Construction of Flocculent Kluyveromyces marxianus Strains Suitable

for High-Temperature Ethanol Fermentation

Sanom NONKLANG,
1
Akihiko ANO,
2
Babiker M. A. ABDEL-BANAT,
1
Yuko SAITO,
2
Hisashi HOSHIDA,
1
and Rinji AKADA
1;y
1
Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine,
Tokiwadai, Ube 755-8611, Japan
2
Iwata Chemical Co., Ltd., Nakaizumi, Iwata 438-0078, Japan
Received December 3, 2008; Accepted February 14, 2009; Online Publication, May 7, 2009
[doi:10.1271/bbb.80853]
Flocculating yeasts are highly useful in fermentation
processes because these cells can be separated easily
from the fermentation mash. However, native yeasts are
usually non-occulating, including Kluyveromyces marx-
ianus, which exhibits a potent high-temperature ethanol
fermentation ability. We describe here the construction
of occulent K. marxianus strains via the introduction of
the FLO1, FLO5, FLO9, and FLO10 genes from
Saccharomyces cerevisiae. The S. cerevisiae FLO genes
were overexpressed by upstream insertion of the con-
stitutive TDH3 promoter, resulting in occulent S.
cerevisiae strains. These TDH3p-FLO sequences were
then amplied by PCR and introduced directly into a
K. marxianus strain. These K. marxianus strains showed
a occulation phenotype, indicating that the introduced
S. cerevisiae TDH3 promoter and all FLO genes were
functional in this strain. Moreover, a occulating
K. marxianus strain showed the same ethanol produc-
tion prole as that of its wild-type parent. The K. marx-
ianus occulating strains we generated should be useful
in the future development of cost-eective fed-batch and
continuous fermentation systems at high temperatures.
Key words: Kluyveromyces marxianus; yeast; occula-
tion; ethanol fermentation
Flocculation is an attractive property in industrial
yeasts because the cells can be easily separated from a
fermentation mash. The application of occulating
strains to industrial fermentation processes removes
the need for extensive and costly cell separation equip-
ment, and these yeasts are thus suitable for both fed-
batch and continuous fermentation.
14)
In addition,
occulating yeast cells are useful in immobilized cell
systems with no inert support materials.
5)
However,
although occulation mechanisms and the introduc-
tion of this property to non-occulent yeast strains have
been extensively studied in S. cerevisiae, few studies of
this nature have been undertaken in other yeast
species.
6,7)
The mechanism of yeast occulation involves the
interaction of lectin-like proteins, called occulins, with
receptors on a neighboring cell wall, resulting in the
formation of aggregates.
811)
Environmental factors such
as pH, calcium, and organic stress also aect occu-
lation.
9,1214)
The ve FLO genes, FLO1, FLO5, FLO9,
FLO10, and FLO11, have been described in S. cerevi-
siae thus far.
1519)
The FLO1, 5, 9, and 10 genes confer
cell-cell adhesion (occulation) ability, whereas FLO11
is responsible for substrate adhesion.
20)
FLO1, FLO5,
and FLO9 are highly similar to each other, but FLO10
has lower similarity. The mechanisms of occulation in
other yeasts appear to be lectin-based but of diering
specicities. For instance, galactose and its derivatives
have been found to inhibit occulation of K. bulgaricus
and K. lactis, suggesting that this interaction involves a
galactose-specic lectin.
21,22)
The advantages of using occulating yeast cells in
fermentation processes have led to many studies on the
construction of occulent strains from non-occulating
S. cerevisiae. The introduction of the FLO1 gene into
non-occulent brewery yeast strains has been found
to confer a occulation phenotype.
23)
Similarly, there
have been many attempts to express FLO genes under
the regulation of heterologous promoters in non-
occulent S. cerevisiae strains, resulting in the gener-
ation of occulating strains,
1,24,25)
but producing oc-
culation strains in other yeast species by introducing
FLO genes of S. cerevisiae has not been previously
reported.
The thermotolerant yeast K. marxianus has received
considerable attention because of its ability to grow and
initiate fermentation at high temperatures, and also
because of its native utilization of xylose, arabinose, and
cellobiose, which are not fermented by S. cerevi-
siae.
26,27)
In particular, the K. marxianus DMKU3-
1042 strain, isolated in Thailand, has shown the most
potent high-temperature growth among 17 strains that
were examined, and displays comparable ethanol fer-
mentation properties to a S. cerevisiae strain used in
Brazil. Moreover, even at 45

C, a highly restrictive
temperature for S. cerevisiae fermentation, the K. marx-
ianus strain produce ethanol.
27)
In addition, the K. marx-
ianus DMKU3-1042 strain has a novel transformation
property that is characterized by random chromosomal
integration of linear DNA.
27)
Conferring occulation
properties on this yeast is thus of great potential utility in
fuel ethanol fermentation. However, the construction of
occulating strains in novel species such as K. marx-
ianus is regarded as problematical due to an absence of
y
To whom correspondence should be addressed. Fax: +81-836-85-9201; E-mail: rinji@yamaguchi-u.ac.jp
Biosci. Biotechnol. Biochem., 73 (5), 10901095, 2009
information on the mechanisms and genes responsible
for occulation in this yeast. In this study, therefore, we
attempted to express S. cerevisiae (Sc) FLO genes in
non-occulent K. marxianus. Surprisingly, we found
that the expression of ScFLO1, 5, 9, and 10 conferred
the occulation phenotype in this strain. To our knowl-
edge, this is the rst study to demonstrate this.
Signicantly, the ethanol fermentation ability of the
K. marxianus ScFLO9 strain was found to be compara-
ble to its non-occulent wild-type parent at 40

C,
indicating its potential application in a more cost-
eective fuel ethanol fermentation system.
Materials and Methods
Yeast strains and growth conditions. The yeast strains used in this
study are listed in Table 1. The S. cerevisiae strains were isogenic to
BY4700,
28)
and the K. marxianus strains were isogenic to DMKU3-
1042.
27,29)
Yeast cells were grown in YPD medium (1% yeast extract,
2% peptone, 2% glucose) and uracil dropout medium (2% glucose,
0.5% ammonium sulfate, 0.17% yeast nitrogen base, and appropriate
nutrients without uracil) were used in transformation selection. YPD10
medium (1% yeast extract, 2% peptone, 10% glucose) was used in
laboratory-scale ethanol fermentation. Molasses-based medium, pre-
pared by mixing 628.8 g of sugar-cane molasses with 2.87 liters of
deionized water and 100 ml of ammonium sulfate buer containing
3.0 g of (NH
4
)
2
SO
4
and 6.0 g of (NH
4
)
2
HPO
4
, pH 6.0, was used in
fermentation in a 5-liter bioreactor. All media were sterilized by
autoclaving at 121

C for 20 min.
PCR. Primers of 40 bases were designed that were specic to
sequences upstream and downstream from the start codons of the
S. cerevisiae FLO genes in order to insert the TDH3 promoter into the
native promoter regions of these genes (Table 2). The URA3-TDH3
promoter cassette was amplied from the pST106 plasmid
30)
using the
following primer pairs: FLO1-401/FLO1-402, FLO5-401/FLO5-402,
FLO9-401/FLO9-402, and FLO10-401/FLO10-402. The PCR reac-
tion was initiated at 94

C for 1 min, followed by 30 cycles of 94

C for
20 s, 55

C for 30 s, and 68

C for 3 min. The reaction mixture (10 ml)

contained 1 KOD plus buer, 0.2 mM of each dNTP, 2 mM MgSO
4
,
0.2 mM of each primer, and 1 U of KOD Plus DNA polymerase
(Toyobo, Osaka, Japan).
To conrm the correct insertion of URA3-TDH3p, colony PCR was
performed
31)
using the following primer pairs: FLO1-517/FLO1.5-
415c, FLO5-413/FLO1.5-415c, FLO9-362/FLO9-455c, and FLO10-
311/FLO10-284c. The thermal setting for the colony PCR reactions
was as follows: initial denaturation for 1 min at 94

C, followed by 30
cycles of 94

C for 20 s, 60

C for 2 s, and 74

C for 4 min. The reaction

mixture (10 ml) contained 1 KOD Dash buer, 0.2 mM of each dNTP,
0.4 mM of each primer, 1 U of KOD Dash DNA polymerase (Toyobo,
Osaka, Japan), and 5 ml of colony DNA template.
31)
The URA3-TDH3p-FLO fragments were amplied from genomic
DNA extracted from occulating S. cerevisiae strains FLO1
(RAK3977), FLO5 (RAK3979), FLO9 (RAK3981), and FLO10
(RAK3983) using primer pairs FLO1-401/FLO1+5037c, FLO5-401/
FLO5+3759c, FLO9-401/FLO9+4454c, and FLO10-401/FLO10+
3980c respectively. The KOD Plus reaction was carried out as
described above with a 5-min extension time.
Transformation. S. cerevisiae transformations were performed
as described previously.
32)
For K. marxianus transformation, the
RAK3605 strain was cultured in 25 ml of YPD in a 250-ml ask with
shaking at 150 rpm for 18 h at 28

C. The cells were then collected by

centrifugation, washed once with 500 ml of TF buer (40% PEG 3350,
0.1 M dithiothreitol, and 0.2 M lithium acetate), and suspended again in
500 ml of TF buer. The cell suspension (100 ml) was next transferred
into a new 1.5-ml tube and mixed with 5 ml of PCR-amplied DNA
fragments. After incubation at 47

C for 15 min, cell suspensions were

spread on uracil dropout plates and incubated at 28

C for 23 d.
Flocculation test. Yeast cells were grown in test tubes containing
YPD with shaking. The culture tube was vortexed vigorously for 1 min
and allowed to settle. Cell sedimentation was then observed.
Ethanol fermentation. The K. marxianus wild-type strain and the
constructed occulating K. marxianus strains were grown in test tubes
containing 5 ml of YPD10 medium and incubated at 30

C or 40

C for
12 h with shaking at 150 rpm. For larger-scale ethanol fermentation,
K. marxianus wild-type and the FLO9 strain (RAK4301) were cultured
in 40 ml of YPD10 at 40

C. For fermentation in a 5-liter bioreactor,

K. marxianus wild-type or FLO9 was pre-cultured in 30 ml of YPD
at 28

C for 24 h with shaking, and the cells were then inoculated

into a 5-liter bioreactor containing 3 liters of molasses-based medium.
The bioreactor was aerated at a ow rate of 0.5 vvm and stirred at
150 rpm. The temperature was controlled at 40

C throughout
fermentation.
Cell-free supernatants were collected by centrifugation and used in
the determination of ethanol and sugar concentrations. Glucose,
sucrose, and ethanol concentrations were determined by Biosensor
BF-5 (Oji Scientic Instruments, Hyogo, Japan) according to the
manufacturers protocol. The specic ethanol production rate was
calculated as described previously.
33)
Table 1. Yeast Strains Used in This Study
Strain Genotype
S. cerevisiae
BY4700 MATa ura30
RAK3977 MATa ura30 URA3-TDH3p-FLO1
RAK3979 MATa ura30 URA3-TDH3p-FLO5
RAK3981 MATa ura30 URA3-TDH3p-FLO9
RAK3983 MATa ura30 URA3-TDH3p-FLO10
K. marxianus
DMKU3-1042 Wild-type
RAK3605 ura3
RAK4299 ura3 Sc[URA3-TDH3p-FLO1]
RAK4300 ura3 Sc[URA3-TDH3p-FLO5]
RAK4301 ura3 Sc[URA3-TDH3p-FLO9]
RAK4302 ura3 Sc[URA3-TDH3p-FLO10]
Table 2. Primers Used in This Study
Name Sequence (5
0
3
0
)
a
FLO1-517 GAATTCTAGCCTTCCTCTGCTC
FLO1.5-415c CTAGGGTTACGTTTGTTGGGGT
FLO1+5037c AAGTTGGCGATGGTTCATTAATTGC
FLO5-413 GGCACCCTCGAGAATTACACTT
FLO5+3759c GTACTGCGTGTGGCATGTAAGCAGC
FLO9-362 GTACATCACACACGACCACAGA
FLO9-455c TAAGAACCCGTCTGTGGTGGTA
FLO9+4454c ACTAGATCTTACGTTAGTACTGCTG
FLO10-311 GTTGTTTGGTATGTATCCGCCG
FLO10-284c GCACAAGTATCTGATGCGCCAT
FLO10+3980c CGCCGGGCAGTAGTAACTATTGTTA
FLO1-401 tatttttaattcttgtcaccagtaaacagaacatccaaaa-
GGCGCGCCCG
FLO1-402 taaagactgccaaaaacatatagcgatgaggcattgtcat-
TTTATGTGAT
FLO5-401 caaatgattttctttaaattgattagcaccactaaaaaaa-
GGCGCGCCCG
FLO5-402 ccaagattaccaaaaatatgcagtggtgtgcaattgtcat-
TTTATGTGAT
FLO9-401 gcaatttaaaaagaacaattgtacaataaaagccccaaaa-
GGCGCGCCCG
FLO9-402 tgacgatggctagtagtaaacaataatgtgccagagacat-
TTTATGTGAT
FLO10-401 tttgttttagggtgcttaatcaaagaacaacaaataaaaa-
GGCGCGCCCG
FLO10-402 ataggccggtcaaaaatatatatcgagcagccacaggcat-
TTTATGTGAT
a
Small letters indicate homologous sequences used in gene targeting.
Construction of Flocculent K. marxianus 1091
Results
Construction of occulating S. cerevisiae strains
A non-occulent S. cerevisiae strain, BY4700, was
used as host in FLO gene manipulation to examine the
eects of FLO gene overexpression. To induce con-
stitutive overexpression of the FLO genes in this strain,
a URA3-TDH3 promoter cassette was inserted upstream
of the FLO1, FLO5, FLO9, and FLO10 open reading
frames (Fig. 1A). The correct insertions were conrmed
by the increase in the band size (2.0 kb), corresponding
to the length of the URA3-TDH3p cassette (Fig. 1B). All
the correct transformants showed a occulation pheno-
type (Fig. 1C), indicating that the BY4700 strain had
acquired this property via constitutive overexpression of
the formerly silent FLO1, 5, 9, and 10 genes. In addition,
the occulation phenotype of all of the transformants
was strong, as the transformed cells always remained at
the bottom of test tubes even with shaking during
cultivation and after vortexing. No signicant pheno-
typical dierences were observed among the S. cerevi-
siae FLO strains.
Introduction of TDH3p-FLO genes from S. cerevisiae
into K. marxianus
As described earlier, the K. marxianus DMKU3-1042
strain has an unusual linear DNA integrating ability.
PCR-amplied DNA can thus be inserted into the
chromosomes of this strain randomly and eciently.
27)
Taking advantage of this property, we attempted to
transform URA3-TDH3p-FLO fragments directly into
K. marxianus. These fragments were amplied by
PCR from S. cerevisiae occulent strains (Fig. 2A)
and transformed into K. marxianus ura3 mutant
(RAK3605).
27)
The resulting transformants were sub-
sequently cultured in 5 ml of YPD overnight. All of the
cultures showed occulation during cultivation (Fig. 2B,
before vortexing), indicating that the S. cerevisiae
FLO1, 5, 9 and 10 genes and the TDH3 promoter were
functional in K. marxianus.
To determine the degree of occulation, overnight
cultures were vigorously vortexed for 1 min, and the cell
TDH3p URA3
FLO genes
Chromosome
TDH3p URA3 FLO genes
B
e
f
o
r
e
v
o
r
t
e
x
i
n
g
A
f
t
e
r

v
o
r
t
e
x
i
n
g
30 s
60 s
1 h
S. cerevisiae-expressing
WT TF WT TF WT TF WT TF
FLO1 FLO5 FLO9 FLO10
FLO1 WT FLO5 FLO9 FLO10
3.0
2.5
2.0
1.5
1.0
kb
A
B
C
Fig. 1. Construction of Flocculating S. cerevisiae.
A, The URA3-TDH3p fragment was amplied from the pST106
plasmid using primers containing targeting sequences and trans-
formed into an S. cerevisiae non-occulent strain. B, Colony PCR
conrmed correct insertion of the fragments. C, Flocculation
phenotype of S. cerevisiae FLO transformants. After culturing, cells
were observed before and after vortexing.
3
8
6
URA3-TDH3p-FLO
5
4
kb
10
FLO1 FLO10 FLO9 FLO5
30 s
60 s
1 h
B
e
f
o
r
e
v
o
r
t
e
x
i
n
g
A
f
t
e
r

v
o
r
t
e
x
i
n
g
WT
A
B
K. marxianus-expressing
FLO1 FLO10 FLO9 FLO5
Fig. 2. Construction of Flocculating K. marxianus by Introduction of
S. cerevisiae FLO Genes.
A, Fragments containing the URA3-TDH3p-FLO genes were
amplied from S. cerevisiae FLO strains and directly transformed
into K. marxianus. B, K. marxianus transformants were observed for
occulation phenotype before and after vortexing.
1092 S. NONKLANG et al.
sedimentation properties were subsequently analyzed.
The cells of K. marxianus containing FLO1, FLO5,
and FLO9 suddenly sedimented after vortexing,
whereas the cells of the FLO10 strain did not settle
even after 1 h (Fig. 2B, after vortexing), indicating that
this gene confers only weak occulation properties in
K. marxianus.
Ethanol production of K. marxianus occulent strains
To examine the ethanol fermentation performance
of the K. marxianus occulation strains, ethanol pro-
duction was compared among these transformants and
wild-type strains (Fig. 3A). When fermentation was
performed in YPD10 medium at 30

C for 12 h, ethanol
concentrations reached 5% in all cases except for the
FLO1 strain (Fig. 3A). The cells of the FLO1 strain
sedimented very strongly at the bottom of the tube,
suggesting that this might aect ethanol fermentation at
30

C. When fermentation was performed at 40

C, the
ethanol concentrations reached approximately 4.5%, but
all four strains, including FLO1, showed similar ethanol
levels to the wild type. However, the cells of K.
marxianus FLO5 strain lost their occulation ability at
40

C, indicating that FLO5 is temperature-sensitive.

In view of the weaker ethanol production at 30

C of
the K. marxianus FLO1 strain (Fig. 3A, 30

C), the loss

of occulation at 40

C in the FLO5 strain (Fig. 3A,

40

C), and the easy dissociation of aggregates of the

FLO10 strain (Fig. 2B), K. marxianus FLO9 appeared
to be the strongest performer among the FLO strains
examined. A time-course fermentation experiment was
next performed at 40

C for this strain (Fig. 3B). The

glucose consumption and ethanol production proles of
K. marxianus FLO9 were found to be comparable with
those of the wild-type parent. These results indicate that
glucose was completely consumed within 10 h and that
the maximum ethanol concentration reached 4.8% at 8 h
(Fig. 3B).
High-temperature ethanol fermentation from mo-
lasses by the K. marxianus FLO9 strain
As described above, the K. marxianus FLO9 strain
showed comparable high-temperature ethanol fermenta-
tion ability when compared with wild-type K. marx-
ianus. In industrial ethanol plants, sugar-cane molasses
is commonly used as the substrate, and hence we
performed ethanol fermentation by K. marxianus wild-
type and FLO9 in a 5-liter bioreactor using molasses-
based medium (Fig. 4). Both strains showed rapid
increases in dry cell weight, which peaked after 14 h,
along with rapid decreases in glucose levels. The ethanol
concentrations increased similarly, and then slightly
decreased until the end of fermentation (Fig. 4, ethanol).
The specic ethanol production rate showed no dier-
ence between the wild-type and FLO9 strains. These
results indicate that the ethanol fermentation perform-
ance of the K. marxianus FLO9 strain is comparable to
the wild-type DMKU3-1042 strain.
Discussion
The construction of occulent strains from non-
occulent strains has been extensively studied in
S. cerevisiae using various genetic tools. In their semi-
nal study, Barney et al.
34)
introduced the FLO1 gene into
spheroplasted S. cerevisiae to confer the occulation
phenotype. The protoplast fusion technique has also
been used in the construction of hybrids between non-
occulent and occulent strains.
3537)
Transformation of
plasmids containing FLO1 under the regulation of a
constitutive ADH1 promoter or a HSP30 promoter was
shown to confer a occulation phenotype on non-
occulent S. cerevisiae strains.
1,23,25)
Moreover, the
FLO5 gene was also used in the construction of
occulating strains in laboratory S. cerevisiae by trans-
forming multicopy plasmids containing an ADH2-FLO5
fusion gene.
11)
However, there have been no previous
reports on the construction of occulent S. cerevisiae
strains using the FLO9 or the FLO10 gene. In this study,
we conferred occulation by engineering constitutive
overexpression of the FLO1, FLO5, FLO9, and FLO10
genes, and found that all were functional but silent in the
S. cerevisiae BY4700 laboratory strain. There were no
signicant dierences in the occulation phenotypes
induced in S. cerevisiae via these dierent genes.
6
5
4
3
2
1
0
WT
30C
A
B
E
t
h
a
n
o
l

%

(
w
/
v
)
FLO1
FLO5
FLO9
WT
FLO10
10
8
6
4
2
0
5
4
3
2
1
0
12 10 8 6 4 2 0
Time (h)
G
l
u
c
o
s
e

%

(
w
/
v
)
E
t
h
a
n
o
l

%

(
w
/
v
)
40C
FLO
1
FLO
5
FLO
9
FLO
10
WT FLO
1
FLO
5
FLO
9
FLO
10
Fig. 3. Ethanol Production by K. marxianus FLO Strains.
A, Ethanol fermentation was performed in YPD10 medium at
30

C or 40

C for 12 h. Final ethanol concentrations were examined

from three independent cultures. The occulation phenotype was
observed without vortexing. B, Glucose consumption (circles) and
ethanol production (triangles) were analyzed in the K. marxianus
wild-type (closed symbols) and FLO9 (open symbols) strains. Cells
were incubated in 40 ml of YPD10 medium at 40

C.
Construction of Flocculent K. marxianus 1093
The thermotolerant K. marxianus yeast strain is an
attractive organism for fuel ethanol production, making
a occulation phenotype highly desirable for this strain
for use in commercial fermentation systems. However,
all of the K. marxianus strains we observed, including
the 17 strains obtained from several yeast culture
collections and many strains isolated in Thailand, were
found to be non-occulent. We assumed therefore that
occulation is not a natural trait of K. marxianus. Due
to the lack of genomic sequence information for
K. marxianus, the presence of FLO homologous genes
cannot be determined at present. Fortunately, however,
we found that a occulation phenotype could be
conferred on the K. marxianus DMKU3-1042 strain by
direct introduction of S. cerevisiae TDH3p-FLO genes.
This indicates that each of the introduced S. cerevisiae
FLO genes and also the TDH3 promoter were functional
in K. marxianus. The degree and stability of occulation
was variable among the K. marxianus FLO strains,
which was not observed in the S. cerevisiae FLO strains.
The occulating K. marxianus FLO1 and FLO9
strains showed strong, stable aggregations even at
40

C, but K. marxianus FLO5 showed temperature-

sensitive occulation. This FLO5 phenotype is thus
attractive, because occulation can be controlled by
temperature. The weak occulation observed for the
K. marxianus FLO10 strain suggests that FLO10 ex-
pression is weak in K. marxianus, or that the occulin
interaction in K. marxianus lacks some factors that are
present in S. cerevisiae, for which the FLO10 over-
expressing strain did not show the same weak pheno-
type. These results for the expression of Flo proteins in a
heterologous host indicate that functional dierences
exist for these proteins.
The ethanol fermentation performances of all the
K. marxianus occulent strains, except for the FLO1
strain, were comparable to that of the wild type at 30

C.
When the temperature was increased, however, the
K. marxianus FLO1 strain produced ethanol as produc-
tively as the other strains tested. This was probably due
to reduced occulation at the higher temperature, which
has also been reported for S. cerevisiae.
38)
Tightly
packed FLO1 aggregates might limit nutrient transfer
from the surface to the interior of the cell.
Flocculating S. cerevisiae strains capable of growing
at high temperatures have been constructed by protoplast
fusion between the occulating S. cerevisiae IR-2 strain
and the thermotolerant S. cerevisiae EP-1 strain.
35)
Moreover, thermotolerant, osmotolerant, occulating
alcohol producing yeast strains have been isolated
from soil samples,
39)
but the temperatures tolerated by
S. cerevisiae were not at the same levels as K. marx-
ianus, for which the DMKU3-1042 strain was able to
grow even at 49

C.
27)
In summary, this is the rst report describing the
construction of occulent K. marxianus strains by
introduction of the S. cerevisiae FLO1, 5, 9, and 10
genes. The resulting K. marxianus FLO9 strain showed
stable occulation properties and ecient ethanol
fermentation ability at 40

C. Hence, this strain has

potential for practical use in fuel ethanol fermentation
at high temperatures. Various occulation behaviors
D
r
y

c
e
l
l

w
e
i
g
h
t

(
g
/
l
)
S
u
c
r
o
s
e

a
n
d

g
l
u
c
o
s
e

c
o
n
c
.

(
g
/
l
)
0
20
40
60
E
t
h
a
n
o
l

(
g
/
l
)
0 6 12 18 24
10
1
S
p
e
c
i
f
i
c

e
t
h
a
n
o
l

p
r
o
d
u
c
t
i
o
n

r
a
t
e
(
g
-
e
t
h
a
n
o
l
/
g
-
c
e
l
l
/
h
)
1.6
1.2
0.8
0.4
0.0
0 6 12 18 24
Time (h)
Time (h)
80
60
40
20
0
Fig. 4. High-Temperature Ethanol Fermentation by K. marxianus Strains Using Molasses as the Substrate in a 5-Liter Bioreactor.
K. marxianus wild-type (open symbols and dotted line) and FLO9 (closed symbols and solid line) strains were cultured in 3 liters of molasses-
based medium at 40

C. Dry cell weights, sucrose (square) and glucose (circle) concentrations, ethanol concentrations, and specic ethanol
production rates were analyzed.
1094 S. NONKLANG et al.
caused by dierent S. cerevisiae FLO genes should also
be useful in the control of fermentation processes.
Investigation of potential applications of these K. marx-
ianus occulent strains in fuel ethanol production is now
in progress.
Acknowledgments
We thank Mrs. Yukie Misumi for technical assistance
and Dr. Kazunobu Matsushita for helpful discussion. We
also acknowledge the technical expertise of the DNA
Core Facility of the Center for Gene Research of
Yamaguchi University. This work was supported in part
by a Ph.D. fellowship from the Scientic Cooperation
Program between the Japan Society for the Promotion of
Science (JSPS) and the National Research Council of
Thailand (NRCT), by the New Energy and Industrial
Technology Development Organization (NEDO), and by
the Program for the Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN) of
Japan.
References
1) Ishida-Fujii K, Goto S, Sugiyama H, Takagi Y, Saiki T, and
Takagi M, J. Gen. Appl. Microbiol., 44, 347353 (1998).
2) Wang FZ, Shen W, Rao ZM, Fang HY, Zhan XB, and Zhuge J,
Biotechnol. Lett., 30, 97102 (2008).
3) Domingues L, Vincente A, Lima N, and Teixeira JA, Bio-
technol. Bioprocess Eng., 5, 288305 (2000).
4) Verstrepen KJ, Derdelinckx G, Verachtert H, and Delvaux FR,
Appl. Microbiol. Biotechnol., 61, 197205 (2003).
5) Andriette SR, Steckelberg C, and Andrietta Mda G, Bioresour.
Technol., 99, 30023008 (2008).
6) Almeida C, Queiros O, Wheals A, Teixeira J, and Moradas-
Ferreira P, J. Microbiol. Methods, 55, 433440 (2003).
7) Bellal M, Boudrant J, Elfoul L, and Bonaly R, Proc. Biochem.,
30, 641648 (1995).
8) Miki BL, Poon NH, James AP, and Seligy VL, J. Bacteriol.,
150, 878889 (1982).
9) Jin YL and Speers RA, Food Res. Int., 31, 421440 (1998).
10) Claro FB, Rijsbrack K, and Soares EV, J. Appl. Microbiol., 102,
693700 (2007).
11) Cunha AF, Missawa SK, Gomes LH, Reis SF, and Pereira GA,
FEMS Yeast Res., 6, 280287 (2006).
12) Soares EV and Seynaeve J, Biotechnol. Lett., 22, 18271832
(2000).
13) Soares EV, Teixeira JA, and Mota M, Can. J. Microbiol., 40,
851857 (1994).
14) Jin YL and Speers RA, J. Am. Soc., Brew. Chem., 58, 108116
(2000).
15) Teunissen AW, Holub E, van der Hucht J, van den Berg JA, and
Steensma HY, Yeast, 9, 423427 (1993).
16) Watari J, Takata Y, Ogawa M, Sahara H, Koshino S, Onnela
ML, Airaksinen U, Jaatinen R, Penttila M, and Keranen S,
Yeast, 10, 211225 (1994).
17) Bidard F, Blondin B, Dequin S, Vezinhet F, and Barre P, Curr.
Genet., 25, 196201 (1994).
18) Teunissen AW and Steensma HY, Yeast, 11, 10011013 (1995).
19) Lo WS and Dranginis AM, J. Bacteriol., 178, 71447151
(1996).
20) Verstrepen KJ and Klis FM, Mol. Microbiol., 60, 515 (2006).
21) Al-Mahmood S, Colin S, and Bonaly R, J. Biol. Chem., 266,
2088220887 (1991).
22) El-Behhari M, Ekome JN, Coulon J, Pucci B, and Bonaly R,
Appl. Microbiol. Biotechnol., 49, 1623 (1998).
23) Watari J, Nomura M, Sahara H, Koshino S, and Keranen S,
J. Inst. Brew., 100, 7377 (1994).
24) Remize F, Schorr-Galindo S, Guiraud JP, Dequin S, and
Blondin B, Biotechnol. Lett., 20, 313318 (1998).
25) Verstrepen KJ, Derdelinckx G, and Delvaux FR, J. Am. Soc.,
Brew. Chem., 59, 6976 (2001).
26) Fonseca GG, Heinzle E, Wittmann C, and Gombert AK, Appl.
Microbiol. Biotechnol., 79, 339354 (2008).
27) Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N,
Hoshida H, Limtong S, Yamada M, and Akada R, Appl.
Environ. Microbiol., 74, 75147521 (2008).
28) Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P,
and Boeke JD, Yeast, 14, 115132 (1998).
29) Limtong S, Sringiew C, and Yongmanitchai W, Bioresour.
Technol., 98, 33673374 (2007).
30) Cha-aim K, Fukunaga T, Hoshida H, and Akada R, Gene, 434,
4349 (2009).
31) Akada R, Murakane T, and Nishizawa Y, Biotechniques, 28,
668674 (2000).
32) Akada R, Kitagawa T, Kaneko S, Toyonaga D, Ito S, Kakihara
Y, Hoshida H, Morimura S, Kondo A, and Kida K, Yeast, 23,
399405 (2006).
33) Ano A, Funahashi H, Nakao K, and Nishizawa Y, J. Biosci.
Bioeng., 88, 5760 (1999).
34) Barney MC, Jansen GP, and Helber GR, J. Am. Soc., Brew.
Chem., 38, 7174 (1980).
35) Kida K, Morimura S, and Sonoda Y, J. Ferment. Bioeng., 74,
169173 (1992).
36) Lima N, Moreira C, Teixeira JA, and Mota M, Microbios, 81,
187197 (1995).
37) Watari J, Kubo M, Nishikawa N, and Kamimura M, Agric. Biol.
Chem., 54, 16771681 (1990).
38) Lindquist W, J. Inst. Brew., 59, 5961 (1953).
39) Kiran Sree N, Sridhar M, Suresh K, Banat IM, and Venkateswar
Rao L, Bioresour. Technol., 72, 4346 (2000).
Construction of Flocculent K. marxianus 1095