Transcriptional analysis of alcohol and aldehyde dehydroge-nase gene families in Pholiota microspora, and estimation of their physiological roles

Surasit SUTTHIKHAMPA1), Yoshiko KAWAI2), Mirai HAYASHI2), Sophon BOONLUE3), Norihiro SHIMOMURA2), Takeshi YAMAGUCHI2) and Tadanori AIMI2)*

1)

The United Graduate School of Agricultural Sciences Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan

2)

Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan

3)

Faculty of Science, Khon Kaen University,Khon Kaen 40002, Thailand

（Received 24 September 2015 / Accepted 10 January 2016）

Abstract

In this study, we analyzed two alcohol dehydrogenase (Adh), eight aldehyde dehydrogenase (Aldh) and two mannitol-1-phosphate dehydrogenase (Mpd) genes in Pholiota microspora. The transcription of Aldh1, 2, 3, Adh1, 2, and Mpd1, 2 was unaffected in liquid medium in the presence of 1 mM ethanol. However, Mpd1 expression was promoted by acetaldehyde. Therefore, Mpd1 is a candidate for an ethanol-producing alcohol dehydrogenase. On the other hand, transcription of Aldh1 and Adh2 was promoted with 3 mM veratryl alcohol; therefore, the role of Aldh1 and Adh2 might have evolved from alcohol metabolism to degradation of aromatic compounds. Transcription of Adh1, Mpd1, Mpd2 and Aldh1 was higher in primordia and fruiting bodies than mycelia. This phenomenon suggested that the response of Adh1, Mpd1, Mpd2 and Aldh1 indicates the presence of oxidative stress during fruiting body development.

Key words: Alcohol dehydrogenase, Aldehyde dehydrogenase, Ethanol, Pholiota microspora, qRT-PCR

Mushroom Science and Biotechnology, Vol. 24 (1) 16-23, 2016Copyright © 2016, Japanese Society of Mushroom Science and Biotechnology

*Corresponding author. E-mail: taimi@muses.tottori-u.ac.jp

Regular Paper

Introduction

The ascomycetous yeast Saccharomyces cerevisiae

is used for efficient bioethanol fermentation. One of

the key enzymes for alcohol fermentation is alcohol

dehydrogenase (Adh). S. cerevisiae Adh1 is expressed

during anaerobic glucose fermentation and converts

acetaldehyde to ethanol1, 2)

. However, S. cerevisiae does

not have a specific enzyme that hydrolyzes the beta-1,

4-linked glucose polymers present in cellulose-rich bio-

mass3). In nature, cellulose is the most abundant biomass,

but is protected from degradation in wood by lignin.

Therefore, lignin is one of the obstacles in development of

a biochemical process for producing lignocellulosic bio-

fuel4). Wood decaying basidiomycetes such as white rot

fungi play the major role in lignin biodegradation5). Bio-

conversion of recalcitrant lignocellulose into bioethanol

using a white rot fungus is attractive and would greatly

enhance cost-effectiveness of bioethanol production in

one step6).

Production of alcohol and Adh activity in mush-

rooms belonging to the basidiomycetous fungi has been

reported. Pleurotus ostreatus, Agaricus blazei, Tricholoma

matsutake and Flammulina velutipes have been used in

wine, sake and beer production, producing 12.2%, 8.0%,

4.6% and 3.0% ethanol, respectively7-9)

. Additionally, F.

velutipes converted 1% D-glucose into ethanol at high

efficiency, up to 88% of the theoretical yield in liquid

medium10)

. On the other hand, Pholiota microspora, a

popular edible mushroom in Japan and widely used as

research material in our laboratory12)

, did not produce

ethanol. Therefore, in order to infer why P. microspora

cannot produce ethanol, we identified and analyzed two

Adh, two Mpd and eight Aldh genes, which include zinc-

containing alcohol dehydrogenases and aldehyde dehy-

drogenases expected to be related to ethanol metabolism.

Moreover, we investigated the effect of ethanol and

acetaldehyde on transcription of these genes, and discuss

the possibility of ethanol fermentation in P. microspora.

Materials and Methods

1.  Fungal strains and culturing conditions

The monokaryotic strains P. microspora NGW19-6

(A4, pdx1), a pyridoxine auxotrophic mutant, and NGW12-

163 (A3, arg4), an arginine auxotrophic mutant,19, 20)

were

used. A dikaryotic strain, obtained by crossing NGW19-6

and NGW12-163 and referred to as NGW19-6/12-163, was

used for experiments.

In order to analyze the effects of different aromatic

compounds on gene expression, the NGW19-6/12-163

strain was grown on M4 agar at 25℃ for 1 week, and then

10 mycelial agar blocks (3 × 3 mm) were transferred into

20 mL of M4 medium13)

(per L: 2.20 g glucose, 0.92 g diam-

monium tartrate, 1.00 g KH2PO4, 0.26 g NaH2PO4•2H2O,

0.50 g MgSO4•7H2O, 1.00 × 10-4 g thiamine hydrochloride,

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MUSHROOM SCIENCE AND BIOTECHNOLOGY

6.60 × 10-3 g CaCl2•2H2O, 5.00 × 10

-3 g FeSO4•7H2O, 3.00 ×

10-4 g MnSO4•H2O, 5.00 × 10

-4 g ZnSO4•7H2O, 6.40 × 10

-4 g

CuSO4, and 15 g agar for a solid formulation) in a 100-mL

Erlenmeyer flask supplemented with test compounds

(final concentration 0.01% lignosulfonate; 0.05 mM

2,5-xylidine; 3 mM veratryl alcohol; 0.1 mM guaiacol;

1 mM ferulic acid; 1 mM veratric acid; 0.1 mM O-anisic

acid; 1 mM ethanol; 1 mM propanol; or 1 mM acetalde-

hyde). After inoculation, the fungus was grown at 25℃

for 10 days and the mycelia were harvested by filtration

for RNA extraction.

Fruiting bodies were cultivated on a sawdust

substrate, which was prepared as follows. Beech sawdust

was mixed with rice bran at a gravimetric ratio of 5:1

and adjusted to 65% moisture content using tap water,

and this medium was placed into a 100-mL Erlenmeyer

flask, followed by autoclaving at 121℃ for 60 min. After

cooling the medium in the air, five mycelial agar blocks

(5 × 5 mm) containing NGW19-6/12-163 were inocu-

lated and incubated at 25℃. When the mycelia had

colonized the substrate (about 40 days after inoculation),

the surface layer was scratched with a spatula, and then

50 mL of sterilized distilled water was poured into the

flask. Water was removed after flasks were incubated at

15℃ overnight, and then cultivation continued at 15℃

until fruiting bodies developed. We defined the day after

water removal as day 0. Samples for RNA extraction

were taken from triplicate cultures at different stages of

the mushroom developmental cycle: mycelia at 30 and 90

days, primordia, and fruiting bodies (1 cm).

2.  Genomic DNA and total RNA preparation

Genomic DNA was extracted from mycelia grown

in liquid medium according to the method of Dellaporta

et al.14)

Harvested mycelia were frozen in liquid nitrogen

and ground to a fine powder in a mortar and pestle. RNA

was extracted using a MagExtractor™

Kit (Toyobo, Osaka,

Japan) according to the manufacturer’s instructions.

cDNA was synthesized using total RNA as a template

with ReverTra Ace® qPCR RT Master Mix with gDNA

Remover kit (Toyobo). PCR was carried out using

Takara Ex Taq® polymerase (Takara Bio, Japan). The

oligonucleotide primers used in this study are listed in

Table 1. Amplified fragments were subcloned into pMD20

T-vector (Takara Bio, Japan) and sequenced.

3. P. microspora genome and retrieved genes

Whole genomic sequences of monokaryon P.

microspora NGW19-6 were determined using Illumina

HiSeq 2000 paired-end technology with CASAVA ver.

1.8.1 software, provided by Hokkaido System Science Co.,

Ltd. (Sapporo, Hokkaido, Japan), as described by Funo

et al.15)

. This sequencing run yielded 30,935,254 high-

quality filtered reads with 101-bp paired-end sequencing.

The genome was assembled using Velvet assembler (hash

length, 85 bp)16)

. The final assembly contained 4,770

contigs with a total length of 33,400,256 bp, and an N50

length of 72,431 bp. The deduced amino acid sequences

Table 1.  Primer sets for cDNA and plasmid construction.

Genes Primers 5’ → 3’

Act1 PnActin1_F1 CGAAATTTCAGCTCTCGTCGT

PnActin1_R1 CTGGAGCACGGAATCGCT

Adh1 PnADH1_F1 CGATGTTATTGTCAAGCTTGCA

PnADH1_R1 CGAGACGTTCTTGTTATAGCACT

Adh2 PnADH2_F1 CCTCCAAGGGCTCATGAAGT

PnADH2_R1 ATCCTTCGCTGATCTCTGACA

Aldh1 ALDHFF2 CTGGACGTCGCATGGTATTTTCTGG

ALDHFR3 CCAACAGGGCCAAAGATCTCC

Aldh 2 PnALDH2_F1 TCATTGCGTCTGTTGTAGCAG

PnALDH2_R1 CGAGTTCCAGCGACACCT

Aldh 3 PnALDH3F2 GTATCATGGAAGCTCGGGC

PnALDH3_R1 TGAGAGACTTGAGGACCGTG

Aldh 4 PnALDH4_F1 CTGGCTGAAGGAAAGGGAGA

PnALDH4_R1 GCCGACGAGAATCTGCGC

Aldh 5 PnALDH5_F1 AGAGTTCGTGGATATTTGCGA

PnALDH5_R1 TGTTGTTCCATTCAATAGCCTGT

Aldh 6 PnALDH6_F1 GTAGCGGCACCCTTGTATTG

PnALDH6_R1 ACGACAGCAAACACCATGAC

Aldh 7 PnALDH7_F1 TGAATATAATGCTCCAGACGCA

PnALDH7_R1 ACGCGATCCCTAAATATGCTAA

Aldh 8 PnALDH8_F1 CTCGCTGCAACTATCCTTGG

PnALDH8_R1 ACGGTCGGTCGAGAAGATTG

Mpd1 Mpd-probe-F GCACATACTGCGAATAGCCC

Mpd-probe-R TAGCAATGACACGGCGACCC

Mpd2 MPD2-F2 ATCCAGTGACTCGACCTGCT

MPD2-R2 CCAAATCCTTGAGGACGTCAT

Pdc1 PD-DiF2 CAAAAGTGGAATACAGGACATCA

PDC-pR AGTGCTCTAGCCGTGCTGTCAAT

Table 2.  Primer sets for qPCR.

Genes Primers 5’ → 3’

Act1 PnActin1_CF1 GCTATGCTATGTCGCGCTTGAT

PnActin1_R1 CTGGAGCACGGAATCGCT

Adh1 PnADH1_CF1 GTACAGCCGTTCTCGAGGTCGT

PnADH1_CR1 GTGGTGCTCCGTGGACTCCA

Adh2 PnADH2_CF1 GTCGGTTGTTGCCGTCAAC

PnADH2_CR1 GCCACAGTCGAGCCCTTGAT

Aldh1 ALDHFF3 GATTTCCCAGATCCAATACGATCGC

ALDHFR3 CCAACAGGGCCAAAGATCTCC

Aldh 2 PnALDH2_CF1 GCGTCTGTTGTAGCAGGTTCT

PnALDH2_CR1 GAGCAACCTAGAGCGTTCCCA

Aldh 3 PnALDH3_CF1 CAACATCGTCAACGGATATGGA

PnALDH3_CR1 CCGCTGACGCTTTCATGACT

Aldh 4 PnALDH4_CF1 CCATCTGCTATGATCACCCGA

PnALDH4_CR1 CCAGCACGTTGTGAGAGTTCA

Aldh 5 PnALDH5_CF1 CTTTACTGGCAGCGAGCACGT

PnALDH5_CR1 CAGGCATGATGATCGAAGCGT

Aldh 6 PnALDH6_CF1 GGTGTTCTGAACTTTCTGCCCA

PnALDH6_CR1 CGCGATCACTTCCAGTAAAGTT

Aldh 7 PnALDH7_CF1 GGACACTGGCAAGACGTTGA

PnALDH7_CR1 CTTGATAGCGTCTTCGCCGTA

Aldh 8 PnALDH8_CF1 GCCGACCTGCATATGACAA

PnALDH8_CR1 CCTTTCGTGTCATCACCTGA

Mpd1 MPDFF1 GCCGCAAGGGATACGAAC

MPDFR1 TACACAGTAAGACCAGCG

Mpd2 PnMPD2-F2 ATCCAGTGACTCGACCTGCT

PnMPD2-CR1 GCCGTGGCTACTTGAGAAGTCA

Pdc1 PD-FF3 CGTTACGGTGGTGTATGTATTATCT

PD-FR3 GTGACGGCGAACGGGGATG

18 Vol.24 No. 1

of known proteins from public databases were searched

against the P. microspora genome using the BLASTp

algorithm. Two alcohol dehydrogenase genes were

assigned as Adh1 and Adh2, two mannitol-1-phosphate

dehydrogenase genes were assigned as Mpd1 and Mpd2,

eight aldehyde dehydrogenase genes were assigned as

Aldh1 to Aldh8, and a pyruvate decarboxylase1 gene was

assigned as Pdc1. The coding sequences of intron-exon

junctions based on GT-AG rules17, 18)

and open reading

frames based on generic rules (start codon, ATG and stop

codon TAA, TAG, or TGA)19)

were then predicted. The

nucleotide sequences of genomic DNA fragments of P.

microspora Adh, Aldh, Mpd and Pdc genes were deposited

in the DDBJ under the following accession numbers:

Adh1 (LC102238); Adh2 (LC102239); Aldh1 (LC102241);

Aldh2 (LC102242); Aldh3 (LC102243); Aldh4 (LC102244);

Aldh5 (LC102245); Aldh6 (LC102246); Aldh7 (LC102247);

Aldh8 (LC102248); Mpd1 (AB686426); Mpd2 (LC102237);

and Pdc1 (LC102240). Protein motifs in deduced amino

acid sequences were identified using the MOTIF search

program (http://motif.genome.jp) and NCBI’s conserved

domain database20)

. Subcellular localization was predicted

using the PSORTII21)

(http://psort.hgc.jp/form2.html),

IntroPro (http://www.ebi.ac.uk/interpro)22)

and SOSUI23)

(http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.

html) online tools.

4.  Quantitative RT-PCR (qRT-PCR) assays

The actin gene (Act1) was used as a reference gene.

Primer pairs for amplification of Adh1-2, Aldh1-8, Mpd1-2,

Pdc1 and Act1 cDNA were designed based on their cDNA

sequences using Genetyx ver. 10.0.3 software (Genetyx,

Tokyo, Japan). Amplification of genomic DNA was

prevented by designing primers for exon-exon junctions.

All primers were tested to ensure that they amplified a

single band with no primer-dimers, as shown in Table 2.

Plasmids with target gene (Adh1-2, Aldh1-8, Mpd1-2,

and Pdc1) and housekeeping gene (Act1) inserts were

extracted as described by Birnboim24)

. Standard curves

were constructed using four ten-fold dilutions of plasmid.

Real-time PCR was performed using a KOD SYBR® qPCR

Mix kit (Toyobo). Thermocycling was carried out using a

PikoReal™ 96 system (Thermo Fisher Scientific) with an

initial incubation for 1 min at 95℃, followed by 40 cycles

of 95℃ for 10 s, 60℃ for 1 min. Each run was completed

with a melting curve analysis to confirm the specificity of

amplification and absence of primer-dimers. Data analysis

was performed in accordance with the manufacturer’s

instructions.

5.  Analysis of sequences and phylogenetic tree 

Nucleotide and protein sequence data were analyzed

using Genetyx software. Protein sequence similarity was

analyzed using the BLASTP algorithm25)

. Peroxidase

genes were retrieved from public domain databases (NCBI

and UniProt). A phylogenetic tree was constructed by

MEGA 6.06 software26)

using the neighbor joining meth-

od with a bootstrap value of 1,000 replicates. Multiple

alignment was performed using ClustalW software27)

.

6.  Statistical analysis

Mean values and standard deviation of the relative

results for each treatment were calculated. Comparisons

between control and treatment groups were made using

Student’s t-test. Differences were regarded as statistically

significant for P values under 5% (P<0.05).

Results

1.  Protein sequences of Adhs and Mpds and phylogenetic 

analysis

A phylogenetic tree constructed using the deduced

amino acid sequences of alcohol dehydrogenases (Adhs)

and mannitol-1-phosphate dehydrogenases (Mpds)

is shown in Fig. 1. Adhs and Mpds were clustered in

different clades. Clustered in the same clade with Mpds

were alcohol dehydrogenases from S. cerevisiae and

Aspergillus nidulans. Adh2 clustered within a group of

class III Adhs including Sfa1 [S-(hydroxymethyl) glutath-

ione dehydrogenase] from S. cerevisiae, which is related to

resistance to formaldehyde. Adh1 was clustered with A.

nidulans Adh (AN8406).

Zinc-binding dehydrogenase (PF00107) and alcohol

dehydrogenase (PF08240) motifs, and the zinc-containing

Adh signature [GHEX2GX5(G, A)X2(I, V, A, C, S), where X

is any other amino acid]28)

, were present in all Adhs and

Mpds. The NAD+-binding domains of several dehydro-

genases were analyzed. A conserved domain [GXGXXG,

where X is any other amino acid)29)

in the medium-chain

alcohol dehydrogenase/reductase) superfamily appeared

in P. microspora Mpd1 and Mpd2, a sequence belonging to

the Class I alcohol dehydrogenase Adh1 from S. cerevisiae,

which converts acetaldehyde to ethanol30)

. However,

P. microspora Mpds were clustered with A. nidulans

alcB (AN3741), which has a lower substrate specificity

to ethanol than to other alcohols such as 2-propanol,

2-pentanol and 2,3-butanediol31)

. This suggests that the

Mpd proteins of P. microspora might have lost activity

toward ethanol, and therefore, transcription of their genes

in liquid medium containing ethanol should be assessed.

2.  Protein sequences of Aldhs and phylogenetic analysis

A phylogenetic tree based on protein sequences of

aldehyde dehydrogenases (Aldhs) is shown in Fig. 2. All

eight P. microspora Aldhs were clustered in different

clades. Aldh1 clustered with Ustilago maydis Iad1

(indole-3-acetaldehyde dehydrogenase), involved in

indole-3-acetic acid metabolism32)

, as well as the ethanol

metabolism related Aldh of S. cerevisiae and A. nidulans,

a predicted Aldh in Arabidopsis thaliana, and retinalde-

hyde dehydrogenase of Homo sapiens. Each cluster of

Aldh2 and Aldh3 grouped with other predicted proteins

of basidiomycetes. However, Aldh2 and Aldh3 clusters

were close to Aldh1, which suggests that Aldh1, 2 and

3 might have similar roles in P. microspora. In addition,

Aldh4 to Aldh8 had predicted functions based on

similarities to succinate semialdehyde dehydrogenase of

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MUSHROOM SCIENCE AND BIOTECHNOLOGY

A. bisporus33)

, Δ1-piperideine-6-carboxylate dehydroge-

nases related to human Aldh7A134)

, a putative salicylalde-

hyde dehydrogenase, a protein involved in meiotic sister-

chromatid recombination of eukaryotic organisms35)

and

Δ-1-pyrroline-5-carboxylate dehydrogenase of A. bisporus

pruA36)

. All Aldh proteins belonged to the aldehyde

dehydrogenase family (PF0017) and were predicted to be

soluble cytoplasmic proteins.

3.  Ethanol was ineffective  in  inducing transcription of 

Adh, Aldh or Mpd

To investigate the relationship between ethanol

metabolism and Adh and Aldh, transcription of the Adh

and Aldh genes in mycelia grown in liquid medium

supplemented with 1 mM ethanol, 1 mM propanol or

1 mM acetaldehyde was analyzed by qRT-PCR. Tran-

scription levels of Aldh1, 2 and 3 are shown in Fig. 3A

and levels of Adh1 and 2 and Pdc1 are shown in Fig. 3B.

Ethanol, propanol and acetaldehyde did not promote the

transcription of Adh, Aldh or Pdc1. Mpd2 also showed

a relatively low level of transcription in liquid medium

supplemented with ethanol, propanol or acetaldehyde.

Moreover, ethanol did not promote transcription of

either Mpd gene (Fig. 3C). However, the presence of 1

mM acetaldehyde significantly promoted transcription

of only Mpd1, which has a protein sequence most similar

to that of Adhs of A. nidulans (AN3741) and S. cerevisiae

Fig. 1. Phylogenetic tree of alcohol dehydrogenases. The tree was

constructed by the neighbor joining method with 1000 bootstrap

replications of P. microspora Adh1, Adh2, Mpd1 and Mpd2 with

corresponding sequences from Agaricus bisporus, Arabidopsis

thaliana, Aspergillus nidulans, Coprinopsis cinerea, Homo sapiens,

Laccaria bicolor, Pleurotus ostreatus and Saccharomyces

cerevisiae. The branches are identified by organism and the gene

name, then protein ID.

Fig. 2. Phylogenetic tree of aldehyde dehydrogenases. The tree was

constructed by the neighbor joining method with 1000 bootstrap

replications of P. microspora aldehyde dehydrogenases (Aldh1-

8) and Aldhs of eukaryotic organisms grouped according to gene

family: white-rot fungi Agaricus bisporus, Coprinopsis cinerea,

Hypholoma sublateritium, Laccaria bicolor and Phanerochaete

carnosa; a single-celled fungus, Saccharomyces cerevisiae;

filamentous fungus Aspergillus nidulans; dimorphic fungus Ustilago

maydis; Animalia Homo sapiens; and Planta Arabidopsis thaliana.

Aldhs fell into six clusters: Aldh1-3 clustered with H. sapiens, A.

thaliana, A. nidulans, U. maydis and S. cerevisiae. Aldh4 was

identified as a succinate semialdehyde dehydrogenase and Aldh5

as related to Δ1-piperideine-6-carboxylate dehydrogenases of A.

thaliana and H. sapiens. Aldh6 was closely related to a putative

salicylaldehyde dehydrogenase of A. nidulans (AN4050). Aldh7

was closely related to a protein involved in meiotic sister-chromatid

recombination in S. cerevisiae. Aldh8 was a Δ1-pyrroline-5-

carboxylate dehydrogenase. The branches are identified by

organism and the gene name, then protein ID.

20 Vol.24 No. 1

(YOL086C). Adhs and Mpd2 from P. microspora do not

seem related to ethanol metabolism. However, based on

these results, if Mpd1 has high enough activity to convert

acetaldehyde to ethanol, it is possible that P. microspora

can become an alcohol producer.

4.  Gene expression of Aldh and Adh in the presence of 

aromatic compounds

In order to predict the roles of Adh1 and 2 and Aldh1,

2 and 3, their transcription in mycelia grown in liquid

medium supplemented with several aromatic compounds

was examined by qRT-PCR. The transcription levels

of Aldh1, 2 and 3 are shown in Fig. 4A. Transcription

of Aldh1 was significantly promoted by 3 mM veratryl

alcohol and 0.1 mM guaiacol. Transcription of Aldh2 was

unaffected by any aromatic compounds tested. Tran-

scription of Aldh3 was promoted significantly by 0.1 mM

guaiacol. On the other hand, the transcriptional levels of

Adh1 and 2 are shown in Fig. 4B. Transcription of Adh1

was unaffected by any tested aromatic compounds, but

transcription of Adh2 was significantly promoted by 3

mM veratryl alcohol. These results indicated that Aldh

and Adh transcription are responsive to the presence of

aromatic compounds; therefore, the role of the Aldh and

Adh genes might have evolved from ethanol fermentation

to degradation of aromatic compounds related to the ones

we tested, such as lignin and lignin-like compounds. More-

over, because veratryl alcohol promotes fruiting body

formation in P. ostreatus, we investigated gene expression

during cultivation of fruiting bodies on sawdust medium.

5. Adh2 was highly transcribed during mycelial growth 

in sawdust medium, but Adh1, Aldh1 and Mpd1 were 

highly transcribed in fruiting bodies

Transcription of Adh1 and 2, Aldh1, 2 and 3, and

Mpd1 and 2 genes during mycelial growth and in

primordia and fruiting bodies was examined by qRT-PCR

(Fig. 5). The expression of the eight aldehyde dehydroge-

nase genes is displayed in Fig. 5A. Transcription of Aldh1

in primordia and fruiting bodies was respectively 130- and

100-fold higher than in mycelia. However, transcription of

the other Aldh genes remained the same in all developmen-

tal stages including mycelia. Transcription of Adhs and

Mpd genes during mycelial growth and in primordia and

fruiting bodies was also examined by qRT-PCR (Fig. 5B).

Only transcription of Adh2 in mycelia was higher than

that of Adh2 (by 6-fold) in primordia and fruiting bodies.

The transcription of Adh1 was 16-fold higher in primordia

Fig. 3. Relative expression of Aldh1-3 (A), Adh1-2 and Pdc1 (B) and

Mdh1-2 (C) in P. microspora mycelia cultured in M4 medium with

ethanol, propanol and acetaldehyde. Total RNA was measured on

day 10 by qRT-PCR in triplicate, with variation denoted by standard

error bars. Asterisks indicate a significant difference in expression

between control basal medium and medium supplemented with

ethanol, propanol or acetaldehyde (t-test, p < 0.05).

Fig. 4. Relative expression of Aldh1-3 (A) and Adh1-2 (B) in P.

microspora mycelia cultured in M4 medium with aromatic

compounds. Total RNA was measured on day 10 by qRT-

PCR in triplicate, with variation denoted by standard error bars.

Asterisks indicate a significant difference in expression between

control basal medium and medium supplemented with aromatic

compounds (t-test, p < 0.05).

21日本きのこ学会誌

MUSHROOM SCIENCE AND BIOTECHNOLOGY

and 12-fold higher in fruiting bodies than in mycelia.

Transcription of Mpd1 was 2-fold higher in primordia and

4-fold higher in fruiting bodies than in mycelia.

Discussion

In order to address why P. microspora cannot produce

ethanol, we analyzed the expression and the predicted

protein sequences of two Adh, two Mpd and eight Aldh

genes. The results suggest that ethanol-related genes in P.

microspora are not related to production of ethanol except

for Mpd1. However, Mpd1 expression was promoted by

acetaldehyde, although P. microspora did not produce

ethanol (data not shown). Therefore, in further study,

we plan to investigate the enzyme activity of Mpd1 to

determine whether it has alcohol dehydrogenase activity

and can convert acetaldehyde to ethanol. However, it is

possible that Mpd1 might have lost alcohol dehydroge-

nase activity during evolution. Therefore, to breed an

alcohol-producing P. microspora for conversion of lignocel-

lulose biomass into bioethanol, molecular breeding, such

as introduction of the alcohol dehydrogenase gene from S.

cerevisiae to the P. microspora genome, might be needed.

Generally speaking, during lignin degradation in

mushrooms, both phenolic acids and phenolic aldehydes

are produced. The ratio of benzoic acid to benzaldehyde

in compost and during growth of Agaricus bisporus

increases until the fourth flush in cultivation37)

. In the

evolution of fungi, oxygen was the most important factor

controlling growth of mycelia and lignin degradation.

Because mushrooms are aerobic microorganisms, the

oxygen required for energy metabolism and lignin

degradation is also oxidized in aerobic conditions38)

.

During lignin degradation in mushrooms, both phenolic

acids and phenolic aldehydes are produced. Mushrooms

are aerobic microorganisms that require oxygen for both

energy metabolism and lignin degradation. The yeast

S. cerevisiae is a fungus with a long history of producing

ethanol due to the proficiency of the ScAdh genes and

the involvement of ScAldh in ethanol metabolism2)

.

However, ethanol fermentation is an anaerobic type of

metabolism. Therefore, during evolution, when white rot

fungi obtained the ability to degrade lignin, the substrate

specificity of alcohol dehydrogenase, which was a protein

involved in anaerobic fermentation, might have changed

and lost its original function.

Furthermore, these results showed that lignin-like

products such as veratryl alcohol significantly promote

Aldh1 transcription. Veratryl alcohol promotes fruiting

Fig. 5. Relative expression of Aldh1-8 (A), Adh1-2 and Mpd1-2 (B) during development of P. microspora in saw-

dust medium. Total RNA was extracted from mycelia cultivated for 1 and 3 months (mo.), primordia, and

fruiting bodies. All samples were triplicates, with variation in measurements denoted by standard error bars.

22 Vol.24 No. 1

body formation and shortens the culture period in P.

ostreatus39)

. Therefore, Aldh1 might have an important

role involving various aromatic compounds in fruiting

bodies. In this study, we investigated Ald, Aldh and

Mpd expression in P. microspora, finding that all were

incapable of producing ethanol. Therefore, we will

further study introduction and expression of S. cerevisiae

Adh in P. microspora to investigate the feasibility of

ethanol production by P. microspora.

Acknowledgment　This work was partially supported by

Grant-in-Aid for Scientific Research (C) 15K07514 by the

Japan Society for the Promotion of Science (JSPS).

和 文 摘 要

Pholiota microspora におけるアルコールと

アルデヒド脱水素酵素遺伝子ファミリー

の転写分析，その生理的役割の推定

Surasit SUTTHIKHAMPA1)・河井　祥2)・林　未来2)

Sophon BOONLUE3)・霜村典宏2)・山口武視2)

• 會見忠則2)

1) 鳥取大学大学院連合農学研究科

　〒680-8553 鳥取県鳥取市湖山町南 4-101

2) 鳥取大学農学部

　〒680-8553 鳥取県鳥取市湖山町南 4-101

3) Faculty of Science, Khon Kaen University,

　Khon Kaen 40002, Thailand

　本研究では，Pholiota microspora での 2 つのアルコール

脱水素酵素（Adh），8 つのアルデヒド脱水素酵素（Aldh）

と 2 つのマンニトール -1- リン酸脱水素酵素（Mpd）遺伝子

の発現を分析した結果，Aldh1, 2, 3, Adh1, 2，及び Mpd1, 2

の転写は，1 mM のエタノールの存在下の液体培養におい

て全く影響を受けなかった．しかし，Mpd1 の発現はアセト

アルデヒドで促進された．したがって，Mpd1 は，エタノー

ル生産のためのアルコール脱水素酵素の候補である．一方，

Aldh1 と Adh2 の転写は，3 mM のベラトリルアルコール

の存在で促進された．したがって，Aldh1とAdh2の役割は，

アルコール代謝から，芳香族化合物の分解に進化した可能

性があると考えられた．Adh1，Mpd1，Mpd2 と Aldh1 の

転写は菌糸より原基および子実体で高かった．この現象は

Adh1，Mpd1，Mpd2 と Aldh1 の応答は子実体形成中に酸

化ストレスが存在することを示唆していた．

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