Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans

Article abstract of DOI:10.1271/bbb.100068

Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant ΔadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. ΔadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting ΔadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on ΔadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

Full text of DOI:10.1271/bbb.100068

Biosci. Biotechnol. Biochem., 74 (7), 1391–1395, 2010
Disruption of the Membrane-Bound Alcohol Dehydrogenase-Encoding Gene
Improved Glycerol Use and Dihydroxyacetone Productivity
in Gluconobacter oxydans
1
;y
1
1
1
Hiroshi HABE, Tokuma FUKUOKA, Tomotake MORITA, Dai KITAMOTO,
2
2
1
Toshiharu YAKUSHI, Kazunobu MATSUSHITA, and Keiji SAKAKI
1
Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science
and Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
2
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
677-1 Yoshida, Yamaguchi, Yamaguchi 753-8515, Japan
1
Received January 28, 2010; Accepted April 5, 2010; Online Publication, July 7, 2010
[doi:10.1271/bbb.100068]
Dihydroxyacetone (DHA) production from glycerol
The acetic acid bacterium Gluconobacter oxydans is
currently used for DHA production, and its membrane-
bound, pyrroloquinoline quinone (PQQ)-dependent
glycerol dehydrogenase (GLDH) involves the oxidative
reaction. Without NADH, GLDH reduces membranous
ubiquinone, which is re-oxidized by a terminal oxidase
using oxygen. Because its reactive center is oriented to
the periplasmic space, DHA is directly released into
and accumulated in the culture medium. Considering
that no energy-consuming substrate transport into the
cell or products out of the cell are required, the use of
GLDH from G. oxydans appears to be an efficient
method for DHA production.
by Gluconobacter oxydans is an industrial form of
fermentation, but some problems exist related to
microbial DHA production. For example, glycerol
inhibits DHA production and affects its biological
activity. G. oxydans produces both DHA and glyceric
acid (GA) from glycerol simultaneously, and mem-
brane-bound glycerol dehydrogenase and membrane-
bound alcohol dehydrogenases are involved in the two
reactions, respectively. We discovered that the G.
oxydans mutant ÁadhA, in which the membrane-bound
alcohol dehydrogenase-encoding gene (adhA) was dis-
rupted, significantly improved its ability to grow in a
higher concentration of glycerol and to produce DHA
compared to a wild-type strain. ÁadhA grew on 220 g/l
of initial glycerol and produced 125 g/l of DHA during
a 3-d incubation, whereas the wild-type did not.
Resting ÁadhA cells converted 230 g/l of glycerol
aqueous solution to 139.7 g/l of DHA during a 3-d
incubation. The inhibitory effect of glycerate sodium
salt on ÁadhA was investigated. An increase in the
glycerate concentration at the beginning of growth
resulted in decreases in both growth and DHA
production.
4
)
However, problems exist concerning microbial DHA
production. Not only glycerol (feedstock) but also
DHA (product) inhibits G. oxydans growth and DHA
production. Claret et al. (1992) demonstrated that
higher initial concentrations of glycerol (31 to 129 g/l)
resulted in an inhibitory effect on cell growth and DHA
5
)
production. Also, the presence of higher concentra-
tions of DHA (0–100 g/l) inhibit the oxidative fermen-
6
,7)
tation process.
Therefore, further improvements in
microbial DHA production yield and efficiency should
be achieved.
From glycerol, G. oxydans simultaneously produces
DHA by GLDH and glyceraldehyde by membrane-
bound alcohol dehydrogenase (mADH), and the latter is
Key words: glycerol use; dihydroxyacetone; glyceric
acid; Gluconobacter oxydans; membrane-
bound alcohol dehydrogenase
8
,9)
subsequently converted to glyceric acid (GA; Fig. 1).
Membrane-bound multimeric quinoprotein GLDH (2
subunits) and membrane-bound quinohemoprotein
mADH (3 subunits) have been found to donate electrons
to ubiquinone, which is embedded in the membrane
Dihydroxyacetone (DHA) is frequently used in the
cosmetics industry as the main active ingredient in all
sunless tanning skincare preparations, and it has also
become a pharmaceutical precursor and a building block
for chemical synthesis. DHA, which has a global market
1
0)
phospholipids, and then to the terminal oxidase.
Hence, as we intended to prevent GA by-production,
DHA production was examined using the G. oxydans
NBRC12528 mutant, in which the mADH-encoding
gene (adhA) was disrupted. We discovered that the adhA
disruptant significantly improved growth and DHA
productivity in high-concentration glycerol as compared
to the wild-type strain.
1
)
of approximately 2,000 tons per year, is produced by
biotechnological conversion of glycerol, a biomass
renewable resource that can be obtained in about 10%
weight as a by-product of biodiesel fuel production
through transesterification of vegetable oils and animal
2)
fats. Many studies and patents involve microbial DHA
production (see review, reference 3).
y
To whom correspondence should be addressed. Fax: +81-29-861-6247; E-mail: hiroshi.habe@aist.go.jp
Abbreviations: DHA, dihydroxyacetone; PQQ, pyrroloquinoline quinone; GLDH, glycerol dehydrogenase; GA, glyceric acid; mADH, membrane-
bound alcohol dehydrogenase; HPLC, high-performance liquid chromatography; vvm, volume of air per volume of medium per min; OD, optical
density

1392
H. HABE et al.
Alcohol
Glycerol
CH OH
dehydrogenase
CH OH dehydrogenase
CHO
COOH
CHOH
2
2
(GLDH)
(mADH)
HC=O
CHOH
CHOH
sldA
adhA
CH OH
CH OH
CH OH
CH OH
2
2
2
2
Dihydroxyacetone
Glycerol
Glyceraldehyde
Glyceric acid
(GA)
(DHA)
Fig. 1. Pathway for the Conversion of Glycerol to Dihydroxyacetone and Glyceric Acid by Gluconobacter oxydans.
sldA and adhA are the genes encoding the catalytic subunits of GLDH and mADH respectively.
Materials and Methods
1
40
20
100
Bacterial strains. G. oxydans NBRC12528 (formerly IFO12528,
obtained from the Institution for Fermentation, Osaka, Japan) and
G. oxydans ꢀadhA9) were precultivated in 5 ml of glucose medium
containing 30 g/l of glucose, 2 g/l of polypepton (Nihon Pharmaceut-
ical, Tokyo), and 5 g/l of yeast extract (Difco Laboratories, Detroit,
1
8
0
ꢀ
MI) at 30 C for 48 h in test tubes (200 mm ꢁ ꢁ18 mm). The seed
6
0
cultures (1.5 ml) were transferred to 300-ml Erlenmeyer flasks
containing 30 ml of glycerol medium (pH 6.5), consisting of 150 g/l
of glycerol, 10 g/l of polypepton, 1 g/l of yeast extract, 0.9 g/l of
40
2
0
.
KH2PO4, 0.1 g/l of K2HPO4, and 1 g/l of MgSO4 7H2O. The cultures
ꢀ
were incubated at 30 C on a BR-23FP rotary shaker (200 rpm; taitec,
Saitama, Japan) for 4 d. After removal of the cells by centrifugation,
the supernatant was filtered with a 0.45-mm cellulose filter. A 20-ml
aliquot of the supernatant was analyzed by high-performance liquid
chromatography (HPLC) to quantify DHA.
0
1
2
3
4
Cultivation time (d)
Fig. 2. Dihydroxyacetone Production by Gluconobacter oxydans
NBRC12528 and the ꢀadhA Mutant.
Symbols: Black and white squares represent wild-type and ꢀadhA
respectively. Error bars represent the standard deviation calculated
from three independent experiments.
Jar fermentor experiments. DHA production by G. oxydans
NBRC12528 and ꢀadhA was conducted in a 1-liter jar fermentor
(Model MDL; B.E. Marubishi, Tokyo). Jar fermentor experiments
were performed as follows: Strains were cultivated in five test tubes,
each containing 5 ml of glucose medium (total, 25 ml of culture) for 2 d
ꢀ
on a rotary shaker (200 rpm) for 4 d. After removal of the cells by
centrifugation, the various supernatants were analyzed by HPLC.
(30 C, 200 rpm). All seed cultures were transferred to a 1-liter jar
fermentor containing 500 ml of glycerol medium (pH 6.5), consisting
of 100–250 g/l of glycerol, 10 g/l of polypepton, 1 g/l of yeast extract,
.
Quantification of glycerol, DHA, and GA. The glycerol, DHA, and
GA concentrations in the culture broth were analyzed by HPLC with
an LC-20AD HPLC pump (flow rate, 1.0 ml/min) and an RID-10A
0
.9 g/l of KH2PO4, 0.1 g/l of K2HPO4, and 1 g/l of MgSO4 7H2O,
and incubated for 3 d. During the jar fermentor experiments, the
aeration rate and agitation speed were set to 0.5 volumes of air per
volume of medium per min (vvm) and 500 rpm. The temperature was
ꢀ
detector (Shimadzu) equipped with a Shodex SC1011 column
(Showa Denko, Tokyo) for glycerol and DHA and a Shodexꢀ
ꢀ
maintained at 30 ꢂ 1 C. pH was controlled with 10 M NaOH to keep it
above 5.
SH1011 column (Showa Denko) for GA. A mobile phase of pure
water and 5 mM H2SO4 solution was chosen as the eluent for the
columns. During analysis, the column temperature was maintained at
In the glycerol-feed experiment, ꢀadhA were precultivated as
described above, and the seed cultures were transferred to a 1-liter jar
fermentor containing 500 ml of medium (pH 6.5), consisting of 50 g/l
of glycerol, 10 g/l of polypepton, 1 g/l of yeast extract, 0.9 g/l of
ꢀ
ꢀ
80 C and 60 C for the two columns, respectively. DL-GA calcium salt
dihydrate (Wako Pure Chemicals, Osaka, Japan) or DL-GA (40% in
water; Tokyo Chemical Industry) and DHA (MP Biomedical, Santa
Ana, CA) was used to determine the standard curve to quantify GA and
DHA, respectively.
.
KH2PO4, 0.1 g/l of K2HPO4, and 1 g/l of MgSO4 7H2O, and
ꢀ
incubated for 1 d at 30 C, 0.5 vvm, and 500 rpm. After removal of
the cells by centrifugation, the cells (2.1 g/l of dry weight) were
resuspended in 500 ml of fresh glycerol medium (pH 6.5), consisting
of 150 g/l of glycerol, 10 g/l of polypepton, 1 g/l of yeast extract,
Chemicals. All chemicals were the purest commercially available
(98–100%; Sigma-Aldrich, Kanto Chemical, Wako Pure Chemicals,
Nacalai Tesque, Tokyo Chemical Industry, MP Biomedical).
.
0
.9 g/l of KH2PO4, 0.1 g/l of K2HPO4, and 1 g/l of MgSO4 7H2O,
ꢀ
and incubated again in a 1-liter jar fermentor for 4 d at 30 C, 0.5 vvm,
and 500 rpm. After a 2-d cultivation, an additional 75 g of glycerol was
added to the fermentor. pH was controlled with 10 M NaOH to keep it
above 5.
Results
In the resting cell reaction experiment, ꢀadhA cells were prepared
as described above, and were resuspended in 500 ml of glycerol
aqueous solution (230 g/l). The reactions were conducted for 4 d at
Differences in DHA productivity between G. oxydans
and its mutant
Gluconobacter oxydans ꢀadhA was examined for its
ability to produce DHA from 150 g/l of initial glycerol,
and DHA productivity was compared to that of the wild-
type strain (Fig. 2). The wild-type strain produced
ꢀ
30 C, 2 vvm, and 700 rpm. pH was controlled with 10 M NaOH to keep
it above 5.
Inhibition of dihydroxyacetone production and growth of G.
oxydans ꢀadhA due to glycerate. To investigate the inhibitory effects
of glycerate on G. oxydans ꢀadhA, seed cultures of ꢀadhA were
transferred to 30 ml of medium (pH 6.5), consisting of 0, 0.2, 0.4, 0.6,
3
3.2 g/l of DHA after a 2-d incubation, and further
incubation had little effect on DHA production. The
wild-type strain accumulated 17.8 and 19.3 g/l of GA in
the culture after 2- and 4-d incubations respectively. In
contrast, ꢀadhA appeared to be a more efficient DHA
producer, as DHA production continued to increase and
0
.8, 1.0, or 2.5% w/v DL-glycerate sodium salt (stock solution: about
.2 mol/l DL-GA in water (Tokyo Chemical Industry, Tokyo)), 10 g/l
5
of polypepton, 1 g/l of yeast extract, 0.9 g/l of KH2PO4, 0.1 g/l of
.
ꢀ
K2HPO4, and 1 g/l of MgSO4 7H2O. Cultures were incubated at 30 C

Dihydroxyacetone Production by a Gluconobacter oxydans Mutant
1393
1
20
4
3
2
1
120
90
4
3
2
1
A
B
9
0
6
0
60
3
0
30
0
0
0
1
2
3
0
1
2
3
Cultivation time (d)
Cultivation time (d)
1
1
1
80
50
20
3
2
3
2
1
1
80
C
D
150
120
90
9
0
6
0
1
0
60
3
0
3
0
0
0
1
2
3
0
1
2
3
Cultivation time (d)
Cultivation time (d)
Fig. 3. Comparison of Dihydroxyacetone (DHA) and Glyceric Acid (GA) Production between Gluconobacter oxydans NBRC12528 and the ꢀadhA
Mutant.
A, initial glycerol (100 g/l), wild type; B, initial glycerol (100 g/l), ꢀadhA; C, initial glycerol (150 g/l), wild type; D, initial glycerol (150 g/l),
ꢀ
ꢀ
adhA. In all the experiments, the reactions were conducted at 30 C, 0.5 volumes of air per volume of medium per min, and 500 rpm. pH was
controlled with 10 M NaOH to keep it above 5. Symbols: Black circles, glycerol concentration; white circles, GA concentration; black diamonds,
dry cell weight; white squares, DHA concentration.
was 3.3 times (120.5 g/l) greater than that of the wild-
type strain (36.6 g/l) after a 4-d incubation (Fig. 2).
ꢀadhA grew on 220 g/l of initial glycerol and produced
125 g/l of DHA, whereas the wild-type did not grow on
such a high glycerol concentration during a 3-d incuba-
tion. GA by-production was just 1.7 g/l in ꢀadhA, and
the remaining glycerol was 63.6 g/l, and therefore the
yield of DHA was 0.81 mol/mol glycerol consumed.
These results indicate that disrupting the adhA gene
significantly improved growth and DHA productivity
in G. oxydans in a high concentration of initial glycerol.
Next, we investigated the DHA productivity of ꢀadhA
with higher cell density and additional glycerol-feed.
Approximately 2 g/l of ꢀadhA cells (dry weight) were
resuspended in glycerol medium and incubated in a 1-
liter jar fermentor. After a 2-d cultivation, results of
3.5 g/l of dry cell weight, no remaining glycerol, and
116.5 g/l of DHA (0.68 mol/mol glycerol consumed) in
the culture were obtained. At this point, an additional
75 g of glycerol was added to the fermentor (132.9 g/l
of glycerol), but this had no significant effect on the
increase in DHA production during a further 2-d
incubation (data not shown).
ꢀ
adhA produced only 0.9 g/l of GA during this period.
Comparison of DHA productivity between G. oxydans
and its mutant using several initial glycerol concen-
trations
We examined growth, DHA production, and GA by-
production in both G. oxydans and ꢀadhA using 100 g/l
of initial glycerol in a jar fermentor (Fig. 3A, B). The
G. oxydans wild-type strain consumed most of the
glycerol within 2 d, and 54.3 g/l of DHA and 24.3 g/l
of GA was produced during a 3-d incubation (Fig. 3A). A
DHA yield of 0.59 mol/mol glycerol consumed was
obtained. In contrast, ꢀadhA accumulated only 1.0 g/l of
GA. Thus 74.8 g/l of DHA was produced during the same
period (0.74 mol/mol glycerol consumed; Fig. 3B).
More differences between the mutant and the wild
type appeared when we used an initial glycerol concen-
tration of 150 g/l (Fig. 3C, D). The wild-type strain
accumulated 38.1 g/l of DHA (0.36 mol/mol glycerol
consumed) and 47 g/l of GA, indicating that a higher
concentration of initial glycerol decreased DHA pro-
duction but increased GA production (Fig. 3C). This
tendency for higher concentrations of initial glycerol
to accumulate more GA corresponded to our recent
results on the effect of initial glycerol concentration on
Time course of DHA production by resting cell
reactions using the G. oxydans mutant
Since ꢀadhA scarcely formed the GA by-product, it
was considered advantageous for further purification.
However, to make DHA purification easier, it is better
not to use medium constituents other than glycerol, such
as polypepton or yeast extract. Hence, we conducted
resting cell reactions using ꢀadhA and 230 g/l of
glycerol aqueous solution. The ꢀadhA preculture was
incubated on 50 g/l of glycerol for 1 d, resulting in
approximately 2 g/l of cells (dry cell weight). After
centrifugation, the cells were resuspended in a glycerol-
water solution and incubated for 3 d. The time course of
9
,11,12)
GA production.
(
(
Besides scarcely producing GA
2.2 g/l), DHA production of ꢀadhA reached 108 g/l
0.68 mol/mol glycerol consumed) within the same
period (Fig. 3D). Poor growth of the wild-type strain
was observed after a 1-d incubation, whereas ꢀadhA
reached the mid-log phase (Fig. 3D) within 1 d.
A further big change was observed when a concen-
tration of more than 200 g/l of initial glycerol was used.

1394
H. HABE et al.
2
2
1
50
5.0
4.0
00
50
3
2
.0
.0
1
00
1.0
50
0
1
2
3
4
0
1
2
3
Cultivation time (d)
Cultivation time (d)
Fig. 5. Changes in Growth in Cultures of Gluconobacter oxydans
ꢀadhA on 150 g/l of Initial Glycerol with 0 ( ), 0.2 ( ), 0.4 ( ),
0.6 ( ), and 0.8% (x; w/v) Sodium Glycerate Concentrations.
Fig. 4. Time Course of Dihydroxyacetone (DHA) Production in
Resting Gluconobacter oxydans ꢀadhA Cells Using 230 g/l of
Glycerol Solution.
ꢀ
The reaction was conducted at 30 C, 2 volumes of air per volume
immobilizing the cells in a polymeric matrix15) also
solved these problems. However, until now, the desired
yield has not been achieved. Therefore, our finding that
the use of an adhA disruptant in the above developed
processes for solving such problems in DHA production
suggests a possibly useful technique. Also, the mutant
strain may be advantageous for applying recent excess
glycerol and excluding the GA by-product.
As shown in Fig. 3A and B, when we used 100 g/l of
initial glycerol, few differences in the bacterial growth
profile or glycerol consumption were found, but a higher
amount of DHA was produced in ꢀadhA (yields, 0.59
and 0.74 mol/mol glycerol consumed for the wild type
and ꢀadhA respectively). Considering that the amounts
of total glycerol derivatives (DHA plus GA) in the
culture were almost the same in the wild type and
ꢀadhA (0.83 and 0.84 mol/l respectively), the amount
of glycerol consumed in GA by-production in the wild
type was used for DHA production in ꢀadhA.
Also, the adhA disruptant was better than the wild type
under 150 g/l of initial glycerol (Fig. 3C and D). Yields
of 0.36 and 0.68 mol/mol glycerol consumed for the wild
type and ꢀadhA respectively were obtained. The yield of
the wild type at 150 g/l of glycerol was about 60% of that
at 100 g/l. The reason is that part of the glycerol for DHA
production is consumed in GA by-production, because
GA accumulation in the culture at 150 g/l of initial
glycerol (47 g/l; Fig. 3C) was much more than that at
100 g/l of initial glycerol (24.3 g/l; Fig. 3A).
of medium per min, and 700 rpm. pH was controlled with 10 M
NaOH to keep it above 5. Symbols: Black circles, glycerol
concentration; white squares, DHA concentration.
DHA production at a 2 vvm aeration rate and 700 rpm
agitation speed showed that approximately 139.7 g/l of
DHA was produced during the 3-d incubation (Fig. 4).
The remaining glycerol and GA accumulated were 28.4
and 2.3 g/l respectively.
Inhibitory effect of glycerate on dihydroxyacetone
production and the growth of G. oxydans ꢀadhA
The inhibitory effect of glycerate on ꢀadhA was
tested in a preliminary way in the presence of 0, 1.0, and
2
.5% w/v DL-glycerate sodium salt. Similarly to Fig. 2,
in no-glycerate conditions, ꢀadhA grew well on
glycerol (OD600, 4.66) and produced 116.5 g/l of DHA
within 4 d. By contrast, in the presence of only 1% w/v
glycerate, very poor growth of the strain was observed
during a 4-d cultivation (OD600, 0.75), and it accumu-
lated DHA to only about one tenth (11.5 g/l) as
compared to the no-glycerate condition. Further addition
of initial glycerate to the medium (2.5% w/v) resulted in
both no growth and no DHA accumulation. We further
investigated ꢀadhA inhibition due to glycerate in detail
with 0, 0.2, 0.4, 0.6, and 0.8% w/v DL-glycerate sodium
salt. As shown in Fig. 5, an increase in the initial
glycerate concentration resulted in a decrease in the
strain’s growth velocity. After a 4-d incubation, 104.7,
7
9.9, 62.9, 48.0, and 29.0 g/l of DHA accumulated in
Concerning bacterial growth, some delay was ob-
served in the wild type at 150 g/l of initial glycerol
(Fig. 3C). Furthermore, only the wild-type strain was
not able to grow on 220 g/l of initial glycerol. Probably,
these phenomena were not due to the inhibitory effect of
the respective cultures. These results indicate that
glycerate has an inhibitory effect on the cells of
G. oxydans, especially at the beginning of its growth.
5
)
Discussion
initial glycerol on the cells, as described previously,
because ꢀadhA exhibited faster growth than the wild
type under the same initial glycerol concentrations
(Fig. 3D). The difference between two strains lies in
whether GA can be produced or not. Hence, we
investigated the inhibitory effect of glycerate sodium
salt on ꢀadhA, and it was found that an increase in the
initial glycerate concentration (0 to 0.8% w/v) resulted
in poorer growth (Fig. 5).
We found that the G. oxydans adhA disruptant had the
ability to grow on a higher concentration of initial
glycerol and to produce more DHA than the wild type. It
has been found that higher concentrations of initial
glycerol and accumulated DHA inhibit cell growth and
3
,5)
DHA production,
and these problems are of great
concern in DHA fermentation. Several process changes
have been made to solve these problems. For example, a
higher amount of DHA was obtained in a fed-batch
culture than in a batch culture,1 and a repeated fed-
As described above, G. oxydans produces both DHA
and GA from glycerol at the same time (Fig. 1), and the
production ratio is determined mainly by aeration
3)
1
4)
8,9)
9)
batch process was more useful. Besides these proc-
7
conditions and the initial glycerol concentration.
We have found that at initial glycerol concentrations
)
esses, a two-stage repeated fed-batch process and

Dihydroxyacetone Production by a Gluconobacter oxydans Mutant
1395
higher than 100 g/l (up to 220 g/l), an increase in the
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do not produce GA, grow well on such higher concen-
trations of glycerol, and this better growth of ꢀadhA than
the wild type results in better DHA productivity as well
as no competition for glycerol between DHA and GA
production.
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This work was supported by the Industrial Technol-
ogy Research Grant Program 08A26202c from the New
Energy and Industrial Technology Development Organ-
ization (NEDO) of Japan.