ABSTRACT The gene encoding mannitol-1-phosphate dehydrogenase, mpd, which has similar function with alcohol dehydrogenase, has been sequenced and characterized from the basiodiomycete Pholiota nameko. The coding region of mpd was composed of 2992 bp and found to encode a polypeptide of 359 amino acids that has similarity with Laccaria bicolor. To evaluate the expression level of mannitol-1-phosphate from P. nameko, mpd cDNA was inserted into pCold shock vector and expressed in host BL21 (DE3) by 24 h induction with 1 mM isopropyl 1-thio-β-Dgalactopyranoside at 15 O C after 2 h growth at 37 O C. The purified protein was detected by SDS-PAGE and western blot

Jia Ning Wan

Norihiro Shimomura

Tadanori Aimi

Yan Li

Yukiko Kinjo

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Proceedings of the 7
th
 International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011 
Section: 
Physiology and Development 
188 
 
HETEROLOGOUS EXPRESSION AND CHARACTERIZATION 
OF MANNITOL-1-PHOSPHATE DEHYDROGENASE FROM 
THE BASIODIOMYCETE PHOLIOTA NAMEKO 
 
YAN LI, JIA NING WAN, YUKIKO KINJO, NORIHIRO SHIMOMURA, TADANORI AIMI
*
 
Faculty of Agriculture,Tottori University, 
Koyamacho Minami 4-101, Tottori-Shi, Tottori, 680-8553, 
 Japan. 
liyan.caas@gmail.com 
 
ABSTRACT 
 
The gene encoding mannitol-1-phosphate dehydrogenase, mpd, which has similar function with 
alcohol dehydrogenase, has been sequenced and characterized from the basiodiomycete Pholiota 
nameko. The coding region of mpd was composed of 2992 bp and found to encode a polypeptide 
of 359 amino acids that has similarity with Laccaria bicolor. To evaluate the expression level of 
mannitol-1-phosphate from P. nameko, mpd cDNA was inserted into pCold shock vector and 
expressed in host BL21 (DE3) by 24 h induction with 1 mM isopropyl 1-thio-β-D-
galactopyranoside at 15 
O
C after 2 h growth at 37 
O
C. The purified protein was detected by SDS-
PAGE and western blot.  
 
Keywords: Mannitol-1-phosphate dehydrogenase; Alcohol dehydrogenase activity 
 
 
INTRODUCTION 
Ethanol as a petroleum substitute has many desirable features that are compatible with 
sustainable development. Most ethanol is produced in large quantities from natural resources 
including corn grain and sugarcane juice. However, there is a need to find an inexpensive and 
widely available source of lignocellulosic biomass. It is well-known that basidiomycete fungi 
cause white rot decay, and are able to degrade the lignin in wood and convert cellulose into 
glucose which is the most desirable sugar for fermentation.  
According to Kalavati [1], mannitol-1-phophate dehydrogenase from Crytococcus 
neoformans is a zinc-containing long-chain alcohol dehydrogenase. Mannitol is a six carbon 
polyol which is abundant in plants, fungi and bacteria. It accumulates in some fungi and may be 
involved in fungal tolerance to environmental stress such as salt stress, and may also function as 
an osmoregulatory compound in supporting turgor and in fruit body development. Mannitol-1-
phosphate dehydrogenase catalyzes the conversion of fructose-6-phosphate to mannitol-1-
phosphate, which plays an important role in mannitol biosynthesis. Based on the similarity 
between mannitol-1-phosphate dehydrogenase and alcohol dehydrogenase, we have attempted to 
mutate the conserved domain in mannitol-1-phosphate dehydrogenase from P. nameko, and have 
investigated the alcohol-producing potential of the mushroom. 
 
 
MATERIALS AND METHODS 
 
Proceedings of the 7
th
 International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011 
Section: 
Physiology and Development 
189 
Fungal strain and plasmids P. nameko NGW19-6 (A4, pdx1), an auxotrophic mutant for 
pyridoxine, was used in this study [19, 20]. Escherichia coli, strain DH5α, (Takara Bio, Shiga, 
Japan) was used as the host for plasmid amplification. Plasmid pMD20 vector (Takara Bio) was 
used to clone polymerase chain reaction (PCR) products. E. coli, strain BL21, (DE3) (Novagen, 
Darmstadt, Germany) was used as the host for recombinant protein gene expression, and pCold 
(Novagen, Darmstadt, Germany) vector was used to clone and express the recombinant protein 
gene. 
 
DNA and RNA preparation. P. nameko mycelium was grown on potato dextrose agar (PDA) at 
25 
O
C for 7 d. After harvesting, the mycelium was frozen in liquid nitrogen and ground in a 
mortar and pestle to a fine power. The genomic DNA extraction kit (Amersham Bioscience, 
Tokyo, Japan) was used to isolate genomic DNA according to the manufacturer’s instructions. 
RNeasy mini kit (Qiagen, Tokyo, Japan) was used to extract RNA. 
 
Genome walking. Genomic DNA from P. nameko NGW19-6 was digested with restriction 
endonucleases (Bgl, Xho, EcoT221, Sal, Xba, Bam, Pst, Hind, Nhe, EcoRI, Fba, Spe) and 
fragments of suitable length were ligated with nucleotide linkers and used as templates for PCR. 
PCR was carried out in 100 μl reaction mixtures containing 1 × Ex Taq buffer, 100 ng extracted 
genomic DNA, 100 pmol of each primer, 0.2 mM dNTPs, and 2.5 U Ex Taq polymerase. PCR 
was carried out using the following cycling parameters: initial denaturation at 94 
O
C for 1 min, 
followed by 30 cycles of 30 s at 94 
O
C and 5 min at 68 
O
C. All amplified DNA fragments were 
subcloned into T-vector pMD20 and all plasmids were sequenced. In order to obtain the full 
sequence of the mpd gene, after genomic walking, four primers were designed for partial 
sequencing. PCR was carried out in 50 μl reaction mixtures containing 1 × Ex Taq buffer; 100 ng 
extracted genomic DNA, 100 pmol of each primer, dNTPs at a final concentration of 0.2 mM, 
and 2.5 U Ex Taq polymerase. PCR was carried out using the following cycling parameters: 
initial denaturation at 94 
O
C for 5 min, followed by 30 cycles of 30 s at 94 
O
C, 30 s at 55 
O
C, and 
5 min at 72 
O
C, followed by 72 
O
C for 10 min. 
 
Reverse transcription-PCR (RT-PCR). Amplification of 3’-ends of cDNA by 3’-rapid 
amplification of cDNA ends (RACE) was performed with a Takara RNA PCR Kit (AMV) 
Version 3.0 (Takara Bio). The 5’RACE was carried out with the 5’-Full RACE Core Set (Takara 
Bio). Reverse transcription and PCR were carried out according to the manufacturer’s 
instructions. 
 
DNA sequencing and computer analysis. All the fragments were cloned into T-vector pMD20 
and sequenced by ABI PRISM
TM
 3100 Genetic Analyser (Applied Biosystems, Tokyo, Japan) 
using the chain-termination procedure with a BigDye Terminator
TM
 Cycle Sequencing Kit 
(version 3.1; Applied Biosystems) according to the manufacturer’s instructions. Sequence data 
editing and assembling were performed using Seqman (DNAStar, Lasergene software, Madison, 
USA); the subcellular location of MPD was predicted by the program SOSUI [2]; the motif 
search program online (http://motif.genome.jp/) was used for motif searching. Amino acid 
sequences of 26 fungal mannitol-1-phosphate and 12 alcohol dehydrogenase amino acid 
sequences were downloaded from the NCBI at the NIH (http://www.ncbi.nlm.nih.gov/). Initially, 
all the amino acid sequences were aligned with the Clustal X program [3]. Then, the PHYLIP 
3.69 package [4] was used to construct a Jones-Taylor-Thornton matrix from the raw data 
Proceedings of the 7
th
 International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011 
Section: 
Physiology and Development 
190 
(PRODIST program) [5]. Cluster analysis was performed using the unweighted pair group 
method with arithmetic averaging (UPGMA) [6]. The robustness of the phylogenetic trees was 
evaluated by bootstrap analysis with 100 replicates [7].  
 
Expression of mannitol-1-phosphate dehydrogenase cDNA in E. coli. For expression of MPD 
cDNA in E. coli, a DNA fragment containing the MPD coding region, framed with restriction 
enzyme sites at both ends, was obtained by PCR with the sequence specific primers designed 
based on the restriction enzyme sites NdeІ and EcoRІ. The PCR fragment was digested with 
NdeІ and EcoRІ and ligated with NdeІ/EcoRІ-digested pCold shock vector; this generated 
plasmid pCold-MPD, which was used to transform BL21 (DE3). The empty vector pCold was 
used as control. Both plasmid and control were grown at 37 
O
C in LB medium supplemented 
with 100 mg/ml ampicillin until the OD600 reached between 0.4-0.6. The culture was adjusted to 
1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and further cultivated for 16 h at 15 
O
C. The 
cells were harvested by centrifugation at 2,600g for 10 min, resuspended in Tris-HCl buffer (pH 
7.5), and disrupted by ultrasound. Following centrifugation at 2,600 g for 10 min, the supernatant 
was used for enzyme assays [8-10]. One unit of the enzyme was defined as the amount that 
catalyzed the formation of 1 μmol of NADH in 1 min under the reaction conditions.  
 
 
RESULTS AND DISCUSSION 
The nucleotide sequence of the mannitol-1-phosphate dehydrogenase gene in P. nameko contains 
2992 bp, and the coding region (from ATG to the stop codon) is 1363 bp and encodes 359 amino 
acids. The locations of the exons and introns in the gene were determined from the nucleotide 
sequences of the RT-PCR products. All the introns started with GT and ended with AG. The 
coding region contained six exons and five introns. In addition, the SOSUI program predicted 
that the MPD protein was a soluble protein, which was located in the cytoplasm. The motif 
search program online revealed that MPD has a zinc-containing alcohol dehydrogenase motif 
from 1916 bp to 2011 bp. A phylogenetic tree was constructed using the deduced amino acid 
sequences of MPD and 27 mannitol-1-phosphate dehydrogenase proteins and 11 alcohol 
dehydrogenase proteins. P. nameko MPD protein had a 73% amino acid identity with MPDH1 of 
Laccaria bicolor [11]. The phylogenetic tree separated into two main parts, one consisting 
entirely of mannitol-1-phosphate dehydrogenases and the other a mixture of alcohol 
dehydrogenases and mannitol-1-phosphate dehydrogenases. In the mixed group, ten fungal 
alcohol dehydrogenases were clustered together, while another alcohol dehydrogenase from 
Saccharomyces cerevisiae clustered with ten fungal mannitol-1-phosphate dehydrogenases. 
MPD was expressed in cultures of engineered E. coli BL21 (DE3), and the crude cell extract had 
alcohol dehydrogenase activity.  
There have been many reports on mannitol-1-phosphate dehydrogenase genes, which 
mainly focused on mannitol metabolism. These include the proposal that mannitol is involved in 
an NADPH-producing cycle involving the utilization of NADH and ATP [12]. However, 
mannitol was still produced by mannitol-1-phosphate dehydrogenase and mannitol 
dehydrogenase double deletion mutants [13] and the existence of a novel mannitol 
phosphorylation pathway for mannitol biosynthesis and metabolism was deduced. Mannitol is 
the main storage material in Agaricus bisporus [14] and also plays important roles in the 
sporulation of Stagonospora nodorum [15]. Mannitol accumulation in fungi may be involved in 
fungal tolerance to environmental stress such as salt stress [16], and function as an 
Proceedings of the 7
th
 International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011 
Section: 
Physiology and Development 
191 
osmoregulatory compound to support turgor and fruit body development [17]. However, there 
are no reports describing the relationship between mannitol-1-phosphate dehydrogenase and 
alcohol dehydrogenase. Here, we report for the first time a mannitol-1-phosphate dehydrogenase 
gene from P. nameko that encodes a protein with the potential to produce alcohol by 
fermentation. The long chain alcohol dehydrogenase from horse liver has approximately 350 
amino acids per subunit, and the short chain alcohol dehydrogenase from Drosophila has 
approximately 250 amino acids per subunit [18]. According to our data, mannitol-1-phosphate 
dehydrogenase from P. nameko has 359 amino acids and is a zinc-containing long chain alcohol 
dehydrogenase.  
Phylogenetic analysis suggests that mannitol-1-phosphate dehydrogenase and alcohol 
dehydrogenase are evolutionary homologous, and some mutations affecting mannitol-1-
phosphate dehydrogenase may lead to the appearance of alcohol dehydrogenase. This may 
explain why some fungi can produce alcohol under anaerobic conditions. Yeasts have the 
capability of alcoholic fermentation but cannot degrade lignocellulose, while basidiomycetes can 
degrade lignocellulose but cannot produce alcohol by fermentation. Mannitol-1-phosphate 
dehydrogenase from P. nameko expressed in E. coli has alcohol dehydrogenase activity and this 
makes mushroom alcoholic fermentation possible. Compared with Agaricus blazei, Flammulina 
velutipes and Tricholoma matsutake [8, 9, 10], the alcohol dehydrogenase activity is very low, 
but genetic engineering methods such as promoter replacement or mutation could be used to 
improve alcohol dehydrogenase activity in P. nameko. 
 
 
CONCLUSIONS 
Mannitol-1-phophate dehydrogenase from P. nameko was cloned and characterized. The 
presence of the zinc-containing long-chain alcohol dehydrogenase suggests that the mushroom 
has the potential to produce ethanol by fermentation. 
 
 
ACKNOWLEDGEMENT 
This work was supported in part by the Global COE Program (Advanced Utilization of 
Fungus/Mushroom Resources for Sustainable Society in Harmony with Nature), MEXT, Japan. 
 
 
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Physiology and Development 
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HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF MANNITOL-1-PHOSPHATE DEHYDROGENASE FROM THE BASIODIOMYCETE PHOLIOTA NAMEKO

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HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF MANNITOL-1-PHOSPHATE DEHYDROGENASE FROM THE BASIODIOMYCETE PHOLIOTA NAMEKO

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