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Microbiological Research 163 (2008) 255266

Isolation and characterization of Candida


membranifaciens subsp. avinogenie W14-3,
a novel riboavin-producing marine yeast
Lin Wang, Zhenmin Chi

, Xianghong Wang, Liang Ju, Zhe Chi, Ning Guo


Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao,
Shandong 266003, China
Received 17 June 2007; received in revised form 3 December 2007; accepted 9 December 2007
KEYWORDS
Riboavin;
Marine yeasts;
C. membranifaciens
subsp. avinogenie;
Molecular
identication
Summary
We found that the marine yeast strain W14-3 isolated from seawater of China Eastern
Sea could produce riboavin. It is interesting to observe that the marine yeast strain
produced a large amount of riboavin in the medium containing xylose, sucrose,
galactose and maltose under the conditions of vigorous shaking. The yeast strain was
found to belong to Candida membranifaciens subsp. avinogenie based on the
results of routine and molecular identication. The protein sequences deduced from
the partial genes encoding GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-
phosphate synthase in the yeast exhibited high identity with those of the
corresponding enzymes for riboavin biosynthesis in other yeasts. Fe
3+
available in
the medium repressed riboavin production and expression of the genes responsible
for riboavin biosynthesis in the yeast. The results have evidenced that a riboavin
synthesis pathway indeed existed in the yeast. This is the rst study to report that
C. membranifaciens subsp. avinogenie W14-3 from the marine environment could
produce riboavin.
& 2008 Elsevier GmbH. All rights reserved.
Introduction
Riboavin, a yellow, water-soluble vitamin
has many physiological roles in human and animals.
The best-known biochemically active coenzymes
formed from riboavin are mononucleotide (FMN)
and avin adenine dinucleotide (FAD), needed as
electron acceptors in oxidoreductases (Stahmann
et al., 2000). In order to avoid deciency symptoms
like dermatitis, a nutritional requirement of
0.31.8 mg riboavin/day for humans and 14 mg
riboavin/kg diet for animals is recommended.
Riboavin can also be used in soft drinks and yogurt
ARTICLE IN PRESS
www.elsevier.de/micres
0944-5013/$ - see front matter & 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2007.12.001

Corresponding author. Tel/fax: +86 532 82032266.


E-mail address: zhenming@sdu.edu.cn (Z. Chi).
(Stahmann et al., 2000). Chemical production still
accounts for the major part of industrial riboavin
synthesis. However, the disadvantages of the
chemical synthesis are a maximum yield of about
60%, thus generating a lot of waste, requiring
organic solvents and 25% more energy in compar-
ison to the fermentation process (Stahmann et al.,
2000). In recent years, the commercial fermenta-
tions for riboavin production have been estab-
lished and it was found that riboavin production
by fermentation has many merits over the chemical
process. For example, mild conditions are used,
less energy is needed, riboavin is more easily
recovered and less wastes are produced during the
fermentation. So far the microorganisms that have
been used for riboavin biosynthesis on a large
scale include the hemiascomycetes Ashbya gossypii
(Stahmann et al., 2000; Wendland and Walther,
2005), a lamentous fungus, Candida famata
(Stahmann et al., 2000), a yeast, Pichia guillier-
mondii (Fayura et al., 2007) and the genetically
engineered Bacillus subtilis, which requires at least
the deregulation of purine synthesis and a mutation
in a avokinase/FAD synthetase (Stahmann et al.,
2000).
During the isolation and identication of the
marine yeasts obtained in this laboratory, we found
that the culture of the marine yeast strain W14-3
isolated from seawater of China Eastern Sea turned
yellow when it was grown in the medium containing
xylose, sucrose, galactose and maltose, at 25 1C for
24 h. Therefore, the main purposes of the present
study are to identify and characterize the marine
yeast strain W14-3. We also purify and identify the
yellow substance produced by the marine yeast
strain W14-3.
Experimental
Yeast strains
Different samples (200 m depth, 8 1C, pH 8.1 and
2.89% salinity) of seawater and sediments of China
Eastern Sea were collected during winter 2006. Two
grams of the sediments and 2.0 ml of the seawater
were suspended in 20.0 ml of YPD medium contain-
ing 2.0% glucose, 2.0% polypeptone and 1.0% yeast
extract and supplemented with 0.05% chloramphe-
nicol (to inhibit bacterial growth) immediately
after sampling and cultivated at natural tempera-
ture for 5 days. Suitable dilutions were prepared
and plated out on YPD agar plates with 0.05%
chloramphenicol and the plates were incubated at
2025 1C for 5 days. Different colonies from the
plates were transferred to the fresh YPD plates.
One isolate, strain W14-2, isolated from seawater
of China Eastern Sea, was selected for further study
based on its production of a yellow substance. The
yeast strain was identied to be Candida membra-
nifaciens subsp. avinogenie according to the
results of routine yeast identication and molecu-
lar methods as described below (MCCC No.
2E00233). C. membranifaciens PYCC2727
T
, the type
yeast strain, was kindly supplied by Dr. Isabel
Spencer-Martins from Centro de Recursos Micro-
biologicos (CREM) Biotechnology Unit, Faculty of
Science and Technology, New University of Lisbon,
Portugal. The yeast strains were maintained in YPD
medium at 4 1C.
Riboavin production
One loop of the cells of the yeast strain was
transferred to 50.0 ml of YPD medium prepared
with distilled water in 250 ml asks and aerobically
cultivated at 25 1C for 24 h. At a point when
the culture reached a high cell density
(OD
600nm
20.0), 0.2 ml of the culture was trans-
ferred to 50.0 ml of the production medium that
contained 2.0% of different sugars (xylose, maltose,
galactose, sucrose, glucose, trehalose, rafnose
and cellobiose), 0.5% of (NH
4
)
2
SO
4
, 0.1% of KH
2
PO
4
,
0.05% of MgSO
4
7H
2
O, 0.01% of CaCl
2
2H
2
O, 0.01%
of NaCl and 0.2% of yeast extract and grown by
shaking at 170 rpm and 25 1C for 5 days. In order to
determine effects of different carbon sources on
riboavin production by the yeast strain, the
different carbon sources were added to the
production medium. The culture was centrifuged
at 14,000g and 4 1C for 10 min and the supernatant
obtained was used as the crude riboavin solution
for quantitative determination of riboavin.
Effects of iron in the production medium on
riboavin production in the yeast
In order to study the effects of iron in the
production medium on riboavin production by the
marine yeast used in this study, iron was removed
from the medium with 8-hydroxyquinoline as
described by Cowart et al. (1980). The iron-
supplemented medium contained 0.005% FeCl
3
.
Quantitative determination of riboavin
The amount of riboavin in the supernatant was
measured quantitatively at 440 nm by using a
spectrophotometer, and riboavin from Sigma
served as standard.
ARTICLE IN PRESS
L. Wang et al. 256
Partial purication of riboavin
Fifty milliliters of 0.1 mol/l hydrochloric acid was
added to the crude riboavin solution (50 ml) in a
250 ml conical ask. The solution was placed in a
water bath at 100 1C for 30 min. After being allowed
to cool, it was adjusted to pH 4.5 with 2.5 mol/l
sodium acetate (Ndaw et al., 2000) and ltered
through lter paper. The ltrate obtained was
applied to an MTcleanup and concentration column
(TEDA FUJI Chromatogram Developing Corp). The
riboavin absorbed on the column was eluted by
using pure methanol. The eluate was ltered
through a MILLEX
s
HV lter (0.45 mm). The ltrate
was used for high-performance liquid chromato-
graphy (HPLC) analysis of riboavin.
Chromatographic analysis
The riboavin in the ltrate was analyzed by
using a HPLC system (Waters, USA). The system
consisted of a pump Waters Delta 600, a detector
Waters 996 and a column YMC-pack ODS-(A),
250 4.6 mm, 5 mm. The elution was performed
by a methanol:water solution 40:60 and the ow
rate was 1.0 ml/min. Other conditions used were
those described by Arella et al. (1996) for the
determination of riboavin. Chromatographic
peaks were quantied using a Star Chromato-
graphic integrator. The riboavin standard was
purchased from Sigma.
DNA extraction, PCR and DNA sequencing
DNA extraction and PCR techniques for ampli-
cation of D1/D2 26S rDNA in the yeast were
performed according to the methods described by
Chi et al. (2007). The common primers for
amplication of the D1/D2 26S rDNA sequence in
the yeast were used, the forward primer NL-1:
5
0
-GCATATCAATAAGCGGAGGAAAAG and the reverse
primer NL-4: 5
0
-GGTCCGTGTTTCAAGACGG (Sugita
et al., 2003). The D1/D2 26S rDNA fragments
inserted on the vector (pMD-19T) were sequenced
by Shanghai Sangon Company.
Phylogenetic analysis of the yeast
The sequences obtained above were analyzed for
similarity by using Clustal X 1.83. For comparison
with currently available sequences, sequences
were retrieved with over 98% similarity belonging
to different genera from NCBI (http://
www.ncbi.nlm.nih.gov). The phylogenetic tree
was constructed by using PHYLIP software package
version 3.56 (Felsenstein, 1995). Distance matrices
were generated by the DNADIST program, based on
Kimuras two-parameter model (Kimura, 1980).
Neighbor-joining analysis of the data sets was
carried out with the program Neighbor of the
PHYLIP package.
Metabolic characterization of the yeast
The yeast strain was also identied by using
BIOLOG system TM (Biolog MicroStation with Micro-
log System, Release 4.20, Biolog, Hayward, CA,
USA), Biolog Universal Yeast Agar (Biolog) and
Biolog Yeast microplate (Biolog) according to the
procedures offered by the manufacturer (Praphai-
long et al., 1997). The routine identication of the
yeast was performed by using the methods as
described by Kurtzman and Fell (2000).
PCR-based cloning of partial GTP
cyclohydrolase II gene and 3,4-dihydroxy-2-
butanone-4-phosphate synthase gene
PCR was used for partial GTP cyclohydrolase
II and 3,4-dihydroxy-2-butanone-4-phosphate synthase
DNA amplication. Genomic DNA was prepared
as described above. The conserved motifs
were usually used to design degenerate primers
to clone these homologs. In this case, amino acid
sequences of GTP cyclohydrolase II and 3,4-dihy-
droxy-2-butanone-4-phosphate synthase from dif-
ferent species of eukaryotic microorganisms were
downloaded from GenBank (http://www.ncbi.
nlm.nih.gov/) and aligned. The degenerate sense
primers and antisense primers for amplication of
partial GTP cyclohydrolase II DNA were rib1_1_2f1:
CAACAGGGAACACTTGGCAAT and rib1_1_4r2:
CGTTCACCACAATCACATCGT, and the degenerate
sense primers and antisense primers for amplica-
tion of partial 3,4-dihydroxy-2-butanone-4-pho-
sphate synthase DNA were rib3_0_1f: GAACGTG-
AAAATGAAGGTGAT and rib3_3_3r:CGACACCGACCT-
CAGTGT. PCR was performed on a GeneAmp PCR
System 2400 made by PerkinElmer using a program
of 94 1C for 1 min, 52 1C for 1 min and 72 1C for 2 min
for 30 cycles, followed by extension for 8 min at
72 1C. PCR products were separated by agarose gel
electrophoresis and recovered by using UNIQ-col-
umn DNA gel recovery kits (BIOASIA, Shanghai). The
recovered PCR products were ligated into pMD19-T
and transformed into competent cells of Escher-
ichia coli DH5a. The transformants were selected
on LB plates with ampicillin. The plasmids in the
transformant cells were extracted by using the
methods as described by Sambrook et al. (1989).
ARTICLE IN PRESS
Isolation and characterization of C. membranifaciens subsp. avinogenie W14-3 257
The cloned DNA fragments inserted on the vector
were sequenced by Shanghai Sangon Company. The
amino acid sequences of the cloned DNA frag-
ments were deduced and protein sequences were
aligned using CLUSTAL X program (Thompson et al.,
1994).
Total RNA isolation and RT-PCR
Total RNA was isolated from strain W14-3 grown
in the production medium without and with iron
supplementation using RNAiso Reagent (TaKaRa,
Japan) according to the manufacturers instruc-
tions. Total RNA in the sample was determined by
spectrophotometry at 260 nm. The same amount of
total RNA (1.0 mg) was used to analyze mRNA levels
by RT-PCR. To synthesize the rst cDNA strand of
the two cloned genes mentioned above, reverse
transcriptase M-MLV (RNase H

) was used according


to the protocols provided by the manufacturer. PCR
amplication was performed using Taq DNA poly-
merase from Promega. Two sets of the specic
primers (rib1_rt_f1: TATTCCCTGGTGGATTACAAGC,
rib1_rt_r1: CACAATCACATCGGGCACT, rib3_rt_f1:
GCGGAATCAATCACCCAAG and rib3_rt_r1: GCGTA-
GTCGCAAGTTATCGTAT designed according to the
sequences of the partial genes cloned above)
were used for RT-PCR. One set of specic primers
for amplication of the 18S rRNA gene (18s_rt_f1:
TACAGTGAAACTGCGAATGGC and 18s_rt_r1: AGCA-
CAAGGTCATGCGATT) was designed according
to the sequence of the 18S rRNA gene (accession
number: EF362753) in this strain. The conditions
for RT-PCR amplication were as follows: initial
denaturation at 94 1C for 5 min, denaturation at
94 1C for 30 s, annealing temperature at 51 1C
for 30 s, extension at 72 1C for 1 min and nal
extension at 72 1C for 10 min. RT-PCR was run for 32
cycles. 18S rRNA gene was used as an internal
standard.
Results
Isolation of yeast strain W14-3 and yellow
substance production
A total of 100 yeast strains from seawater and
sediments in China Eastern Sea were obtained. We
found that the culture of the marine yeast strain
W14-3 turned into yellow color when it was
aerobically grown in the medium containing mal-
tose at 25 1C for 24 h (Figure 1B). The yeast strain
was isolated from China Eastern Sea. However, the
type yeast strain C. membranifaciens PYCC2727
T
did not produce such a yellow substance in the
same medium under the same conditions (Figure 1A).
In order to know if the yeast strain could produce
the yellow substance when it was grown on
other carbon sources, different carbon sources
(glucose, sucrose, fructose, maltose, trehalose,
xylose, D-rafnose, D-galactose) were added to
the production medium. The results in Table 1
indicate that the yeast strain produced more yellow
substance only in the medium containing xylose,
sucrose, galactose and maltose.
Analysis of the yellow substance by HPLC
The supernatant from the culture with a yellow
color was treated as described in Materials and
ARTICLE IN PRESS
Figure 1. The culture color occurred in the cultures of the marine yeast strain W14-3 (B) and type yeast strain
PYCC2727
T
(A) within 72 h. The sugar concentrations were 2.0%.
Table 1. The yellow substance production from differ-
ent carbon sources
A B C D E F G H
+ ++ ++++ ++ ++++ + +++ +++
A: D-trehalose; B: D-glucose; C: D-xylose; D: fructose; E: sucrose;
F: D-rafnose; G: D-galactose; H: maltose. The sugar concentra-
tions were 2.0%.
++++: the most colorful solution; +++: more colorful solution;
++: the colorful solution; +: trace yellow color in the solution.
L. Wang et al. 258
ARTICLE IN PRESS
200.00
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00
5.75
440.7
364.7
256.8
218.8
5.75
440.7
364.7
265.8
218.8
nm
200.00
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00
nm
0.003
0.002
0.001
0.000
-0.001
A
U
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Minutes
Minutes
0.012
0.010
0.008
0.006
0.004
0.002
0.000
A
U
2.00 4.00 6.00 8.00 10.00 12.00 14.00
Figure 2. HPLC spectra of riboavin standard (A) and the partially puried yellow substance (B).
Isolation and characterization of C. membranifaciens subsp. avinogenie W14-3 259
methods. The treated supernatant was applied to
the column and the yellow substance in the eluate
was analyzed by using HPLC. The HPLC spectra of
the partially puried yellow substance show that
there was only one main peak that was identical to
that of the riboavin standard (Figure 2A and B). It
can also be seen from the results in Figure 2 that
ultraviolet spectra of the riboavin standard
and the sample were the same (upper panel of
Figure 2A and B). These results demonstrate that
the yellow substance produced by the marine yeast
strain W14-3 was riboavin. However, further work,
using analytical techniques (such as NMR, MS, etc.)
is needed to obtain structural characterization that
will ultimately provide conclusive evidence that
the compound of interest is indeed a riboavin, or a
riboavin-like compound.
Cloning of the partial genes encoding GTP
cyclohydrolase II and 3,4-dihydroxy-2-
butanone-4-phosphate synthase
In order to conrm that the RIB1 gene encoding GTP
cyclohydrolase II and the RIB3 gene encoding 3,4-
dihydroxy-2-butanone-4-phosphate synthase exist in
strain W14-3 and type yeast strain C. membranifaciens
PYCC2727
T
used in this study, the conserved motifs
ARTICLE IN PRESS
Figure 3. Multiple alignment of protein sequences encoding yeast GTP cyclohydrolase II. The GTP cyclohydrolase II are
referenced by their databases. Multiple sequence alignment of proteins was carried out using the method of Clustral X
1.83 based on amino acid sequences of GTP cyclohydrolase II from Candida albicans-EAK96830 (1), Candida famata-
CAH17652 (2), Candida glabrata-XP_446157 (3), Debaryomyces hansenii-XP_456888 (4), Kluyveromyces lactis-
XP_452081 (5), Pichia guilliermondii-CAA88916 (6), Pichia stipitis-XP_001383311 (7), Saccharomyces cerevisiae-
NP_009520 (8) and amino acid sequence (9) deduced from the cloned partial DNA fragment of the yeast strain used in
this study.
L. Wang et al. 260
were used to design degenerate primers to clone the
partial genes encoding GTP cyclohydrolase II and 3,4-
dihydroxy-2-butanone-4-phosphate synthase in the
yeast strain W14-3 and C. membranifaciens
PYCC2727
T
, respectively. In this case, amino acid
sequences of GTP cyclohydrolase II and 3,4-dihy-
droxy-2-butanone-4-phosphate synthase from differ-
ent species of eukaryotic microorganisms were
downloaded from GenBank (http://www.ncbi.nlm.
nih.gov) and aligned. The PCR-generated fragments
obtained from genomic DNA of the yeast strain W14-3
were sequenced. The alignment and comparison of
the protein (GTP cyclohydrolase II and 3,4-dihydrox-
y-2-butanone-4-phosphate synthase) sequences de-
duced from the partial genes (accession numbers
were EU239888 and EU275163) with sequences in the
protein databases using BLAST program are shown in
Figures 3 and 4, respectively. The deduced proteins
showed very high identity with GTP cyclohydrolase II
and 3,4-dihydroxy-2-butanone-4-phosphate synthase
from other yeasts (Figures 3 and 4).
Routine identication of the marine yeast
strain W14-3
On yeast extract and malt (YM) medium the
colonies were white to chalky, dull, powdery, dry,
and wrinkled with a rough border (Figure 5A).
Single cells were oval, producing daughter cells
by single polar budding in liquid YM medium
(Figure 5B). Pseudomycelia occurred, but no sexual
reproduction was seen. It is very interesting to note
that the colonies of the marine yeast strain W14-3
on the plate containing maltose are white,
although it is common to all other microorganisms
that riboavin production is recognizable by the
yellow color of the colonies. We found that the
yeast strain produced riboavin only by vigorous
shaking (Figure 1B). The yeast strain could not
ferment maltose, galactose, lactose and rafnose,
but could ferment glucose and sucrose (data not
shown). Biolog analysis shows that it could assimilate
ARTICLE IN PRESS
Figure 4. Multiple alignments of protein sequences of yeast 3,4-dihydroxy-2-butanone-4-phosphate synthases. They
are referenced by their databases. Multiple sequence alignment of proteins was carried out using the method of Clustral
X 1.83 based on amino acid sequences of of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Saccharomyces
cerevisiae-AAB64927 (1), Pichia stipitis-XM_001386886 (2), Kluyveromyces lactis-XP_451875 (3), Saccharomyces
cerevisiae-Z21619 (4), Yarrowia lipolytica-XM_500663 (5), Candida glabrata-CAG60514 (6), Schizosaccharomyces
pombe-CAA18874 (7) and amino acid sequence (8) deduced from the cloned partial DNA fragment of the yeast strain
used in this study.
Figure 5. Photographs of colonies (A) and microphoto-
graph of vegetable cells (B) of the marine yeast strain
W14-3. Medium: YPD medium; incubation temperature:
25 1C; time: 2 days.
Isolation and characterization of C. membranifaciens subsp. avinogenie W14-3 261
glucose, galactose, L-sorbose, sucrose, maltose,
cellobiose, trehalose, melibiose, rafnose, inulin,
D-xylose, L-arabinose, D-arabinose and L-rhamnose,
but could not assimilate L-rhamnose (Table 2).
Based on the fermentation spectra and carbon
source assimilation spectra of the marine yeast and
those of the type strain (C. membranifaciens) listed
in The yeast: a taxonomic study, 4th revised and
enlarged edition (Kurtzman and Fell, 2000), we
found that the yeast strain W14-3 was closely
related to C. membranifaciens.
Phylogenetic analysis of partial sequences of
the D1/D2 26S rDNA sequence
According to Kurtzman and Fell (2000), tradi-
tional and routine identication methods, which
depend on phenotype, usually lead to uncertain
and inaccurate interpretations of species interac-
tion. Sequence analysis of phylogeny for microbial
taxonomy is a more accurate method for determin-
ing inter- and intra-specic relationships. There-
fore, a partial sequence of D1/D2 26S rDNA of the
yeast strain was determined and aligned by using
BLAST analysis (http://www.ncbi.nlm.nih.gov/
BLAST). A phylogenetic tree was constructed by
using PHYLIP software package version 3.56
(Felsenstein, 1995). Distance matrices were gener-
ated by the DNADIST program based on Kimuras
two-parameter model (Kimura, 1980). Neighbor-
joining analysis of the data sets was carried out
with the program Neighbor of the PHYLIP package.
Ricciocarpos natans was used as the out-group
during the construction of a consensus tree of the
ARTICLE IN PRESS
Table 2. Assimilation of carbon sources of strain W14-3 by Biolog analysis
Carbon sources Results Carbon sources Results
Acetic acid D-Galactose +
Formic acid D-Psicose +
Propionic acid L-Rhamnose
Succinic acid L-Sorbose +
Succinic acid mono-methyl ester a-Methyl-D-glucoside +
L-Aspartic acid b-Methyl-D-glucoside +
L-Glutamic acid Amygdalin
L-Proline w Arbutin +
D-Gluconic acid + Salicin +
Dextrin Maltitol
Inulin + D-Mannitol +
Fumaric acid w D-Sorbitol +
L-Malic acid w Adonitol +
Succinic acid mono-methyl ester Lactose +
Bromosuccinic acid Amidulin +
L-Glutamic acid + D-Arabitol +
g-Aminobutyric acid w Xylitol +
a-Ketoglutaric acid i-Erythritol +
2-Keto-gluconic acid + Glycerol +
D-Gluconic acid + Tween 80
Dextrin L-Arabinose +
D-Cellobiose + D-Arabinose +
Gentiobiose + D-Ribose
Maltose + D-Xylose +
Maltotriose + Succinic acid mono-methyl ester plus D-xylose w
D-Melezitose + N-acetyl-L-glutamic acid plus D-xylose
D-Melibiose + Quinic acid plus D-xylose
Palatinose + D-Glucuronic acid plus D-xylose
D-Rafnose + Dextrin plus D-xylose
Stachyose + a-D-Lactose plus D-xylose
Sucrose + D-Melibiose plus D-xylose +
D-Trehalose + D-Galactose plus D-xylose +
Turanose + m-Inositol plus D-xylose
N-Acetyl-D-glucosamine + 1,2-Propanediol plus D-xylose
D-Glucosamine W Acetoin plus D-xylose
a-D-glucose +
W: weak; +: positive; : negative.
L. Wang et al. 262
ARTICLE IN PRESS
Figure 6. Consensus tree of the isolate based on D1/D2 26S rDNAs obtained in this study and 23 previously published
sequences obtained from GenBank. The outgroup we used was Ricciocarpos natans. Numbers on tree branches indicate
the percentages of bootstrap samplings derived from 1000 samples that supported the internal branches by 50% or
higher. All the strains shown are type strains.
Isolation and characterization of C. membranifaciens subsp. avinogenie W14-3 263
isolate based on the D1/D2 26S rDNA sequence. The
search for the similarity between the D1/D2 26S
rDNA sequence of the isolate and that in the NCBI
database shows that many phylogenetically related
yeast species were similar to the marine yeast
strain obtained in this study. Phylogenetic relation-
ships of the D1/D2 26S rDNA sequence of the marine
yeast strain are shown in Figure 6 and its GenBank
accession number is EF362752. The topology of the
phylogram in Figure 6 conrms that 100% bootstrap
values were detected between the D1/D2 26S rDNA
sequence of strain W14-3 and that of C. membra-
nifaciens. Therefore, the yeast strain W14-3 was
closely related to C. membranifaciens.
Discussion
As shown in Figures 1 and 2 and Table 1, the
marine yeast strain W14-3 produced a large amount
of riboavin, whereas the type yeast strain
C. membranifaciens PYCC2727
T
did not produce
any riboavin under the same conditions. The
results (Figure 7) also show that the marine yeast
strain W14-3 also produced a large amount of
riboavin in the production medium prepared with
seawater, but Fe
3+
available in the medium
completely repressed riboavin production. Finally,
the yeast strain W14-3 was identied to be
C. membranifaciens subsp. avinogenie (Table 2,
Figure 6), meaning that it was different from the
type yeast strain C. membranifaciens due to its
production of riboavin and the presence of genes
responsible for riboavin biosynthesis (Figures 3
and 4). In addition, the yeast strain W14-3 grew
well in the medium containing 60% glucose, while
C. membranifaciens PYCC2727
T
grew poorly in the
same medium (data not shown).
The biosynthetic pathway of riboavin has been
studied in considerable detail in bacteria, fungi and
yeasts (Humbelin et al., 1999; Stahmann et al.,
2000; Karos et al., 2004). It has been well
documented that GTP cyclohydrolase II is encoded
by the RIB1 gene, while the 3,4-dihydroxy-2-
butanone-4-phosphate synthase is encoded by the
RIB3 gene (Humbelin et al., 1999; Stahmann et al.,
2000; Karos et al., 2004). The deduced amino acid
sequences from the partial genes encoding GTP
cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-
phosphate synthase in the marine yeast strain
W14-3 showed very high identity with those of
GTP cyclohydrolase II and 3,4-dihydroxy-2-buta-
none-4-phosphate synthase from different eucar-
yotic microorganisms (Figures 3 and 4), suggesting
that a riboavin synthesis pathway indeed existed
in the yeast.
Among the terrestrial microorganisms, A. gossy-
pii, a lamentous fungus, C. famata, a yeast, and
the genetically engineered B. subtilis have been
commercially used to produce riboavin by fer-
mentation (Stahmann et al., 2000; Wendland and
Walther, 2005). In addition, Sabry et al. (1989) used
C. guilliermondii Wickerham to produce riboavin
and Buzzini and Rossi (1998) used the immobilized
Candida tropicalis cells to produce riboavin.
Eremothecium ashbyii, a fungus was also found to
have the ability to produce a large amount of
riboavin (Kalingan and Liao, 2002). It has been
reported that P. guilliermondii is capable of
riboavin overproduction under iron deciency
(Fayura et al., 2007). Therefore, this is the rst
study to report that C. membranifaciens subsp.
avinogenie W14-3 isolated from the marine
environment can produce riboavin. Usually, iron
in the medium will repress riboavin production
by the terrestrial yeasts and fungi. The results in
Table 3 also show that added iron in the production
medium completely repressed riboavin production
by the marine yeast. However, removal of iron only
weakly affected riboavin production by the
marine yeast. Therefore, it is more suitable and
economical to apply the marine yeast strain to
riboavin production on a large scale than any
other riboavin producers. The results in Figure 8
ARTICLE IN PRESS
Table 3. Effect of iron in the production medium on riboavin production by the marine yeast
Media Medium treated with
8-hydroxyquinoline
Production medium
without any treatment
Medium supplemented
with 0.005% FeCl
3
Riboavin yield (mg/ml) 16.370.1 14.770.3 070.1
Figure 7. Riboavin production in the production media.
1: The production medium prepared with distilled water;
2: the production medium supplemented with iron; 3: the
production medium prepared with seawater.
L. Wang et al. 264
indicate that added iron in the medium repressed
expression of RIB1 and RIB3 genes in the yeast
cells. This means that iron also negatively affects
riboavin production by the marine yeast strain
W14-3 at the transcriptional level. In order to
enhance riboavin production, optimization of the
medium and cultivation conditions for riboavin
production by C. membranifaciens subsp. avino-
genie W14-3 is being undertaken in the laboratory.
Acknowledgments
This research was supported by the Hi-Tech
Research and Development Program of China
(863), and the grant number is 2006AA09Z403.
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