Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Journal of Japanese Society for Extremophiles (2006), Vol. 5, 21-26

ORIGINAL PAPER

Minegishi Ha, Miura Tb, Yoshida Ya, Usami Ra and Abe Fb

Phylogenetic analysis of pectin degrading yeasts from deep-sea environments
aDepartment of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe 350-0815, Japan,
Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15
Natsushima-cho, Yokosuka 237-0061, Japanb

Corresponding author: Fumiyoshi Abe, abef@jamstec.go.jp
Phone: +81-46-867-9678, Fax: +81-46-867-9715
Received: November 23, 2005 / Revised: November 24, 2005 / Accepted: November 25, 2005
and high hydrostatic pressure, and therefore
microorganisms inhabiting such environments havedeveloped the ability to survive under such extremeconditions. Mud samples are expected to be valuable
resources for isolating extremophiles and useful enzymes
that can function under extreme conditions5,6).
Pectic compounds are polysaccharides originating inplants, with α-1,4-glycoside linkage of polymers ofgalacturonic acids. Bacteria and filamentous fungi
produce pectin-degrading enzymes, which are essential
in phytopathogenesis. Fungal polygalacturonases(PGases) have mainly been used in food industries,
especially in the extraction and clarification of fruit
juices. Today, the main source of PGases in industry isfrom the fungus Aspergillus niger7). However,commercial preparations of fungal origin contain acomplex mixture of different enzymes with pectinolytic
activity including endo- and exo-PGase, pectin lyase, andthe undesirable pectin esterases and other nonspecificenzymes. In this sense, yeast PGases may haveadvantages over fungal enzymes and could offer analternative to fungal ones for the supply of pure enzymes.
Attempts have been made to isolate PGases from yeasts.
The biochemical properties of the PGases were reviewed
by Blanco et al.8) The first PGase-producing yeast was
Saccharomyces fragilis reported by Luh and Phaff9). APGase produced by Cryptococcus albidus has been
purified and biochemically characterized10). The
biochemical characterizations of several PGases fromyeasts have now been reported and these genes havebeen successfully expressed in Saccharomyces
cerevisiae11,12). They are usually extracellular enzymes,
showing differences in molecular mass, and most are
glycoproteins13,14). In some species, the presence of

Abstract Deep-sea yeast strains were isolated frommud samples collected in Sagami Bay (1,100-1,400 m)
and the Japan Trench (4,500-6,500 m). All of the 46
yeast isolates were capable of growth at 24°C andatmospheric pressure, suggesting that these strains might
originate neritic regions or land but survived in deep sea.
Based on sequencing of 26S rRNA gene, 15 strains wereclassified into basidiomycetous yeasts including
Cryptococcus
liquefaciens,
Kondoaaeria,
Rhodosporidium
diobovatum,
Rhodosporidium
sphaerocarpum, Rhodotorula mucilaginosa, Rhodotorula
dairenensis and Rhodotorula slooffiae, and 8 strains wereclassified into ascomycetous yeasts including
Aureobasidium
pullulans,
Candidazeylanoides,
Kluyveromyces
nonfermentans,
Metschnikowia
bicuspidate and Williopsis saturnus. Screening of the 46
isolates appeared to yield a high frequency ofpolygalacturonase (PGase) producers capable ofdegrading pectin. We suggest that deep-sea yeasts are
new sources of PGase producers.
Keywords: Deep-sea yeasts, Polygalacturonase, Pectin
Introduction
Studies on yeasts in aquatic environments have focusedon their application as organic pollution indicators. In
general, the yeast population in oceans is lower than thatin fresh water or on land1,2). The frequency of yeast
occurrence is known to decrease with increasing ocean
depth, except in sites colonized by marine invertebrates3).
Nevertheless, new yeast species have been successfully
isolated from deep-sea mud samples3, 4), and some maybe useful in industrial applications. The deep-sea ischaracterized by low nutrient levels, low temperature,21

Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

several isozymes has been demonstrated8).
In our previous studies, two PGases designated p36
and p40 were successfully purified from deep-sea yeast
strain N6 and biochemical properties of the enzymes
were analyzed with respect to obtaining kinetic
parameters and protein glycosylation15). However, theremainder of PGase producers and PGases have not beencharacterized. In this paper, we describe phylogenetic
analysis of PGase-producing yeasts isolated from Sagami
Bay and the Japan Trench.

72°C for 2 min, and denaturation at 94°C for 1 min.
After PCR, the reaction mixture was treated with SAP(Amersham Biosciences, Corp., Piscataway, NJ, USA)and subjected to DNA sequencing using an ABI377 DNA
sequencer. A phylogenetic tree was constructed using theneighbor-joining (NJ) method using Clustal X ver. 1.83
and NJ Plot17).
The accession numbers for the D1/D2 domain of 26S
rRNA gene of the deep-sea yeast strains were obtained
from the DNA Data Bank of Japan (DDBJ) and are listed
in legends of Fig. 1 and Fig. 2.

Materials and methods
Identification of PGase-producing yeasts
To detect PGase production by yeasts, cells wereincubated on SDP (0.67% Difco yeast nitrogen base,
1.25% polygalacturonic acid, 50 mM potassium
phosphate, 0.5% agar, pH 5.5) plates at 24°C for 2 days.
Then the plates were stained with 0.1% ruthenium red18).
A deep purple halo is observed around a colony if thestrain produces extracellular PGase.

Isolation of deep-sea yeasts
The procedure for the isolation of deep-sea yeasts has
been described previously3,4,15). Deep-sea sediment
samples were obtained using core samplers installed inthe submersibles Shinkai 2000 and Shinkai 6500 at a
depth of 1,100–1,400 m in Sagami Bay and 4,500–6,500
m in the Japan Trench, respectively. Approximately 0.1 gof the sediment was directly spread on each YPD plate(1% w/v bacto yeast extract, 2% w/v bacto peptone, 2%w/v glucose) or YPP plate (1% w/v bacto yeast extract,2% w/v bacto peptone, 1% w/v pectin) containing
penicillin (50 mg/L) and streptomycin sulfate (50 mg/L).
The plates were first incubated at 8°C for 2 weeks. Aftercolonies appeared, incubation was continued at 15°C andthen at 26°C, after which the colonies were collected.

Results
Phylogenetic relationships among PGase-producing
deep-sea yeasts and related species
Isolation of deep-sea yeast strains was successfully
performed using YPD medium containing glucose as acarbon source, although no colony was obtained using
pectin-based YPP medium. Forty-six strains wereisolated. Strains designated by “S” were obtained from
Sagami Bay and those by “N” were obtained from theJapan Trench. The D1/D2 region of 26S rRNA gene wasdetermined for all of the 46 strains and compared withpublished sequences of related species. The deep-sea
yeast strains were distributed in basidiomycetous yeast
genera Cryptococcus, Kondoa, Rhodosporidium, andRhodotorula (Fig. 1), and ascomycetous yeast genera
Kluyveromyces, Williopsis, Candida, Metschnikowia, andAureobasidium (Fig. 2). Red yeasts such as the genera
Rhodosporidium and Rhodotorula commonly occur indeep-sea mud samples3,4). In our preliminary observation,
the deep-sea yeasts were capable of growth on poor
nutrition, e.g., in 100-times diluted YPD medium.
Accordingly, the high occurrence of red yeasts indeep-sea might be concerned with the ability to grow onpoor nutrition.

DNA isolation and PCR amplification of 26S rRNA geneFor genomic DNA extraction, cells were grown on YM(0.5% bacto peptone, 0.3% bacto yeast extract, 0.3%
bacto malt extract, 1% glucose) agar and suspended in 1
ml of extraction buffer (50 mM TrisHCl, 50 mM EDTA,3% SDS, pH 8.0). The cells were disrupted byhomogenization for 2 min in the presence of alumina.
The resulting homogenate was extracted with an equalvolume of phenol, followed by phenol-chloroform
extraction. After ethanol precipitation, DNA wasresuspended in 50 µl of TE buffer (10 mM TrisHCl, 1mM EDTA, pH 8.0).
The divergent D1/D2 domain of 26S rRNA gene wasamplified with primers F63 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and LR3 (5'-GGTCCGTGTTTCAAG ACGG-3')16). Amplification was performed for 30
cycles with annealing at 58°C for 1 min, extension at22

Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1Fig. 2. Phylogenetic tree of 8 deep-sea ascomycetous yeasts and related species. The tree was constructed basedon a total of 644 aligned nucleotide sites in the D1/D2 region of 26S rRNA gene. Numbers at branches arebootstrap values, derived only for the nodes supported by greater than 50% probability (100 replicates). APGase-producing yeast-like fungus strain N10 is underlined. The accession numbers for the D1/D2 region of26S rRNA gene are: AB217513 (S4), AB217514 (S8), AB217515 (S25), AB217516 (S27), AB217517 (S3),AB217518 (S26), AB217519 (S24) and AB217520 (N10).

(Fig. 3). The result suggests that the production of PGase
is likely to be a distinctive property of these yeast species
that inhabit deep-sea environments. We noticed that avery small dilute halo was formed around the colony of
Crytptococcus liquefaciens NBRC0434T within 1mm(Fig. 3). This suggests that the type strain NBRC0434T
may produce a small amount of membrane bound- or cellwall associated-PGase.

All 46 yeast isolates together with some type strains
were subjected to a PGase assay using ruthenium red
staining to visualize degradation of PGA (Fig. 3). This
yielded a high proportion of PGase-producing yeasts
comprising 15 strains (Figs. 1 and 2, underlines). The
PGase-producing deep-sea yeasts were classified into 3
species closely related to Cryptococcus liquefaciens,
Rhodosporidium diobovatum and Aureobasidium
pullulans. Notably, the type strains of C. liquefaciens
NBRC0434T and R. diobovatum NBRC1830T, and a typestrain of common marine yeast, Crytptococcus albidus
IFO0378T did not produce extracellular PGase as far as
detectection using ruthenium red staining is concerned24Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

Fig. 1. Phylogenetic tree of 38 deep-sea basidiomycetous yeasts and related species. The tree wasconstructed based on a total of 644 aligned nucleotide sites in the D1/D2 region of 26S rRNA gene.
Numbers at branches are bootstrap values, derived only for the nodes supported by greater than 50%
probability (100 replicates). Fourteen PGase-producing yeasts designated by “S” or “N” are underlined.
The accession numbers for the D1/D2 region of 26S rRNA gene are: AB217475 (N13), AB217476 (N1),
AB217477 (N2), AB217478 (N3), AB217479 (N4), AB217480 (N5), AB217481 (N7), AB217482 (N8),AB217483 (N9), AB217484 (S13), AB217485 (S15), AB217486 (S16), AB217487 (S17), AB217488(S21), AB217489 (S22), AB217490 (S23), AB217491 (S12), AB217492 (N14), AB217493 (N15),AB217494 (N18), AB217495 (N20), AB217496 (N31), AB217497 (N34), AB217498 (N36), AB217499(N40), AB217500 (N51), AB217501 (N52), AB217502 (N53), AB217503 (N54), AB217504 (S1),
AB217505 (S2), AB217506 (S9), AB217507 (S10), AB217508 (S11), AB217509 (S18), AB217510 (N49),
AB217511 (S19) and AB217512 (N6).

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Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

S13

S15

S16

S21

S22

S23

N1N2N3N4N5N6N7N9N10C. albidus

C. liquefaciens

R.diobovatum

IFO0378T

NBRC0434T

NBRC1830T

Fig. 3. Identification of PGase-producing yeasts on ruthenium red staining. Cells were incubated on SDP plates
at 24°C for 2 days. A halo was visualized by staining the plate with 0.1% ruthenium red.

characterization of a 41-kDa PGase from C. albidus var.
albidus strain IMAT-473510). Type strain of C. albidus
IFO 0378T did not produce PGase detectable using
ruthenium red staining (Fig. 3). Therefore, production ofPGase may be a distinctive property of strain
IMAT-4735.
C. liquefaciens strain N6 was originally identified asa copper-tolerant yeast, and was characterized withrespect to the role of superoxide dismutase22,23). In ourpreliminary result, a halo is formed on pectin-based agar
containing 10 mM CuCl2. Accordingly, the PGase ofstrain N6 might be copper tolerant. Cloning of the genes
encoding both PGases is in progress. In our preliminary
experiments, the supernatant from the culture of strain
N6 efficiently dissolved natural orange peel. Therefore
PGases of strain N6 are likely to be useful in foodprocessing such as the clarification of juices. We suggestthat deep-sea yeasts are new sources for the supply ofindustrially useful enzymes.

Discussion
Recently, the idea has emerged that PGases from avariety of yeasts could offer an alternative to fungal
PGases for industrial applications, potentially providing
pure and safe enzyme preparations8). Deep-sea sediment
samples are new sources for the isolation of novel
microorganisms and useful enzymes produced by theisolates. Of 46 deep-sea yeast isolates investigated, 15
strains were identified as PGase producers including C.
liquefaciens, R. diobovatum, and A. pullulans. However,such a high occurrence of PGase-producing yeasts underdeep-sea environments is still inexplicable. The deep-sea
yeast isolates are capable of growth on poor nutrition(our unpublished observation). The availability of low
nutrients for growth might be related to the availabilityof pectin, which is less favorable for growth of general
fermentation yeast species.
In our previous study, two PGases designated p36
and p40 were successfully purified from C. liquefaciens
strain N615), and were further characterized with respect
to effects of high hydrostatic pressure and low
temperature19). The production of PGase by Rhodotorulasp.20) and A. pullulans ATHUM 291521) has been reported.
It is worthwhile to analyze the phylogenetic relationships
and PGases between our isolates and previously reported
PGase producers. Federici reported the purification andAcknowledgements
We thank K. Horikoshi for valuable comments, theShinkai 2000 and Shinkai 6500 teams for the collectionof deep-sea mud samples and T. Nagahama forphylogenetic analysis.

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Journal of Japanese Society for Extremophiles (2006) Vol.5, No.1

characterization of the enzyme. Enzyme Microb. Technol.

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