1 Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile
2 USDA Forest Products Laboratory, Madison, WI 53705, USA
Correspondence
Rafael Vicuña
rvicuna{at}genes.bio.puc.cl
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY219235 and AY219236.
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INTRODUCTION |
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The active site of laccases possesses four copper ions, which can be classified according to their spectrophotometric properties. Type I copper (blue copper) exhibits an intense absorption at about 600 nm, owing to the charge transfer between Cu(II) and a cysteine residue. Type II copper shows a very weak absorption and functions as a one-electron acceptor. Type III copper contains two copper centres absorbing at 330 nm and functions as a two-electron acceptor (Jonsson et al., 1995; Reinhammer, 1984
).
Several laccase isoforms can be identified in C. subvermispora cultures depending on the composition of the medium (Salas et al., 1995). Low-stringency Southern blots (Karahanian et al., 1998
) and N-terminal sequences of laccases isoenzymes (Salas et al., 1995
) suggest a single laccase gene, and its expression is strongly regulated by copper at the transcriptional level (Karahanian et al., 1998
). The phenomenon of isoenzyme multiplicity is commonly observed among ligninolytic fungi, although its physiological significance is not known.
To further biochemical analysis of the C. subvermispora laccase we describe here the cloning and expression of Lcs-1 cDNA in Aspergillus nidulans and Aspergillus niger. The A. nidulans system produced multiple isoforms in a pattern that was similar to the native C. subvermispora enzyme, and copper was found to play a post-transcriptional role in laccase expression.
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METHODS |
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Cloning and sequencing.
RT-PCR was used to isolate a full-length cDNA as described by Karahanian et al. (1998). Design of primers was based on the genomic sequence of lcs-1 (GenBank accession number AF053472). The upstream primer was located at position -30 from the ATG, whereas the downstream primer was at position +7 with respect to the stop codon. The PCR product, designated Lcs-1, was directly cloned in pBluescript KSII+ as described by Lobos et al. (1998)
. To obtain genomic clones, lcs-1 was amplified from C. subvermispora homokaryons 3 and 8 (Tello et al., 2001
). Nucleotide sequence was determined with the ABI-Prism Dye terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems) with an ABI373 DNA sequencer. Sequence editing and analysis were done with DNAstar software.
Aspergillus expression.
The expression vector pexLcs-1 was constructed by placing the Lcs-1 cDNA coding region, including the sequence for its signal sequence, under the control of the TAKA amylase promoter (Andersen et al., 1992) and the Aspergillus awamori glucoamylase terminator (Kersten et al., 1995
) by the PCR overlap extension technique (Horton et al., 1989
) using proofreading polymerase Pfu (Stratagene). The coding region and junctions of the expression cassette were sequenced. The selection marker, ppyrG, was obtained from the Fungal Genetic Stock Center.
A. nidulans A722 and A. niger A969 cotransformations were as described by Larrondo et al. (2001), except that A. niger protoplasts were prepared from germinated conidia after an overnight incubation at 300 r.p.m. at 30 °C. Protoplasts were cotransformed with 5 µg ppyrG and 5 µg pexLcs-1. Selection was based on complementation of uridine auxotrophy by the selectable marker pyrG. Transformants carrying the genetic construction pexLcs-1 were confirmed by PCR.
One hundred millilitres of Aspergillus minimal medium (AMM) (Cullen et al., 1987) (5 % maltose) or YEM (0·5 % yeast extract, 5 % maltose) was inoculated with 1x107 spores and incubated for 3 days at 30 °C in an orbital shaker (250 r.p.m.). In the case of A. niger A969, the medium was also supplemented with 1 mM methionine. Mycelium was harvested by filtration through Miracloth, snap frozen in liquid nitrogen and stored at -70 °C. For routine cultivations, the medium was supplemented with CuSO4 up to a final concentration of 100 µM. In copper-deficient cultures, only traces of copper were present (13 µM).
We screened rLcs-1 production by plate assay. Isolated transformants were inoculated and selected in agar-minimal medium plates containing maltose (5 %) and 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS; Sigma). Characteristic ABTS oxidation could be observed after 23 days. Transformants were also evaluated for rLcs-1 production in liquid media by spectrophotometric measurements of ABTS oxidation
Enzyme purification and characterization.
One litre of extracellular fluid from A. nidulans Lcs-1 transformant (AnidL-6) culture was directly fractionated by chromatography on Q-Sepharose. One litre of supernatant from A. niger Lcs-1 transformant (AnigL-2) was concentrated 10-fold by filtration in a 185 ml Amicon cell possessing a 10 kDa cut-off membrane, and then dialysed twice against 500 ml 25 mM sodium acetate pH 4·5 and fractionated by chromatography on Q-Sepharose (Lobos et al., 1998). Enzyme activity was measured at 30 °C in a Shimadzu 160 UV-visible recording spectrophotometer. Reaction mixtures (1 ml) contained 50 mM glycine, pH 3·0, and 4·33 mM ABTS as substrate. Km values for ABTS were determined with 0·01 U laccase, using EadieHofstee plots. For endoglycosidase treatment, 5 µg enzyme was treated as described by Larrondo et al. (2001)
.
Zymograms were as previously reported (Larrondo et al., 2001). Briefly, samples were applied with nonreducing denaturing loading buffer without boiling and subjected to SDS-PAGE at 4 °C. Gels were then fixed in a solution containing 10 % acetic acid and 40 % methanol for 10 min followed by incubation in 50 mM glycine buffer, pH 3·0, and 5 mM ABTS for 10 min, or until activity could be detected.
Reconstitution assays.
Q-Sepharose-purified laccase (0·5 µg) from low-copper-containing media was incubated at 25 °C with various concentrations of copper as indicated in the corresponding figure legend. Thereafter, samples were withdrawn at the indicated times to measure laccase activity.
Other methods.
Analytical IEF and protein concentration measurements were as described by Larrondo et al. (2001); RNA extraction and Northern blot hybridization were as described by Karahanian et al. (1998)
. The DNA probe corresponding to the entire coding region of Lcs-1 was labelled with [
-32P]dCTP by nick translation (Gibco-BRL).
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RESULTS |
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Cloning of lcs-1 cDNA
A unique and abundant product was obtained when RT-PCR was conducted with RNA extracted from dikaryotic strain FP105752 of C. subvermispora grown in liquid culture containing 100 µM CuSO4. The cDNA was cloned and sequenced, and based on the nucleotide difference mentioned above, it corresponded to lcs-1B (accession number AY219236).
Production of rLcs-1 in Aspergillus spp.
Transformants confirmed by PCR were screened for extracellular laccase activity when grown in minimal medium. Similar levels of enzyme activity were obtained for both Aspergillus hosts. No major variations in enzyme activity were detected when AMM or YEM media (both supplemented to 100 µM CuSO4 final) were used. Laccase activity appeared on day 2, reaching a peak of 0·23 U ml-1 on day 4 and then detected without major variation up to day 5 or 6. For purification purposes, cultures were routinely collected on day 3. The levels of rLcs-1 produced were about 1·5 mg per litre of culture.
Enzyme purification.
Q-Sepharose purification of laccase from A. nidulans-Lcs-1 cultures yielded one major protein (Anid-rLcs-1) with the same molecular mass and immunoreactivity as the native C. subvermispora enzyme (Fig. 1A). Zymograms showed the presence of only one major band, with a higher electrophoretic mobility than the enzyme under reducing conditions (Fig. 1B
). The enzyme purified from A. niger-Lcs-1 cultures (Anig-rLcs-1) also reacted with antibodies against C. subvermispora laccase although its molecular mass was higher than expected. It was also detected as a single band in zymograms, with a lower electrophoretic mobility than Anid-rLcs-1 (Figs 1A and 1B
, respectively).
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Effect of copper on rLcs-1 production
Copper dramatically affected laccase titres in YEM cultures of A. nidulans Lcs-1. YEM cultures supplemented with 100 µM CuSO4 yielded laccase levels up to 200 U l-1, as compared to less than 20 U l-1 obtained in unsupplemented growth medium. When normalized by biomass, this represents a sixfold stimulation by copper (Fig. 4A). A similar result was observed when A. niger transformants were analysed (data not shown). This effect was not exerted at the transcriptional or the translational levels, as determined by Northern (Fig. 4C
) and Western (Fig. 4B
) blots, respectively. This suggests that apoprotein is efficiently produced and secreted at low copper levels. The effect of copper was also assessed in AMM, with and without added copper. In the absence of externally added copper (copper levels of 13 µM), three and five times less activity was observed in A. nidulans and A. niger transformants, respectively.
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DISCUSSION |
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We were able to successfully express Lcs-1 cDNA in A. nidulans and A. niger. To our knowledge, this is the first report of expression of a white-rot fungus laccase in A. nidulans and the second instance of using A. niger (Record et al., 2002) as a host. Enzyme titres were substantially lower than those reported for other laccases in Aspergillus oryzae systems (Wahleithmer et al., 1995
; Yaver et al., 1996
, 1999
; Record et al., 2002
; Berka et al., 1997
).
The molecular mass obtained for Anid-rLcs-1 was similar to that of the native enzyme, whereas Anig-rLcs-1 had a higher molecular mass. After treatment with endoglycosidase H, the molecular mass of Anig-rLcs-1 remained higher than that of the native enzyme, which suggests that modifications other than N-glycosylation may alter molecular mass. This was also associated with an altered pI, although the Km for ABTS remained unchanged.
The IEF pattern of Anid-rLcs-1 was strikingly similar to that obtained with laccase activity from C. subvermispora. The phenomenon of isoform multiplicity was previously observed with recombinant Cs-MnP1, as well as recombinant Phanerochaete chrysosporium MnP1, both expressed in A. nidulans (Larrondo et al., 2001). We have also observed multiple isoforms for P. chrysosporium multicopper oxidase when expressed in Aspergillus (L. F. Larrondo and others, unpublished). Thus, our experimental data clearly show that multiple isoforms can arise from a single cDNA, which is consistent with the presence of a single laccase gene in C. subvermispora. The precise nature of isoform modifications in A. nidulans and C. subvermispora remains unknown. Possibilities of post-translational modification include glycosylation and phosphorylation. Notably, IEF of A. niger-produced laccase revealed a single isoform, indicating that post-translational modifications differ among species of Aspergillus. Further work is required to understand the biochemical basis of this divergence. Prior to this work, there had been a single report of laccase expression in A. niger. In that study, the recombinant protein had the same characteristics as the native enzyme (Record et al., 2002
).
It has been reported that the expression of the Trichoderma reesei -mannanase gene in yeast gives rise to two proteins with different pIs, which match the pIs described for the same activity in T. reesei (Stalbrand et al., 1995
). A detailed study of T. reesei cellobiohydrolase I (CBHI) secretion revealed several pI forms during processing in the secretion pathway (Pakula et al., 2000
). Both N- and O-glycans might be responsible for the final IEF pattern, but at least in the case of CBHI, it seems plausible that isoform multiplicity is due to phosphorylation of O-glycans. In addition, sulfatation of the glycopeptide region, as described in CBHI produced by the T. reesei strain ALKO2788 (Harrison et al., 1998
), can not be excluded.
Previous investigations have stressed the importance of copper in the regulation of laccase at the transcriptional level (Karahanian et al., 1998; Soden & Dobson, 2001
; Palmieri et al., 2000
; Galhaup et al., 2002
). We previously demonstrated that under low-copper conditions, lcs-1 is not efficiently transcribed in C. subvermispora (Karahanian et al., 1998
). Here, we assessed the influence of copper on the laccase titres in a foreign host. Copper limitation decreased production of fully active laccase, but transcript levels remained constant. At low concentrations of this metal, laccase activity was barely detectable. Interestingly, when AMM medium was used, the effect of copper limitation was not as striking as in YEM medium. Biomass and the total extracellular protein produced in YEM cultures were not drastically affected by supplementation with 100 µM CuSO4. We corroborated this observation by partially recovering activity from supernatants by incubation with copper concentrations in the millimolar range. This implies that the apoprotein lacks some or all of the structural copper atoms necessary for its activity.
There has been some interest in the study of laccase copper centres, by depletion and reconstitution after addition of this metal (Hanna et al., 1988). Usually, this has been achieved by extensive dialysis against cyanide ion. In one case, apoprotein was produced under low copper levels (Bligny et al., 1986
), but the activity could not be reconstituted by incubation with copper. On the other hand, reconstitution of laccase from the Chinese lacquer tree requires the addition of Cu(I) and not Cu(II) (Hauenstein & McMillin, 1978
). Omura (1961)
also reported that Cu(I), but not Cu(II), could be used to reconstitute the apoprotein, results that were confirmed by Ando (1970)
. In the case of C. subvermispora laccase produced by A. nidulans, spectroscopic studies may help to clarify whether all three types of copper or only a single one are affected in cultures with limiting concentrations of this metal.
The expression systems reported here should be useful for an array of biochemical investigations, including studies of post-transcriptional modification. The results also highlight the importance of adequate copper levels in the production of fully active laccase.
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ACKNOWLEDGEMENTS |
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Received 26 November 2002;
revised 13 February 2003;
accepted 14 February 2003.