RT-PCR analysis of gene transcrip-tion in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1was expressed at every stage
Trang 1Biochemical and molecular characterization of a laccase
Shicheng Chen, Wei Ge and John A Buswell
Department of Biology and the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, NewTerritories, Hong Kong SAR, China
We have isolated a laccase (lac1) from culture fluid of
Vol-variella volvacea, grown in a defined medium containing
150 lMCuSO4, by ion-exchange and gel filtration
chroma-tography Lac1 has a molecular mass of 58 kDa as
deter-mined by SDS/PAGE and an isoelectric point of 3.7
Degenerate primers based on the N-terminal sequence of
purified lac1 and a conserved copper-binding domain were
used to generate cDNA fragments encoding a portion of
the lac1 protein and RACE was used to obtain full-length
cDNA clones The cDNA of lac1 contained an ORF of
1557 bp encoding 519 amino acids The amino acid sequence
from Ala25 to Asp41 corresponded to the N-terminal
sequence of the purified protein The first 24amino acids are
presumed to be a signal peptide The expression of lac1 is
regulated at the transcription level by copper and various
aromatic compounds RT-PCR analysis of gene
transcrip-tion in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1was expressed at every stage of the mushroom devel-opmental cycle defined in this study, although the levels
of transcription varied considerably depending upon the developmental phase Transcription of lac1 increased shar-ply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body mor-phogenesis Gene expression then declined to 20–30% of peak levels throughout the subsequent stages of sporophore development
Keywords: Volvariella volvacea; laccase; edible mushroom; gene expression
Volvariella volvacea, the edible straw mushroom, produces
multiple forms of laccase (benzenediol:oxygen
oxidoreduc-tase, EC 1.10.3.2) when grown in submerged culture on
defined media containing copper or various aromatic
compounds, or in solid-state systems representative of the
conditions used for industrial cultivation ([1]; S Chen,
W Ge and J A Buswell, unpublished results) Whereas, in
many other basidiomycetes enzyme biosynthesis is normally
associated with primary growth [2–5], laccase production in
V volvaceahas the relatively novel feature of occurring only
in the later stages of primary growth i.e., when fungal
biomass production has reached a maximum [1]
Further-more, when the fungus is grown on cotton waste composts
[6], the very low levels of laccase observed throughout the
substrate colonization phase increase sharply at the onset of
fruit body initiation This increase in laccase activity was
observed only in those composts that produced fully
developed sporophores [1]
Laccases have been assigned several different biological roles In higher plants, laccases are involved in lignification
of xylem tissues [7] The enzyme has also been linked to pigment biosynthesis during conidial development and maturation in Aspergillus nidulans [8], the pathogenicity of the chestnut blight fungus Cryphonectria parasitica [9] and
in the biosynthesis of cinnabarinic acid, a fungal metabolite produced by Pycnoporus cinnabarinus that exhibits anti-microbial activity against various bacterial species [10] Of particular importance to our own research are the assigned roles of laccases in lignin degradation [11–13], in rendering phenolic compounds less toxic via oxidative coupling and polymerization [14] and in sporophore formation [15,16] As all these latter three functions are of fundamental import-ance for the colonization of the various lignocellulosic substrates used in mushroom cultivation systems and for mushroom fruiting body development, we sought to learn more about the laccase component(s) of V volvacea This commercially important edible mushroom is grown and consumed in many parts of Asia, and currently ranks fifth among the major cultivated species in terms of annual production worldwide [17]
Our earlier studies established that two laccase isoforms were induced in submerged cultures of V volvacea in response to addition of copper or various aromatic com-pounds to the culture medium [1] In this study, we have purified and characterized one of the laccase isoforms, cloned and sequenced the cDNA encoding the enzyme protein, and examined the effect of copper and various
Correspondence to J.A Buswell, Edible Fungi Institute,
35 Nanhua Road, Shanghai 201106, China.
Fax: + 86 21 62207544, Tel.: + 86 21 62208660,
E-mail: jbuswell@saas.sh.cn
Abbreviations: lac1, laccase; 2,6-DMP, 2,6-dimethoxyphenol; HBT,
1-hydroxybenzotriazole; XYL, 2,5-xylidine; FA, ferulic acid.
Enzyme: benzenediol:oxygen oxidoreductase (EC 1.10.3.2).
(Received 26 August 2003, revised 5 November 2003,
accepted 18 November 2003)
Trang 2aromatic compounds on gene expression We have also
determined the transcription pattern for the laccase gene
in V volvacea grown on paddy-straw throughout various
stages of the mushroom developmental cycle and detected
large increases in lac1 gene transcription late in the substrate
colonization phase and during the early stages of fruit body
morphogenesis A good correlation existed between total
laccase activity and lac1 expression under these growth
conditions A better understanding of the role(s) played
by individual laccase isoforms in sporophore development
should aid the development of strategies for improving
mushroom growth yields
Experimental procedures
Organism and growth conditions
V volvaceaV14was obtained from the culture collection
of the Centre for International Services to Mushroom
Biotechnology (accession no CMB 002) [1]
The fungus was cultivated at 32C in stationary 250 mL
Erlenmeyer flasks containing 50 mL basal medium [18]
Nitrogen was added as NH4NO3 and L-asparagine at
concentrations equivalent to 26 mM-N [19] The effects of
ferulic acid (FA; Sigma, St Louis, MO, USA) on lac1
transcription were studied by growing the fungus on basal
medium without FA for 6 days before supplementing
cultures with different concentrations of FA as indicated
Total RNA was extracted from mycelia after 24h further
incubation A basal medium prepared with a modified trace
element solution [18] lacking the Cu component (equivalent
to 1.5 lMCu) was used to examine the effect of Cu on
laccase gene expression The effect of different aromatic
compounds on laccase production was determined after 36 h
following supplementation of 6 day-old cultures with 2 mM
(final concentration) of the test compound For purification
of laccase 1 (lac1), the fungus was grown in 2 L flasks
containing 600 mL basal medium with 150 lMCuSO4
Gene expression during the mushroom developmental
cycle was determined in rice-straw compost cultures
prepared as follows: 150 g rice-straw was soaked overnight
in 450 mL of distilled water After draining off any
remaining free water, the straw was mixed with lime
(15 g) and wheat bran (15 g) and the material distributed
into cellophane bags and autoclaved at 121C for 30 min
After cooling, the compost was inoculated with fungal
mycelium (from 2 week-old compost cultures) and
incuba-ted at 32C and 90–95% relative humidity The bags were
removed after 14days to promote fruiting Samples were
taken from duplicate cultures at different stages of the
mushroom developmental cycle: early, middle and late
substrate colonization stages (4, 8 and 12 days); pinhead
stage (day 14), button stage (day 18), egg stage (day 21),
elongation stage (day 22) and mature stage (day 23)
After collection, compost material was stored immediately
at)70 C prior to analysis
Enzyme assay
Laccase activity was determined using
2,2¢-azinobis-(3-ethylbenzo-6-thiazolinesulfonic acid) (ABTS) as described
previously [1,20]
Protein determination Protein in culture supernatants was determined by the method of Bradford [21] with bovine serum albumin as standard, and in column effluents by measuring A280 Purification of lac1
The following procedures were all performed at 4C Culture fluid obtained after filtration of 7-day cultures of
V volvacea was centrifuged (10 000 g, 30 min) and con-centrated 40-fold with the Pellicon ultrafiltration system (Millipore) using a 10-kDa molecular mass cut-off mem-brane Solid ammonium sulfate was added and the fraction precipitating at 80% saturation was collected by centrifu-gation, redissolved in 20 mL 10 mM phosphate buffer,
pH 5.8, and dialysed overnight against two changes of fresh buffer Precipitated material was removed by centrifugation (10 000 g, 30 min) and the supernatant was applied to a column (2.5· 20 cm) of DEAE/Sepharose pre-equilibrated with the same buffer After washing with 350 mL of 10 mM
phosphate buffer, the enzyme was eluted with a linear gradient of 0–1.0MNaCl in 500 mL of this buffer at a flow rate of 0.5 mLÆmin)1 The active fractions ( 60 mL) were pooled, concentrated to 10 mL by ultrafiltration using a Centriprep YM-10 centrifugal filter (Millipore) and applied
to a Sephacryl-S300 column (1.5· 90 cm) pre-equilibrated with 10 mM potassium phosphate buffer, pH 6.5 The enzyme was eluted with the same buffer at a flow rate of 0.5 mLÆmin)1and the combined active fractions ( 50 mL) concentrated to 5 mL by ultrafiltration This pooled material was applied to a Sephacryl-S100 column (1.5·
90 cm) pre-equilibrated with 10 mMpotassium phosphate buffer, pH 6.5, and the enzyme was eluted with the same buffer at a flow rate of 0.5 mLÆmin)1 Pooled active fractions ( 30 mL) were concentrated with a Centriprep YM-10 centrifugal filter (Millipore) and stored at)20 C Enzyme characterization
The molecular mass of purified laccase was determined by SDS/PAGE (15% w/v acrylamide gels) using low molecular mass standards (Bio-Rad) The isoelectric point of the enzyme was determined with the Phastsystem using Phast-Gel IEF 3–9 operated for 410 Vh and standard pI markers (Pharmacia) The standard assay conducted in 0.1MNaAc buffer (pH 5.0) over the range 30–65 C was used to determine the optimal temperature, and the optimal pH was established using 0.1MNa2HPO4-citrate (pH 2.2–7.0) and 0.1Macetate (pH 4.0–7.0) buffer systems Substrate speci-ficity of purified lac1 was determined spectrophotometri-cally in sodium citrate buffer (0.1M, pH 5.0) using the specific wavelength of each substrate Michaelis–Menten constants for ABTS, 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine were determined from Lineweaver–Burk plots of data obtained by measuring the reaction rate under optimal conditions using substrate concentration ranges of 0.005–1M, 0.01–1Mand 0.0025–0.025M, respectively The effect of putative laccase inhibitors was determined in standard assay reaction mixtures following incubation of lac1 with individual inhibitors (0.1 mMor 1.0 mMin sodium citrate buffer, pH 5) at 32C for 5 min
Trang 3N-terminal sequencing of laccase
The N-terminal amino acid sequence of purified laccase was
determined by electroblotting the enzyme on to an
Immo-bilon poly(vinylidene difluoride) (PVDF) Millipore
mem-brane (LKB Multiblot apparatus, Bio-Rad) followed by
Edman degradation performed with a Hewlett-Packard
G1005A Protein Sequencer coupled to a HPLC
(Hewlett-Packard, Model 1090) for analysis of the phenylthiodantoin
amino acids
RNA manipulations, cDNA synthesis and cloning
Mycelium from V volvacea cultures grown for 12 days in
defined medium with 200 lMcopper was harvested, frozen
with liquid nitrogen and ground to a fine powder with a
mortar and pestle Total RNA was isolated from this
material using the Tri-Reagent (Molecular Research Center,
Inc Cincinnati, OH, USA) and used to synthesize cDNA
Reverse transcription was carried out at 42C for 2 h in a
10-lL reaction volume containing: 2 lL
diethylpyrocarbo-nate-treated H2O, 2 lL 5· First Strand Buffer (Gibco
Invitrogen), 0.01M dithiothreitol, 0.5 mM dNTPs, 0.5 lg
oligo(dT), 2 lg total RNA and 100 U SuperScript II (Gibco
Invitrogen) The cDNA from the reaction was kept at
)70 C and used for PCR amplification using degenerate
primers designed on part of the N-terminal amino acid
sequence and a conserved copper-binding region The
sequences and primers were: primer 1 (upper primer):
5¢-(CT)T(AGCT)AC(AGCT)AA(CT)GG(AGCT)TT(CT)
GC-3¢ (encoding LTNGFA); primer 2 (lower antisense
primer): 5¢-(AG)(AG)TG(AGCT)(GC)(AT)(AG)TG(AG)
TACCA(AG)AA-3¢ (encoding FWYHSHL) Different
primers were designed with any one of the bases shown in
parentheses
PCR amplification of the cDNA fragment encoding a
portion of lac1 was carried out using a PTC-100 (MJ
Research, Watertown, MA, USA) in 50 lL reaction
volumes containing 1.25 U Taq DNA polymerase, 5 lL
10· Mg-free reaction buffer, 200 lMdNTP, 2.5 mMMgCl2,
1 lMprimer 1 or primer 2 and 0.5 lL template
Amplifi-cation conditions were: 1 cycle of 94C for 3 min, 50 C for
30 s and 72C for 1 min; 30 cycles of 94 C for 30 s, 54 C
for 30 s, and 72C for 1 min; then a final extension at 72 C
for 10 min before storage at 4C Amplification products
were fractionated by electrophoresis in 2.0% agarose/Tris
borate/EDTA gels and appropriate bands excised and
purified from the gel using the NucleoTrap Gel Extraction
Kit (Clontech) The purified DNA was precipitated with
ethanol and resuspended in 10 lL H2O An aliquot (4 lL)
was incubated at 4C overnight with 3 U T4DNA ligase
(Promega), 1 lL 10· buffer with 10 mMATP (Promega)
and 1 lL pGEM T-vector in a total volume of 10 lL and
transformed into E.coli DH5a Plasmids encoding the lac1
fragment were isolated using the Wizard Miniprep Kit
(Promega) and sequenced by the dideoxy chain-termination
method using an automated ABI310 sequencer (Perkin
Elmer) according to the manufacturer’s instructions
RACE was performed with the SMART RACE cDNA
Amplification Kit (Clontech) to obtain full-length cDNA
clones Using the 309-bp fragment sequence of lac1
obtained above, the gene-specific primer (5¢-GGCACT
GAGTGACGAAGGCAGGACCATC-3¢), was designed for the 5¢-RACE reaction to generate the 5¢-cDNA end fragment of lac1 The 5¢-cDNA end fragment was cloned into pGEM T-vector and sequenced as above The full-length cDNA of lac1 was then generated by 3¢-RACE using the primer 5¢-TCTCAACCGTCGACAGCAG TGTTCGTG-3¢ designed from the sequence of the extreme 5¢ end of lac1 The full-length cDNA of lac1 was cloned and sequenced as above
RT-PCR for semiquantification of thelac1 expression levels
Total RNA extracted from liquid cultures and mRNA extracted from compost cultures using the polyA TRACT mRNA isolation system II kit (Promega), was reverse transcribed into cDNA at 42C for 1 h in a total volume of
10 lL reaction solution containing 1 lg total RNA or
30 ng mRNA, 1· First Strand Buffer, 10 mM dithiothrei-tol, 0.5 mM each dNTP, 0.5 lg oligo-dT and 100 U SuperScript II (Gibco Invitrogen) PCR was performed in
a volume of 25 lL consisting of PCR buffer, 0.2 mMeach dNTP, 2.5 mM MgCl2, 0.2 lM each primer, and 0.5 U of Taqpolymerase One microlitre of RT reaction was used in each PCR reaction
As a control for RNA loading, a 330 bp fragment of the
V volvacea, V14glyceraldehyde-3-phosphate dehydro-genase (gpd) gene was amplified with primers PGPDF (5¢-TAATGACGGCAAACTCGTGATC-3¢) and PGPDR
(5¢-TGTATGACTTTGGCCAGAGGTG-3¢) (accession number AY280633) The PCR cycle programme was:
94C for 2 min; 23 cycles of 94 C for 20 s, 52 C for 20 s and 70 C for 2 min; then a final extension at 72 C for 10 min The primers for lac1 PLAC1F (5¢-AGCTTT CATTCCCAGTGATTG-3¢) and PLAC1R (5¢-AACGAG CTCAAGTACAAATGACT-3¢) were designed according
to our cloned cDNA (GenBank Accession No AY249052) The PCR cycle programme was: 94C for 2 min; 28 cycles of
94C for 20 s, 52 C for 20 s and 70 C for 2 min; then a final extension at 72C for 10 min To validate the semiquanti-tative RT-PCR reactions, a series of RT-PCR reactions were sampled at different cycles and analysed by electrophoresis
to ensure that product abundance was evaluated in the exponential phase of the reaction (half of the maximum product) To further validate the assays, the optimized cycle number (23 and 28 for gpd and lac1, respectively) was used to amplify serially diluted gpd and lac1 DNA templates After electrophoresis on 2% agarose gel, the PCR products were stained with ethidium bromide and visualized in a UV-transilluminator The signal intensity was quantified with the Gel-DOC 100 system using the MOLECULAR ANALYST SOFTWARE(Bio-Rad) The specificity of PCR and RT-PCR amplification was confirmed by cloning the products into pGEM T-vector (Promega) followed by sequencing
Results
Purification of lac1 Lac1 was separated as a single peak by a final gel filtration step using Sephacryl S-100 and shown to be homogeneous
Trang 4by SDS/PAGE and by isoelectric focusing combined
with silver staining (data not shown) After a five-step
purification protocol, the specific activity of lac1 was
increased 14-fold with a 22% recovery yield (Table 1)
Enzyme characterization
The molecular mass of purified lac1 was estimated by SDS/
PAGE to be 58 kDa and the isoelectric point of the enzyme
was 3.7 In addition to ABTS, syringaldazine and 2,6-DMP
were also oxidized by lac1 ( 17% of the activity observed
with ABTS) Guaiacol, catechol and 2,6-DMP were poor
substrates for the enzyme (< 5% compared with ABTS)
and no activity was detected with tyrosine, FA, L
-3,4-dimethoxyphenol and dihydroxyphenylalanine (L-DOPA)
With ABTS as the substrate, lac1 displayed a pH optimum
of 3.0 corresponding to a specific activity of 13.5 UÆmg
protein)1 Corresponding pH optima and specific activities
for syringaldazine and 2,6-DMP were pH 5.6 and 3.4UÆmg
protein)1and pH 4.6 and 2.8 UÆmg protein)1, respectively
In standard assay mixtures, the velocity of ABTS oxidation
was maximal at 45C The dependence of the rate of ABTS
oxidation by lac1 on substrate concentration at pH 3.0 and
45 C followed Michaelis–Menten kinetics A reciprocal
plot revealed an apparent Kmvalue of 0.03 mMand a Vmax
of 16.4ÆU mg protein)1 Corresponding values for
syring-aldazine and 2,6-DMP under optimal conditions were
0.01 mM and 4.9 UÆmg protein)1, and 0.57 mM and
5.6 UÆmg protein)1, respectively Lac1 is inhibited (100%)
by thioglycollic acid (1 mM), dithiothreitol (0.1 mM), azide
(0.1 mM) and cysteine (0.1 mM) but less so by 1 mMEDTA
(20%) The N-terminal sequence of native protein was
N-ALSSHTLTLTNGFASPD
Cloning of full-length cDNA oflac1
The cDNA contained a predicted ORF of 1557 bp encoding
519 amino acids (Fig 1) The amino acid sequence from
Ala25 to Asp41 corresponded to the N-terminal sequence of
the purified protein, and the putative presequence of 24amino
acids is a hydrophobic signal peptide as predicted by the
computer programPLOT.AHYDusing the method described by
Kyte and Doolittle [22] The remaining 495 amino acids are
considered to constitute the lac1 mature protein giving a
calculated molecular mass of 53 212 Da Two putative
polyadenylation signals (TATAAA and CATAAA) were
identified near the 3¢-end, suggesting possible differential
splicing over the 3¢-untranslated region after transcription
Alignment of the deduced amino acid sequence of lac1
with deduced amino acid sequences of other fungal laccases
showed highest overall identity with laccase 1 (BAB84354) from Lentinula edodes (58%), laccase (AF170093) from Pycnoporus cinnabarinus (57%), laccase LCC3-1 (AF176230) from Polyporus ciliatus (56%) and laccase 1 (AAC498287) from Trametes versicolor (56%) (Fig 2) The lac1 protein contains only one potential N-glycosylation site (Asn-X-Thr/Ser in which X is not proline) The calculated isoelectric point for the cloned cDNA product is 4.5 All the amino acid residues that serve as Cu2+ ligands (10 His residues and one Cys residue) are present in the lac1 coding sequence (Fig 2)
Validation of semiquantitative RT-PCR assay forlac1 andgpd
A series of RT-PCR reactions for lac1 (Fig 3: left panel) and gpd were performed at different cycles and analysed by electrophoresis Using the cycle number that generated the half-maximal PCR amplification, PCR reactions were performed on serially diluted lac1 and gpd template DNA
A clear linear relationship between the amount of template inputs and PCR amplification was obtained for both lac1 (Fig 3: right panel) and gpd (data not shown) demonstra-ting the workability of the RT-PCR assay for quantitademonstra-ting lac1mRNA levels
Regulation oflac1 expression by copper The effect of copper concentration on lac1 expression in cultures of V volvacea is shown in Fig 4 Lac1 transcrip-tion increased with increasing concentratranscrip-tions of CuSO4in the culture medium up to 200 lM Higher copper concen-trations were inhibitory and approximately 70% fewer transcripts were observed in hyphae grown in the presence
of 300 lMcopper sulfate No lac1 transcripts were detected
in fungal cells grown in the absence of copper A constant level of control gpd fragment amplification was observed in each reaction thereby confirming the uniform efficiency of PCR amplification of RT reaction products In time-course experiments, transcripts of lac1 were detected in fungal hyphae after 6 days growth in cultures supplemented with
200 lMcopper sulfate and reached peak levels after 14days (data not shown)
Regulation oflac1 expression by aromatic compounds Laccase induction in V volvacea by various aromatic compounds occurred at the level of gene transcription Figure 5 shows the effect of different aromatic compounds
on lac1 expression in V volvacea grown in
nitrogen-Table 1 Summary of the purification procedure for V volvacea lac1.
Volume (ml)
Total activity (IU)
Protein (mg)
Specific activity (IUÆmg)1)
Yield (%)
Purification fold
Trang 5Fig 1 Nucleotide and deduced amino acid sequences of V volvacea lac1 Dashed underline, signal peptide; solid line, N-terminal sequence; double solid line, amino acid sequences used to design degenerate primers The putative N-glycosylation site is boxed; *; stop codon The putative polyadenylation signals (TATAAA and CATAAA) are in white on a black background.
Trang 6sufficient medium without copper Highest levels of gene
transcription were seen with FA, veratric acid,
4-hydroxy-benzoic acid and 2,5-xylidine (XYL) Amounts of lac1
mRNA in the cultures increased with increasing
concentra-tions of FA (1–10 mM) although transcript levels in
mycelium grown with 5 mM and 10 mM of the aromatic
compound were not significantly different (data not shown)
No transcription was detectable in controls without added
FA
Transcriptional regulation oflac1 during growth
and fruiting ofV volvacea on straw
The duration of the developmental cycle of V volvacea when
the mushroom was grown on rice-straw composts was
approximately 23 days For the purpose of this study, eight
separate stages were identified as follows: half-colonization
of substrate (day 4), full-colonization (day 8), formation of
primordia (day 12), appearance of pinheads (day 14), button stage (day 18), egg stage (day 21), elongation stage (day 22), and mature fruiting body (day 23) The results of RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw and analysed at various stages of the developmen-tal cycle are shown in Fig 6A No transcripts were detected
in the early stages of substrate colonization and lac1 was first expressed only when substrate colonization was virtually complete A large increase in the number of transcripts was seen at the stage of primordia formation and transcription levels were still high when pinheads appeared Transcription levels declined at the button stage and then remained relatively stable throughout the remaining stages of fruiting body development Approximately 70–80% fewer tran-scripts were detected throughout these stages compared with peak levels observed in the initial stages of sporophore formation Extracellular laccase activity in the same compost samples are shown in Fig 6B
Fig 2 Alignment of deduced amino acid sequences of lac1 and other fungal laccases *, Amino acid conserved among all of the sequences Ten His residues and one Cys residue represent the amino acids that serve as Cu 2+ ligands and are shown in white on a black background.
Trang 7Basidiomycetes typically produce multiple laccase isoforms
[5,23–29] and V volvacea produces at least two protein
bands with laccase activity when grown in submerged
culture under different conditions [1] Previous physiological
studies have shown that, as in other basidiomycetes, laccase
production by V volvacea is induced by copper and by
various aromatic compounds However, unlike most other
basidiomycetes, enzyme activity can be detected in the
extracellular culture fluids of V volvacea only during the
latter stages of primary growth In order to better
under-stand these effects at the molecular level, we have now
purified and characterized one of these laccases, lac1, and cloned and sequenced the cDNA encoding the protein RT-PCR was used to study the regulation of lac1 gene expression in V volvacea when the fungus was grown in submerged culture in the presence of known laccase inducers, and in solid-state systems representing conditions used for industrial cultivation To our knowledge, this
Fig 3 Validation of semiquantitative RT-PCR assays for lac1 Left panel: Kinetics of PCR amplification with the electrophoretic image shown at the top The cycle number (28·) that generates half maximal reaction was used to analyse the expression of the gene Right panel: PCR amplification of the cloned lac1 cDNA using the cycle number obtained from the left panel Each value represents the mean ± SD of three PCR reactions.
Fig 4 RT-PCR analysis of transcription patterns of lac1 induced by
different concentrations of copper Total RNA was isolated from
V volvacea, V14, grown in defined medium with various
concentra-tions of copper sulfate PCR products were electrophoresed in a 2%
agarose gel and stained with ethidium bromide The expression levels
were normalized by using the relative mRNA ratio (lac1/gpd).
Fig 5 RT-PCR analysis of total RNA isolated from V volvacea, V 14, grown in defined medium supplemented with 2 m M aromatic compounds Fungal mycelia were cultured for 6 days prior to addition of aromatic compound and harvested after a further 36 h incubation PCR pro-ducts were electrophoresed in a 2% agarose gel and stained with ethidium bromide The expression levels were normalized by using the relative mRNA ratio (lac1/gpd) A, Vanillic acid; B, syringic acid;
C, 4-hydroxybenzoic acid; D, veratric acid; E, 4-hydroxybenzaldehyde;
F, ferulic acid; G, 3,4,5-trimethoxybenzoic acid; H, 2,5-xylidine;
I, vanillin; J, 3,4-dimethoxybenzaldehyde; K, 3,4-dimethoxybenzyl alcohol; L, p-coumaric acid.
Trang 8represents the first report on the cloning of a laccase gene
and the factors affecting its transcription in this
economi-cally important mushroom
The purified lac1 protein is unusual in itself in that
concentrated solutions lack the typical blue colour and the
spectral maxima near 600 nm that characterize all the blue
oxidases Furthermore, guaiacol is a poor substrate for the
enzyme In both cases, lac1 resembles the white laccase (POXA1), isolated earlier from Pleurotus ostreatus [30] and the laccase produced by Phellinus ribis [31] Furthermore, the N-terminal sequence of the lac1 protein exhibits very low homology with sequences of other basidiomycete laccases (Fig 7)
Although the spectral characteristics of the lac1 protein suggest the absence of a type 1 copper site, this is not borne out by analysis of the deduced amino acid sequence of the enzyme Thus, the 10 histidines and one cysteine residue required to coordinate the four copper atoms at the active site of the enzyme [32] were all conserved in the V volvacea gene (Fig 4) It is possible that depletion of type 1 copper may have occurred during purification However, attempts
to reconstitute the copper to the purified enzyme were unsuccessful Laccases also have an additional residue involved in the coordination of type 1 copper atoms, located
10 residues downstream of the single cysteine This residue, which appears to have a role in determining the redox potential of the enzyme [33], can be methionine, leucine or phenylalanine Therefore, lac1 with a leucine residue at position 458 should be assigned to class 2 according to the categorization proposed by Eggert et al [34] Other class 2 enzymes include laccases from the basidiomycetes, Agaricus bisporus[27], Podospora anserina [35], Phlebia radiata [36], the ascomycetous fungi, G graminis [23], Neurospora crassa [37], Cryphonectria parasitica [9] and the yeast, Cryptococcus neoformans[38] Two laccase genes described in Lentinula edodes also have a leucine residue in the analogous downstream position However, the cysteine residue believed to be critical for coordination of the copper atoms
is apparently not conserved in these genes and is replaced by tryptophan [39]
Although V volvacea produces at least two laccase isoforms [1], the primers used in this study appear to be specific for lac1 as in every case, only one band was amplified from each of the different samples and Southern blot analysis using the probe prepared with the lac1 primers also revealed a single band Further-more, all of these generated PCR products were found to comprise of sequences identical to those present in the lac1 cDNA
Copper regulates lac1 transcription in V volvacea and the correlation between copper concentration and lac1 transcription corresponds well with previously observed effects of copper on total extracellular laccase activity in copper-supplemented cultures of V volvacea [1] A similar regulatory role for copper has been proposed for some laccasegenes in several other basidiomycete fungi including Pleurotus spp [5,26,40,41]., Trametes spp [2,42]., Podos-pora anserina[35] and Ceriporiopsis subvermispora [43] In
P ostreatus, copper not only regulated laccase gene expression but also positively affected the activity and stability of the enzyme [40] The effect of copper on enzyme stability may be related to the inhibitory effect of the metal on the activity of an extracellular protease produced by P ostreatus (PoSl) that is reported to degrade laccase [44]
Copper does not appear to be essential for the activation
of lac1 transcription in V volvacea as several aromatic compounds also induce gene transcription in copper-deficient cultures Stimulation of laccase production by
Fig 6 Transcription analysis of lac1 by RT-PCR (A) and total laccase
activity (B) during various stages of V volvacea fruitbody development.
mRNA was extracted from the mycelium harvested at the following
stages: substrate colonization stages (4, 8, 12 days), pinhead (day 14),
button stage (day 18), egg stage (day 21), elongation stage (day 22) and
mature stage (day 23) The expression levels were normalized by using
the relative mRNA ratio (lac1/gpd) Extracellular protein was
extrac-ted from rice-straw composts by suspending the substrate in 50 m M
KHPO 4 buffer (pH 6.5) and shaking (150 r.p.m.) for 2 h at room
temperature.
Trang 9aromatics is well-documented and has led to the suggestion
that one role of the enzyme is as a defence mechanism
against oxidative stress caused by oxygen radicals
origin-ating from aromatic compounds [14,45] However, wide
variations are seen with respect to the induction of laccase
gene transcription by aromatic compounds with both
inducible and noninducible forms described [46], suggesting
that only certain isoenzymes serve in a protective capacity
Transcription of one laccase gene, lcc1, from Trametes
villosawas induced 17-fold by the addition of XYL but a
second gene (lcc2) was constituitive under the conditions
tested [29] Amounts of laccase mRNA and laccase activity
in 10-day-old cultures of T versicolor were a direct function
of concentrations of the laccase inducers,
1-hydroxybenzo-triazole (HBT) and XYL but no induction was observed
after the addition of FA and veratric acid (VA) [2] Two
laccase genes, lcc1 and lcc2 present in an unidentified
basidiomycete I-62 were both inducible by veratryl alcohol
but at different stages of growth, whereas transcriptional
levels of a third gene, lcc3, were unaffected [24] HBT, XYL,
FA and VA all induced extracellular laccase production in
P sajor-cajuand transcription levels of three laccase genes
were increased by FA and XYL [5] Higher levels of laccase
mRNA were also reported in cultures of three white-rot
fungi supplemented with aromatic compounds [47]
Tran-scription of only one of two laccase genes in T versicolor
was induced by 2,5-dimethylaniline [30] Several aromatic
compounds increase transcription of V volvacea lac1 to
varying degrees FA was the most effective inducer of
transcription and correspondingly high levels of
extracellu-lar laccase activity were observed in cultures supplemented
with this aromatic compound [1] Ferulic acid is toxic to
V volvacea and radial mycelial growth on agar plates
containing 1, 5 and 10 mMFA is inhibited by 37.6%, 71.8%
and 100%, respectively (data not shown) suggesting that
induction of this laccase isoform may in part, at least, be a
detoxification response
Both laccase activity and lac1 gene transcription in
compost cultures of V volvacea were detected late in the
substrate colonization phase when a sharp increase in both
parameters was recorded (Fig 6A,B) There was also
good correlation between total laccase activity and lac1
expression although, as V volvacea produces at least one
other laccase isoform in addition to lac1 [1], this and
possibly other laccase isoforms may be contributing to the
total laccase activity detected at the various developmental
stages The pattern of laccase gene expression observed in
compost grown cultures of V volvacea contrasts sharply
with activity profiles reported for other mushroom species
In straw cultures of Pleurotus cornucopiae var citrinopile-atus, laccase activity was maximal during the vegetative growth and declined rapidly at the onset of fruiting [48] Laccase activity in cultures of A bisporus grown under standard composting conditions increased in the compost until just after the pinning stage (height of sporophores, 1.0 cm) of development and was correlated strongly with the loss of lignin from the compost [49] Enzyme activity then declined rapidly during the later stages of fruit body development [16,50,51], decreasing by 87% in the 7 days between the appearance of the first fruit body initials and fruit body maturation [50] Moreover, laccase gene expres-sion measured as mRNA levels was maximal at the fully colonized stage prior to fruiting and then declined to very low levels during fruiting [52] Similarly, the activity of laccase was regulated strongly during the development of
L edodes fruit bodies [53,54] Laccase gene expression (measured as mRNA levels) during growth of the fungus on
a sawdust-based substrate was maximal at the fully colonized stage prior to fruiting and declined to very low levels during fruiting [53,54]
While the higher levels of laccase gene expression observed during vegetative growth of A bisporus and
L edodes, together with supporting biochemical data, indicate that laccases are involved directly in lignin biocon-version by these fungi [49,53,54], such a function in
V volvaceaseems unlikely Indeed, there is little evidence showing that V volvacea is actually capable of degrading the lignin polymer and it has been suggested that an inability
to do so accounts for the relatively poor mushroom yields achieved on lignified growth substrates [55] Instead, the appearance of extracellular laccase activity in submerged cultures of V volvacea at the onset of secondary metabolism [1], the temporal correlation observed between laccase production and sporophore formation when the mushroom was grown on cotton waste composts which were virtually devoid of any lignin component [1] and the pattern of lac1 transcription reported here, all provide strong evidence indicating that at least one enzyme isoform plays a key role
in the morphogenesis of the V volvacea fruit body It has been proposed that phenoloxidases such as laccase could crosslink hyphal walls into coherent aggregates during primordium initiation [56] and may continue to act on the hyphal surfaces throughout fruit body development [57] Further studies on a possible link between laccase and sporophore development in V volvacea are underway as part of our overall aim to develop strategies for improved mushroom production through the control of fruit body growth, flush yield and flush timing [52]
Fig 7 Comparison of N-terminal sequences of various fungal laccases.
Trang 10This work was supported by a grant from the Hong Kong Research
Grants Council (grant CUHK 4163/01 M) We thank Dr Shao-jun
Ding for providing the solid-state samples for transcriptional analysis.
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