nonfungal systems and, especially in S. cerevisiae, reasonable expression levels have proven to be very difficult to achieve. The laccase gene from Trametes trogii used in this work was previously expressed in P. pastoris (Colao, 2006) and K. lactis (Camattari, 2007), but the levels of enzyme activity obtained with the heterologous systems (2.5 U mL−1) were lower than those obtained with the fungus (60 U mL−1) (Colao, 2006). In this paper, we describe the construction of stable integrative recombinant strains of K. lactis and optimization steps for the production of fungal laccase in a bioreactor. We also tested the ability of the recombinant enzyme to decolorize different synthetic dyes. Materials and methods Strains, plasmids and media The yeast strains used are listed in Table 1. The elements of pRDLCi were assembled into pSKBluescriptII (Stratagene, Cedar Creek, TX) as follows: the S. cerevisiae PHO5 terminator from plasmid pYG81 (Fleer, 1991) was cloned into the EcoRI and PstI sites. The S. cerevisiae URA3 gene (EcoRI–SalI fragment from plasmid pYG68, a gift from R. Fleer) was cloned into the NotI site, after end blunting with the Klenow enzyme. Then the KlPDC1 promoter (SalI–HindIII fragment from pMD12; Destruelle, 1999) was cloned into the corresponding plasmid sites, and finally the HindIII fragment containing the cDNA of the Lcc1 gene (Colao, 2003) was inserted. Kluyveromyces lactis strains were transformed using the frozen cells (Dohmen, 1991) or the electroporation protocol (Salani & Bianchi, 2006). The oligonucleotides used to verify the locus of chromosomal integration (KlPDC1 gene) were UPF (upstream of the KlPDC1 promoter 5′-GGTTTCAACAATCTCGGCGTA-3′) and PDR (downstream of the KlPDC1 promoter 5′-TAAGAACCGGCAATACCGTT-3′), which paired with the chromosome, and SKF (upstream of the cloned KlPDC1 promoter 5′-TAAAACGACGGCCAGTGA-3′) and LCR (in the Lcc1 gene 5′-AACGAGCGAGAGGGTGATGAA-3′), which paired with the plasmid. 1 Kluyveromyces lactis strains Strain  Genotype  References  MW98-8C  MATαlysA argA ura3 rag1 HGT1 rag2  Bianchi (1987)  MW270-7B  MATametA leu2 ura3 RAG1 HGT1  Billard (1996)  JA6  MATαade1-600 adeT-600 trp1 ura3 KHT1 KHT2  Breunig & Kuger (1987)  HF1987  MATametA ura3 RAG1 HGT1  Salani & Bianchi (2006)  MW179-1D  MATαade trpA metA leu2 ura3 lac4-8 KHT1 KHT2  This work  PM6-7A  MATaadeT-600 ura3 RAG1 HGT1  (Wésolowski-Louvel 1992a, 5  MATαlysA leu2 ura3 lac4-8 RAG1 HGT1  Destruelle (1999)  MW278-8C  MATαade2 leu2 ura3  Uccelletti (2006)  CPK1  MATαade2 leu2 ura3 KlPMR1::KanR  Farina (2004)  Strain  Genotype  References  MW98-8C  MATαlysA argA ura3 rag1 HGT1 rag2  Bianchi (1987)  MW270-7B  MATametA leu2 ura3 RAG1 HGT1  Billard (1996)  JA6  MATαade1-600 adeT-600 trp1 ura3 KHT1 KHT2  Breunig & Kuger (1987)  HF1987  MATametA ura3 RAG1 HGT1  Salani & Bianchi (2006)  MW179-1D  MATαade trpA metA leu2 ura3 lac4-8 KHT1 KHT2  This work  PM6-7A  MATaadeT-600 ura3 RAG1 HGT1  (Wésolowski-Louvel 1992a, 5  MATαlysA leu2 ura3 lac4-8 RAG1 HGT1  Destruelle (1999)  MW278-8C  MATαade2 leu2 ura3  Uccelletti (2006)  CPK1  MATαade2 leu2 ura3 KlPMR1::KanR  Farina (2004)  * Formerly CBS2359/152F. † A gift from M. Wésolowski-Louvel. View Large 1 Kluyveromyces lactis strains Strain  Genotype  References  MW98-8C  MATαlysA argA ura3 rag1 HGT1 rag2  Bianchi (1987)  MW270-7B  MATametA leu2 ura3 RAG1 HGT1  Billard (1996)  JA6  MATαade1-600 adeT-600 trp1 ura3 KHT1 KHT2  Breunig & Kuger (1987)  HF1987  MATametA ura3 RAG1 HGT1  Salani & Bianchi (2006)  MW179-1D  MATαade trpA metA leu2 ura3 lac4-8 KHT1 KHT2  This work  PM6-7A  MATaadeT-600 ura3 RAG1 HGT1  (Wésolowski-Louvel 1992a, 5  MATαlysA leu2 ura3 lac4-8 RAG1 HGT1  Destruelle (1999)  MW278-8C  MATαade2 leu2 ura3  Uccelletti (2006)  CPK1  MATαade2 leu2 ura3 KlPMR1::KanR  Farina (2004)  Strain  Genotype  References  MW98-8C  MATαlysA argA ura3 rag1 HGT1 rag2  Bianchi (1987)  MW270-7B  MATametA leu2 ura3 RAG1 HGT1  Billard (1996)  JA6  MATαade1-600 adeT-600 trp1 ura3 KHT1 KHT2  Breunig & Kuger (1987)  HF1987  MATametA ura3 RAG1 HGT1  Salani & Bianchi (2006)  MW179-1D  MATαade trpA metA leu2 ura3 lac4-8 KHT1 KHT2  This work  PM6-7A  MATaadeT-600 ura3 RAG1 HGT1  (Wésolowski-Louvel 1992a, 5  MATαlysA leu2 ura3 lac4-8 RAG1 HGT1  Destruelle (1999)  MW278-8C  MATαade2 leu2 ura3  Uccelletti (2006)  CPK1  MATαade2 leu2 ura3 KlPMR1::KanR  Farina (2004)  * Formerly CBS2359/152F. † A gift from M. Wésolowski-Louvel. View Large YPD medium contained 1% yeast extract (Becton Dickinson, Sparks, MD), 2% peptone (Becton Dickinson) and 2% glucose. SD medium contained 0.67% yeast nitrogen base (Becton Dickinson), 2% glucose and auxotrophic requirements as needed; 2% Bacto casamino acids (Becton Dickinson) were added to the SDA medium. Agar 2% was added to solid media. ABTS plates contained 2,2′ azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Fluka, Buchs, Switzerland), 0.2 mM and CuSO4 0.1 mM. The corn steep liquor (CSL)-based medium was composed of 5% glucose, 1% CSL (Cargill, Italy), pH 7, 0.05% MgSO4·7H2O, 0.05% KH2PO4 and 0.01% CaCl2·2H2O. Fermentations Fermentation processes were carried out using the BiostatQ four-vessel bioreactor (B-Braun, Melsungen, Germany). Typical parameters were 700 mL of medium/vessel, 28 °C, 250–300 r.p.m. and 0.2 L min−1 of air supply. Cells were precultivated in SD (centromeric transformants) or YPD (integrative strains) and inoculated into BiostatQ at a starting concentration of 105 or 106 cells mL−1. Fermentations were conducted for 4 days and monitored every 24 h. Samples were used to determine OD600 nm and laccase activity in the supernatant after centrifugation. The centrifuged cells were used for dry weight measurements and RNA or protein extraction. Aliquots of culture samples were plated, after dilution, onto YPD and SD plates to measure the CFU and plasmid stability. Expression analysis Total RNAs were extracted from sampled biomass using a hot-phenol procedure (Köhrerk & Domdey, 1991). After fractionation by agarose/formaldehyde electrophoresis, RNAs were transferred to Nytran-N membranes (Schleicher and Schuell, Dassel, Germany) by capillary blotting and hybridized with 32P-labeled DNA probes. Probes were labeled using the Random Primed DNA labeling kit (Roche, Mannheim, Germany). The Lcc1 probe was the HindIII fragment of the cDNA. The actin probe was the 1.3-kbp HindIII fragment containing exon II of the KlACT1 gene (Deshler, 1989). Probed membranes were exposed for autoradiography. Crude extracts were prepared by resuspending cells in 20% glycerol, 0.1 M Tris-HCl, pH 8, and 1 mM dithiothreitol, and then breaking with glass beads. After extract separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide 29 : 1), proteins were detected by Coomassie staining. Laccase activity determination and enzyme characterization The laccase activity toward ABTS was measured by means of a spectrophotometric assay carried out as described previously (Colao, 2006), and the enzymatic activity was expressed as nkat mL−1 (1 nkat corresponds to 0.06 IU). Fermentation broths for laccase characterization were harvested at the end of the processes (96 h) and the biomass was separated by centrifugation (8000 g at 4 °C for 10 min). The exhausted media containing the recombinant laccase Lcc1 were concentrated by ultrafiltration and equilibrated in imidazole/HCl 10 mM and CuSO4 100 μM. The samples were fractionated on a Vivapure Q Mini H (Sartorius) anion-exchange spin column according to the manufacturer's instructions. Recombinant Lcc1 was eluted with NaCl 0.5 M and then equilibrated again with imidazole/HCl 10 mM and CuSO4 100 μM. The corresponding native enzyme from T. trogii and recombinant Lcc1 produced from P. pastoris and S. cerevisiae were also obtained as reported previously (Garzillo, 1998; Colao, 2006). Native PAGE was carried out on 7.5% polyacrylamide gels at pH 8.8 under nondenaturating conditions; laccase activity was visualized in the gel with 10 mM p-phenylenediamine as a substrate in 0.1 M acetate buffer, pH 5.0. Dye decolorization Dyes representing different chemical classes were purchased from Sigma-Aldrich: carmoisine λmax=515 nm, patented blue λmax=625 nm, blue indigo carmine λmax=610 nm and remazol brilliant blue R λmax=592 nm. The enzymatic treatment of textile dyes was performed in a microtiter plate assay in a reaction volume of 200 μL per well. The reaction mix contained 0.17 nkat laccase, 1 mM 1-hydroxybenzotriazole and dye (0.025 mg mL−1 patented blue, 0.050 mg mL−1 blue indigo and carmoisine, 0.250 mg mL−1 remazol brilliant blue R) in Na phosphate buffer, pH 5.0. Samples were incubated at room temperature and decolorization activity was determined spectrophotometrically as the relative decrease of absorbance at the maximal absorbance wavelength of each dye. Results Strain selection We performed a preliminary selection of strains (listed in Table 1) for the secretion of Lcc1 laccase of T. trogii by transformation with the centromeric vector pLC12 (Camattari, 2007). This vector contained the Lcc1 gene under the transcriptional control of the KlPDC1 promoter. The transformed strains were inoculated in synthetic glucose medium supplemented with 2% casaminoacids (SDA) for parallel fermentations with the four-vessel bioreactor BiostatQ. The results of the maximal secreted activity (nkat mL−1) measured in the fermentation processes, together with the time points and OD of the cultures corresponding to the highest activity values, are shown in Table 2. Specific activities (nkat mL−1× OD) and other strain and growth parameters, such as vector stability and maximal growth, are also given in Table 2. The results clearly indicated that the strain MW98-8C produced one to two orders of magnitude more laccase than other strains in terms of activity and specific activity. The tested strains differed especially in terms of glucose transporters, as shown in Table 1: the low-affinity glucose transporter genes RAG1 (Wésolowski-Louvel, 1992a) and KHT1/KHT2 (KHT2 encodes a glucose transporter with intermediate affinity; Milkowski, 2001) or the high-affinity transporter HGT1 (Billard, 1996). No correlation could be found between laccase production and assortment of glucose transporters, except that the best producing strain, MW98-8C, was defective in the low-affinity transport of glucose (rag1 mutation). This strain harbored an additional rag mutation (rag2) in the phospho-glucose isomerase gene, which also impaired fermentative growth. 2 Selection of the best laccase-producing replicative strain Strain  Maximal activity (nkat mL−1)  Time (h)  OD600 nm  Maximal specific activity (nkat mL−1× OD)  CFU (× 108)  Stability (%± SD)  Maximal growth (OD600 nm)  MW98-8C  1.80  72  9.1  0.198  5.4  79 ± 18  10.2  MW270-7B  0.26  96  9.7  0.027  ND  ND  9.7  JA6  0.19  48  4.1  0.046  1.6  94 ± 9  8.9  HF1987  0.17  72  5.9  0.029  2.5  84 ± 7  10.0  MW179-1D  0.12  72  4.3  0.028  1.8  79 ± 12  10.2  PM6-7A  0.04  42  4.0  0.010  ND  ND  10.0  MW341/5  0.02  68  8.3  0.002  ND  ND  8.3  MW278-8C  0.24  96  9.5  0.025  3.1  ND  9.5  CPK1  0.18  72  2.1  0.085  ND  ND  4.4  Strain  Maximal activity (nkat mL−1)  Time (h)  OD600 nm  Maximal specific activity (nkat mL−1× OD)  CFU (× 108)  Stability (%± SD)  Maximal growth (OD600 nm)  MW98-8C  1.80  72  9.1  0.198  5.4  79 ± 18  10.2  MW270-7B  0.26  96  9.7  0.027  ND  ND  9.7  JA6  0.19  48  4.1  0.046  1.6  94 ± 9  8.9  HF1987  0.17  72  5.9  0.029  2.5  84 ± 7  10.0  MW179-1D  0.12  72  4.3  0.028  1.8  79 ± 12  10.2  PM6-7A  0.04  42  4.0  0.010  ND  ND  10.0  MW341/5  0.02  68  8.3  0.002  ND  ND  8.3  MW278-8C  0.24  96  9.5  0.025  3.1  ND  9.5  CPK1  0.18  72  2.1  0.085  ND  ND  4.4  Fermentations in the bioreactor with SDA medium at 300 r.p.m., 28°C and 0.2 L min−1 aeration. Time, OD600 nm, CFU and stability values at the maximal activity points are reported. Maximal growth was observed at 96 h. ND, not determined. View Large 2 Selection of the best laccase-producing replicative strain Strain  Maximal activity (nkat mL−1)  Time (h)  OD600 nm  Maximal specific activity (nkat mL−1× OD)  CFU (× 108)  Stability (%± SD)  Maximal growth (OD600 nm)  MW98-8C  1.80  72  9.1  0.198  5.4  79 ± 18  10.2  MW270-7B  0.26  96  9.7  0.027  ND  ND  9.7  JA6  0.19  48  4.1  0.046  1.6  94 ± 9  8.9  HF1987  0.17  72  5.9  0.029  2.5  84 ± 7  10.0  MW179-1D  0.12  72  4.3  0.028  1.8  79 ± 12  10.2  PM6-7A  0.04  42  4.0  0.010  ND  ND  10.0  MW341/5  0.02  68  8.3  0.002  ND  ND  8.3  MW278-8C  0.24  96  9.5  0.025  3.1  ND  9.5  CPK1  0.18  72  2.1  0.085  ND  ND  4.4  Strain  Maximal activity (nkat mL−1)  Time (h)  OD600 nm  Maximal specific activity (nkat mL−1× OD)  CFU (× 108)  Stability (%± SD)  Maximal growth (OD600 nm)  MW98-8C  1.80  72  9.1  0.198  5.4  79 ± 18  10.2  MW270-7B  0.26  96  9.7  0.027  ND  ND  9.7  JA6  0.19  48  4.1  0.046  1.6  94 ± 9  8.9  HF1987  0.17  72  5.9  0.029  2.5  84 ± 7  10.0  MW179-1D  0.12  72  4.3  0.028  1.8  79 ± 12  10.2  PM6-7A  0.04  42  4.0  0.010  ND  ND  10.0  MW341/5  0.02  68  8.3  0.002  ND  ND  8.3  MW278-8C  0.24  96  9.5  0.025  3.1  ND  9.5  CPK1  0.18  72  2.1  0.085  ND  ND  4.4  Fermentations in the bioreactor with SDA medium at 300 r.p.m., 28°C and 0.2 L min−1 aeration. Time, OD600 nm, CFU and stability values at the maximal activity points are reported. Maximal growth was observed at 96 h. ND, not determined. View Large Construction of integrative strains The best producing strains, MW98-8C and MW270-7B, the ‘Crabtree-positive’ strain JA6 and strain MW179-1D, which have never been used for basic or applied research to date, were chosen to construct strains with the expression cassette for Lcc1 stably integrated into the chromosome. Integrative transformants can eliminate problems related to plasmid loss, which can cause reduction of product yield and is not admissible for a validated and scaled-up process. In addition, integrative transformants can be cultured on complete and/or rough media, which are more suited to industrial applications. A drawback of integrative transformation in K. lactis is that homologous recombination is rare and the randomly integrated recombinant clones that can be obtained might be inactivated in genes critical for process perspectives. The map of the vector constructed for the integrative transformation, named pDRLCi, is shown in Fig. 1: the Lcc1 gene transcription was under the control of the KlPDC1 promoter and the S. cerevisiae PHO5 terminator. Selected Ura+ transformants – designated DR98, DR270, DRJA6 and DR179, obtained with circular pDRLCi from strains MW98-8C, MW270-7B, JA6 and MW179-1D, respectively – were repeatedly subcloned on YPD and finally plated on YPD plates, selective plates and plates containing ABTS. Two independent transformed clones for each strain, showing 100% stability of the Ura+ phenotype and laccase activity, were tested by PCR to determine the locus of integration. Only one DRJA6 transformed clone had the plasmid pDRLCi integrated in the KlPDC1 promoter, which is the only sequence homologous to the host chromosome present in the plasmid. The subsequent analysis did not show any significant difference between the homologous and the random integrative clones in our experiments. 1 View largeDownload slide Map of the integrative plasmid pDRLCi. The thin line denotes the plasmid pSKBluescript II; the black arrow denotes the the URA3 selection marker; the white arrow denotes the cDNA of the Lcc1 gene and the squared boxes denote the regulatory elements of the expression cassette: the KlPDC1 promoter and the PHO5 terminator. Restriction sites are B, BamHI; E, EcoRI; H, HindIII; N, NheI; P, PstI; and S, SalI. 1 View largeDownload slide Map of the integrative plasmid pDRLCi. The thin line denotes the plasmid pSKBluescript II; the black arrow denotes the the URA3 selection marker; the white arrow denotes the cDNA of the Lcc1 gene and the squared boxes denote the regulatory elements of the expression cassette: the KlPDC1 promoter and the PHO5 terminator. Restriction sites are B, BamHI; E, EcoRI; H, HindIII; N, NheI; P, PstI; and S, SalI. Laccase production in different integrative strains Production of laccase from the integrative transformants was preliminarily measured in Erlenmeyer flasks containing YPD medium (at 28 °C and 175 r.p.m. shaking) and incubated up to 4 days. Secreted laccase activity and growth were measured daily. Figure 2a shows the best results of specific activity (nkat mL−1× OD) registered for each strain. Except for a few cases with DR179 and DRJA6 strains, in which maximal activity was reached at 48 h, maximal values were measured at 72 or 96 h from the inoculum. The best producing strain was DR98, derived from MW98-8C, which also showed the maximal activity per milliliter and growth values (not shown). 2 View largeDownload slide Laccase expression in different strains. (a) Comparison of maximal specific laccase production (nkat mL−1× OD) of the four integrative DR98 strains. DR270, DR179 and DRJA6 were grown in shake-flask cultures with YPD medium. Values are averages of four experiments with SDs reported. (b, c) Laccase productions of strains DR98, DR270 and DRJA6 in the bioreactor with SDA (b) and YPD (c) media. Specific laccase activities (U mg−1dry weight) are reported in the top panels; transcription analyses (Lcc1 and KlACT1 transcription and total RNA loading: LCC, ACT and rRNA, respectively) are shown in the bottom panels. Samples of RNA preparations from identical numbers of cells have been loaded onto gels of the experiments shown in (b) and (c) to analyze the representative RNA content per cell. (d) SDS-PAGE analysis of crude extracts from cells grown on SDA and YPD. Extracts from a 48-h YPD culture of the parental strain MW98-8C have been loaded as a reference (wt). M indicates the ladder lane with relevant molecular weights (kDa) marked on the right. 2 View largeDownload slide Laccase expression in different strains. (a) Comparison of maximal specific laccase production (nkat mL−1× OD) of the four integrative DR98 strains. DR270, DR179 and DRJA6 were grown in shake-flask cultures with YPD medium. Values are averages of four experiments with SDs reported. (b, c) Laccase productions of strains DR98, DR270 and DRJA6 in the bioreactor with SDA (b) and YPD (c) media. Specific laccase activities (U mg−1dry weight) are reported in the top panels; transcription analyses (Lcc1 and KlACT1 transcription and total RNA loading: LCC, ACT and rRNA, respectively) are shown in the bottom panels. Samples of RNA preparations from identical numbers of cells have been loaded onto gels of the experiments shown in (b) and (c) to analyze the representative RNA content per cell. (d) SDS-PAGE analysis of crude extracts from cells grown on SDA and YPD. Extracts from a 48-h YPD culture of the parental strain MW98-8C have been loaded as a reference (wt). M indicates the ladder lane with relevant molecular weights (kDa) marked on the right. The three strains DR98, DR270 and DRJA6, were further compared in the bioreactor for laccase production in SDA and YPD media. These strains had different assortments of the glucose transporters that might affect the fermentative metabolism and/or the expression from the KlPDC1 promoter. Four-day processes were performed. Laccase activity in supernatants, OD600 nm and biomass dry weight mL−1 were measured. The correlations (R2) between dry weight and OD were >0.95. Crude extracts and total RNAs were also prepared. The results of specific laccase activity (mg−1 dry weight) in the supernatant and RNA analysis are shown in Fig. 2b (SDA medium) and Fig. 2c (YPD medium). For each time point, identical fractions of the RNA preparations that were obtained from identical amounts of biomass (RNA yield per cell) were loaded onto gels for Northern blot analysis. Strain DR98 confirmed its higher efficiency in the production of extracellular laccase (Fig. 2b and c, upper panels). However, the transcription of the laccase gene in DR98 (Fig. 2b and c, lower panels) was lower than in the other strains, especially in the YPD medium at 24 h, when compared with the intensities of the reference signals of the actin gene and of the ribosomal rRNAs genes, although it was (somewhat) more extended over time. SDS-PAGE analysis of crude extract from SDA or YPD cultures did not reveal the presence or the accumulation of a protein corresponding in size (55 kDa) to the recombinant laccase. Figure 2d shows SDS-PAGE analysis of DR98 extracts: strains DR270 and DRJA6 yielded similar results (not shown). The laccase activity was also measured in the crude extracts. The pattern of activities (not shown) was similar to that measured in the supernatants (Fig. 2b and c); the highest values were for DR98 and they progressively increased along the fermentation process for all strains. Activities in SDA-grown cells ranged between 10 and 250 pkat mg−1 and were barely detectable in YPD-grown cells. Cultural conditions: medium composition The results reported above suggested that the production of secreted laccase was more efficient on SDA medium than on YPD. Therefore, we assayed in more detail laccase production by the DR98 strain in parallel bioreactor fermentations with SDA, YPD and a CSL-based medium, a component frequently used in industrial practice. Fermentation runs were conducted with air inflow at 0.2 L min−1. The results of specific activity (nkat mL−1× OD) are shown in Fig. 3a. Laccase was accumulated in the culture supernatants to different extents and at different rates by varying the composition of the medium. In SDA, laccase started accumulating from the beginning of the process (24 h) and its concentration progressively increased. In YPD, the enzyme secretion also began at 24 h, but its rate of production and concentration were lower, even taking into account the developing biomass (data reported as OD600 nm in Fig. 3b). In CSL, the laccase was entirely produced between 24 and 48 h, but the amount was low and did not increase further. Biomass growth also ceased in CSL after 24 h. 3 View largeDownload slide Laccase production on different media. (a) Comparison of laccase production of the integrative DR98 strain in SDA (black blocks) vs. YPD (gray blocks) and CSL (striped blocks) media. Values are averages of specific laccase productions (nkat mL−1× OD) obtained from repeated experiments of parallel fermentations at the indicated process times. SDs are reported. (b) Average growth of the corresponding processes, expressed as OD600 nm values, at the indicated times. 3 View largeDownload slide Laccase production on different media. (a) Comparison of laccase production of the integrative DR98 strain in SDA (black blocks) vs. YPD (gray blocks) and CSL (striped blocks) media. Values are averages of specific laccase productions (nkat mL−1× OD) obtained from repeated experiments of parallel fermentations at the indicated process times. SDs are reported. (b) Average growth of the corresponding processes, expressed as OD600 nm values, at the indicated times. The transcription profiles of the laccase gene in one of these typical fermentations with SDA and two with YPD media are shown in Fig. 4a. In this Northern blot analysis, gel lanes were loaded with the same amounts of total RNAs. Lcc1 transcription was similarly intense in both media at 24 h, but, in contrast to the laccase detection in the supernatants reported above, it decreased progressively in SDA at the following time points. In contrast to SDA, on YPD medium, Lcc1 transcription showed a peak of intensity at 48 h and then decreased. These results suggest that Lcc1 transcription and the process of extracellular enzyme accumulation might follow different temporal patterns and/or have different efficiencies when the medium composition is changed. 4 View largeDownload slide Transcription analysis of the integrated Lcc1 gene. Transcription analysis was performed using Northern blotting. Filters have been hybridized with the Lcc1 probe (upper panels). Ethidium bromide fluorescence of rRNA genes before capillary transfer is reported as a loading control (lower panels). RNA samples were extracted at 24, 48, 72 and 96 h. (a, b) Comparative analysis of media composition (one SDA process and two independent YPD fermentations) and of the aeration rate (0.2, 0.4 and 1 L min−1), respectively. 4 View largeDownload slide Transcription analysis of the integrated Lcc1 gene. Transcription analysis was performed using Northern blotting. Filters have been hybridized with the Lcc1 probe (upper panels). Ethidium bromide fluorescence of rRNA genes before capillary transfer is reported as a loading control (lower panels). RNA samples were extracted at 24, 48, 72 and 96 h. (a, b) Comparative analysis of media composition (one SDA process and two independent YPD fermentations) and of the aeration rate (0.2, 0.4 and 1 L min−1), respectively. Cultural conditions: aeration In shake-flask cultures, one limiting factor is air supply, in particular at a high cell density and if strains that are unable to ferment, such as MW98-8C or DR98, are used. In addition, the KlPDC1 promoter used to express the Lcc1 gene is regulated by oxygen (Camattari, 2007). For this reason, we tested in the bioreactor whether air supply could affect laccase production of strain DR98. The inlet airflow was regulated at 0.2, 0.4 and 1.0 L min−1, and processes were carried out as usual in the SDA medium. A very clear repression by air supply of Lcc1 transcription could be observed, but only at 24 h (Fig. 4b). Interestingly, after 48 h, Lcc1 expression in the three assays converged to an intermediate, but relevant, level, accompanied by a steady state of cellular metabolism, as indicated by the OD increase (not shown). Consistent with this finding, the overall profiles of enzyme production at the different air supplies were almost similar (not shown). Residual dissolved oxygen at 72 and 96 h was as high as 20–30% of the starting value in all vessels, suggesting a physiological correlation in this strain of the expression of the KlPDC1 promoter with the growth phase rather than with hypoxia. Comparison of replicative vs. integrative strains Next, we evaluated the production of the integrative strains in the bioreactor and compared them with the centromeric transformants. The production processes were performed with the BiostatQ bioreactor in duplicate for each of the two integrative and centromeric transformed clones tested. Four days of fermentations in SDA medium were carried out; laccase activity in the medium (nkat mL−1), cell OD, CFU and stability (Ura+ colonies) were determined and, in addition, RNA was extracted. The results of laccase activity are reported in Fig. 5a and showed a continuous enzyme accumulation up to 96 h with an approximately double the final production (3.2±1 vs. 1.8±0.4 nkat mL−1) by the integrative strain DR98 with respect to the MW98-8C[pLC12]-transformed clones. The DR98 strain also showed a slightly better growth (Fig. 5b), more pronounced at 24 and 48 h, and slightly stronger Lcc1 transcription throughout the process (Northern blotting; data not shown). Stability data (not shown) indicated that integration was maintained until the end of the process, without marker loss (Ura+ phenotype). The comparison in the bioreactor between integrative and centromeric strains, under identical conditions as above, was also extended to DR270 and MW270-7B[pLC12] strains, which produced 0.43 and 0.26 nkat mL−1 at 96 h, respectively, thus confirming the superiority of the integrative strains and, in particular, of DR98. 5 View largeDownload slide Laccase production in replicative and integrative strains. (a) Comparison of laccase production between the integrative DR98 strains (black blocks) and the replicative transformants MW98-8C[pLC12] (gray blocks). Laccase activities (nkat mL−1) in the supernatants sampled at 24, 48, 72 and 96 h of production processes performed in the bioreactor (SDA medium, 300 r.p.m., 28°C and 0.2 L min−1) are reported. Values are averages of three independent processes with SDs reported. (b) Growth (OD600 nm) of the integrative DR98 strains (black blocks) and the replicative transformants MW98-8C[pLC12] (gray blocks) in the bioreactor processes reported in (a). 5 View largeDownload slide Laccase production in replicative and integrative strains. (a) Comparison of laccase production between the integrative DR98 strains (black blocks) and the replicative transformants MW98-8C[pLC12] (gray blocks). Laccase activities (nkat mL−1) in the supernatants sampled at 24, 48, 72 and 96 h of production processes performed in the bioreactor (SDA medium, 300 r.p.m., 28°C and 0.2 L min−1) are reported. Values are averages of three independent processes with SDs reported. (b) Growth (OD600 nm) of the integrative DR98 strains (black blocks) and the replicative transformants MW98-8C[pLC12] (gray blocks) in the bioreactor processes reported in (a). Laccase characterization Supernatants of K. lactis-integrative and -replicative transformants of different strains and from different media were withdrawn from fermentation vessels for enzyme characterization. A zymogram analysis was performed to compare the electrophoretic mobility of the enzyme with that of native and recombinant Lcc1 laccases produced in other yeast species such as P. pastoris and S. cerevisiae. The results (Fig. 6a) showed that native and recombinant enzymes produced in yeast species behave differently. In particular, Lcc1 secreted by K. lactis exhibited the lowest mobility, followed by S. cerevisiae and P. pastoris Lcc1. We demonstrated previously that the different electrophoretic mobility of the recombinant laccase by P. pastoris as compared with the native enzyme can be ascribed to a higher level of glycosylation and that the two proteins, after treatment with endoglycosidase H, showed similar molecular masses (Colao, 2006). 6 View largeDownload slide Electrophoretic analysis of laccases. (a) Electrophoretic analysis of native and recombinant laccases expressed in three different yeast species. Lane 1, native Lcc1; lane 2, Pichia pastoris Lcc1; lane 3, Kluyveromyces lactis Lcc1; lane 4, Saccharomyces cerevisiae Lcc1. The native-PAGE is stained with p-phenylenediamine as a substrate. (b) Electrophoretic mobility of laccase from the wild-type strain MW278-8C (lane 1), the O- and N-glycosylation-defective mutant CPK1 (lane 2) and the native laccase (lane 3). 6 View largeDownload slide Electrophoretic analysis of laccases. (a) Electrophoretic analysis of native and recombinant laccases expressed in three different yeast species. Lane 1, native Lcc1; lane 2, Pichia pastoris Lcc1; lane 3, Kluyveromyces lactis Lcc1; lane 4, Saccharomyces cerevisiae Lcc1. The native-PAGE is stained with p-phenylenediamine as a substrate. (b) Electrophoretic mobility of laccase from the wild-type strain MW278-8C (lane 1), the O- and N-glycosylation-defective mutant CPK1 (lane 2) and the native laccase (lane 3). Recombinant Lcc1 secreted by K. lactis showed two different bands of different intensity, suggesting heterogeneity in glycosylation. We transformed the mutant strain CPK1, defective in O- and N-glycosylation (Farina, 2004), and the parental wild-type strain MW278-8C, with plasmid pLC12 and the produced laccase enzyme from the transformant strains cultivated in the bioreactor on SDA medium. Fermentation data are summarized in Table 2. The results of activity staining of laccase after electrophoresis on native gels are shown in Fig. 6b. The laccase from the mutant strain, defective in both glycosylation pathways, showed a unique active form with increased mobility with respect to those of the wild-type strain, confirming that both the major and the minor forms of the laccase produced by K. lactis were due to the heterogeneity of the glycosylation pattern. On the other hand, the mobility difference of the enzyme produced by CPK1 with the native laccase could be ascribed to modifications occurring in the endoplasmic reticulum, as CPK1 is a Golgi mutant (Farina, 2004). Decolorization of synthetic dyes Laccases expressed in different yeasts were compared for their ability to decolorize four synthetic dyes: azo dyes (carmoisine), triarylmethane (patented blue), blue indigo and the anthroquinonic dye remazol brilliant blue R, in the presence of the redox mediator 1-hydroxybenzotriazole. As shown in Fig. 7, the tested dyes were decolorized to different extents depending on the structure and the complexity of the molecule. After 2 h of treatment, K. lactis Lcc1 showed the best performance, yielding a decolorization rate >46% for all the tested dyes, whereas decolorization of the native laccase exhibit was always <37%. Kluyveromyces lactis and P. pastoris Lcc1 always oxidized dyes more efficiently than did the native protein. Saccharomyces cerevisiae Lcc1 was superior only to remazol brilliant blue and blue indigo. After 6 h, the decolorization of the four dyes by K. lactis Lcc1 was complete, and it was significantly more efficient than other laccases in the decolorization of remazol brilliant blue R, a compound representative of an important class of anthraquinone-type dyes. These results demonstrated that K. lactis Lcc1 could be a useful catalyst for the oxidative degradation of several recalcitrant dyes. 7 View largeDownload slide Decolorization analysis of laccases. Enzymatic decolorization of some industrial dyes with recombinant Lcc1 (0.17 nkat) secreted by different yeast species. Data points represent decolorization percent after 2 h (a) and 6 h of treatment (b) in the presence of the redox mediator 1-hydroxybenzotriazole (1 mM) and are averages of triplicate measurements with SD within 10% of the mean. 7 View largeDownload slide Decolorization analysis of laccases. Enzymatic decolorization of some industrial dyes with recombinant Lcc1 (0.17 nkat) secreted by different yeast species. Data points represent decolorization percent after 2 h (a) and 6 h of treatment (b) in the presence of the redox mediator 1-hydroxybenzotriazole (1 mM) and are averages of triplicate measurements with SD within 10% of the mean. Discussion The different genetic backgrounds of natural K. lactis isolates and those occurring among laboratory strains, derived from crosses and mutagenesis, drastically affect heterologous protein production. The specific genetic locus or loci influencing this complex and multistep process are usually not known and are difficult to identify. In addition, different proteins may be produced with different efficiencies in the same host strain. The common practice is thus to test a certain number of strains for the production of each protein. In our study, we used the KlPDC1 promoter, which is strongly regulated by the carbon source and oxygen (Bianchi, 1996; Camattari, 2007), for the expression of the T. trogii Lcc1 gene. We demonstrate that the production of extracellular fungal laccase in K. lactis is strongly strain dependent, with 100-fold differences among strains, and integrative transformants are more efficient than replicative low-copy number (centromeric) transformants. Integration did not occur frequently at the locus (the KlPDC1 gene) with sequence homology with the vector; however, none of the (about 10) nonhomologous recombinant clones tested showed impaired growth or reduced laccase production, suggesting that random integration is an efficient system for obtaining stable and vigorous recombinant strains for heterologous protein production. The best laccase-producing strains are MW98-8C and the integrative derivative DR98, which are respiratory obligated (Rag−) strains, mutated in the low-affinity glucose transporter gene (RAG1) and in the glycolytic gene RAG2, coding for the phospho-glucose isomerase. These strains did not suffer from impaired growth and/or laccase production either in flask cultures or in the bioreactor, indicating that the high-affinity glucose transport and the pentose phosphate pathway are sufficient for high biomass yield and recombinant protein production. On the other hand, high biomass is not sufficient per se to guarantee a high product yield, as reported in Table 2. In addition, the rag1 and rag2 mutations were shown to negatively regulate KlPDC1 transcription (Bianchi, 1996), suggesting that, in theory, this strain might not be optimal for the expression of a heterologous gene from the KlPDC1 promoter. Indeed, Lcc1 transcription was significantly decreased in the DR98 strain with respect to the other fermentative strains, especially on YPD medium. However, the regulation of the expression of KlPDC1 depends on many other elements (Destruelle, 1999; Salani & Bianchi, 2006; Tizzani, 2007), and because the production and secretion of heterologous protein is a complex pathway, other factors specific for the MW98-8C background are likely to come into play to favor laccase production. Besides strain specificity, our present results indicate that the production of active and secreted laccase does not seem to be necessarily correlated with the level of laccase mRNA in the producing strain. In fact, strain DR98, in which Lcc1 transcription is the weakest, produces more laccase. This might be the consequence either of a hypothetically minor protease production by this strain or of a more efficient pathway of protein synthesis and secretion, or it could be the effect of a more prolonged phase of active metabolism competing with protease degradation, as suggested by transcript analysis in YPD. A similar phenomenon of extended metabolic activity has also been demonstrated for other strains expressing heterologous proteins with the KlPDC1 promoter (Salani & Bianchi, 2006). Finally, the combination of the KlPDC1 promoter and the strain (MW98-8C) background seems to be quite flexible and yields good production under different process conditions. For example, air supply is not a critical process parameter in a wide range of values in our laccase-producing strain when prolonged processes are used. In fact, transcription analysis reveals a composite expression pattern of the Lcc1 gene under the KlPDC1 promoter, where the hypoxic regulation is evident only in the first phase of the process, when cells are in the log or in the late-log phase. When the growth rate declines and the culture enters the stationary phase, oxygen regulation is lost and other phase-dependent, but still unknown, factors regulate transcription. Interestingly, this factor allows quite a high level of expression. This stationary-phase regulation might depend on the impairment of the fermentative metabolism, because it has been observed to date only in Rag− strains (Salani & Bianchi, 2006; and the present work). However, how a fermentation defect could affect the expression of the KlPDC1 gene in a phase-dependent manner is still an open question. The decolorization of model dyes is a simple method to assess the bioremediation potential of ligninolytic enzymes (Lorenzo, 2006). In an attempt to verify the decolorization ability of recombinant laccases, assays were carried out with Lcc1 in the presence of a suitable redox mediator such as 1-hydroxybenzotriazole, because, according to Claus (2002), the addition of 1–2 mM of 1-hydroxybenzotriazole significantly improves or facilitates the decolorization of synthetic dye by these enzymes. Under these conditions, the extracellular laccase produced by K. lactis DR98 is very effective in the oxidative degradation of several polluting dyes. The diversity between recombinant laccases produced by yeasts might be explained by variations in post-translational modification, such as glycosylations, among different hosts. These modifications could lead to differences in the global folding of the protein and may affect the catalytic properties of laccase in unpredictable ways. The final decolorization capacity of recombinant Lcc1 from K. lactis was also higher than that of a pure fungal laccase obtained from a commercial formulation used in the textile industry (Soares, 2001), which exhibits a threefold lower decolorization of remazol brilliant blue in a reaction mix containing a higher concentration of enzyme and mediator (2 nkat and 2.2 mM, respectively) and a lower concentration of dye (0.020 mg mL−1). Here, we show that expression of the Lcc1 laccase gene in different yeast species makes it possible to obtain alternative isoforms with different decolorization capacity toward azo, triarylmethane, blue indigo and anthraquinonic dyes. Choosing the appropriate heterologous host for laccase production can enhance the potential technological applications for this enzyme. 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TI - Optimization of recombinant fungal laccase production with strains of the yeast Kluyveromyces lactis from the pyruvate decarboxylase promoter
JO - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2009.00532.x
DA - 2009-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/optimization-of-recombinant-fungal-laccase-production-with-strains-of-bzgteYGrQL
SP - 892
EP - 902
VL - 9
IS - 6
DP - DeepDyve
ER -