Biosci. Biotechnol. Biochem.
, 69 (3), 659–662, 2005
Communication
Microbial Production of L-Ascorbic Acid from D-Sorbitol, L-Sorbose,
L-Gulose, and L-Sorbosone by Ketogulonicigenium vulgare DSM 4025
Teruhide S UGISAWA,1; y Taro M IYAZAKI,2 and Tatsuo H OSHINO3
1
Biotechnology R&D, DSM Nutritional Products Ltd., Bldg 203/854, CH-4002 Basel, Switzerland
2
Department of Genome Antibody Product Research, Chugai Pharmaceutical Co., Ltd.,
Fuji Gotemba Research Labs, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
3
Tamagawa University Research Institute, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan
Received November 10, 2004; Accepted January 4, 2005
Ketogulonicigenium vulgare DSM 4025, known as a 2-
keto-L-gulonic acid producing strain from L-sorbose via
L-sorbosone, surprisingly produced L-ascorbic acid
from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone
as the substrate under a growing or resting condition.
As the best result, K. vulgare DSM 4025 produced 1.37 g
per liter of L-AA from 5.00 g per liter of L-sorbosone
during 4 h incubation time at 30  C under the resting
cell condition having 5.70 g per liter of wet cells. The
precursor of L-AA formation from D-sorbitol and L-
sorbose, except for L-gulose, was thought to be the
putative furanose form of L-sorbosone. This is the first
time it is reported that bacteria can produce vitamin C
via L-sorbosone.
Key words: Ketogulonicigenium vulgare; L-sorbosone;
vitamin C
Ketogulonicigenium vulgare DSM 4025,1,2) recently
renamed (formally Gluconobacter oxydans DSM4025),
is known as a 2-keto-L-gulonic acid (2KGA) producing
strain from L-sorbose, in which L-sorbosone is an
intermediate in the fermentative production of 2KGA,
the latter being an L-ascorbic acid (L-AA) precursor. In
this context, we isolated and characterized the enzyme,
L-sorbose/L-sorbosone dehydrogenase,3) which is re-
sponsible for the sequential conversion of L-sorbose to
2KGA involved in this pathway. On the other hand, we
also isolated and characterized L-gulono-
-lactone de-
hydrogenase as the enzyme converting L-gulono-
-
lactone to L-AA.4)
Until now, we did not notice that L-AA formation
occurs in the fermentation broth of K. vulgare DSM
4025 using L-sorbose without any addition of L-gulono-

-lactone. Surprisingly, it was observed that K. vulgare
DSM 4025 can produce L-AA from both substrates, D-
sorbitol and L-sorbose, under growing conditions.
Furthermore, L-gulose and L-sorbosone, being possible
precursors of L-gulono-
-lactone and L-AA respectively,
can serve as well as D-sorbitol and L-sorbose under
y
To whom correspondence should be addressed. Tel: +41-61-687-3185; Fax: +41-61-687-1847; E-mail: teruhide.sugisawa@dsm.com
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resting cell conditions. Regarding the enzyme catalyzing
the oxidation reaction of L-sorbosone, several enzymes
have been reported: (1) membrane-bound L-sorbosone
dehydrogenase of the Acetobacter strain,5) (2) NAD(P)-
dependent L-sorbosone oxidoreductase of Gluconobact-
er oxydans UV106) and Gluconobacter oxydans T-100,7)
and (3) L-sorbose/L-sorbosone dehydrogenase, men-
tioned above. But the oxidative product is 2KGA. Only
the existence of an NADP-dependent L-sorbosone
dehydrogenase converting L-sorbosone to L-AA, located
in spinach leaf8) has been reported. The general
metabolic pathway of L-AA production in plants and
mammals goes via L-galactono-
-lactone and L-gulono-

-lactone respectively. In this context, we think L-gulose
is a precursor of L-gulono-
-lactone.
In the growing culture, one loopful of K. vulgare
DSM 4025 grown on agar medium consisting of 5.0% D-
.
mannitol, 0.25% MgSO4 7H2 O, 1.75% corn steep
liquor, 5.0% baker’s yeast, 0.5% urea, 0.5% CaCO3 ,
and 2.0% agar, cultivated at 27
C for 4 d, was
inoculated into 5 ml of seed culture medium consisting
.
of 8% L-sorbose, 0.25% MgSO4 7H2 O, 1.75% corn
steep liquor, 5.0% baker’s yeast, 0.05% glycerol, 0.5%
urea, 1.5% CaCO3 , and one drop of antifoam in a test
tube, and then incubated at 30
C at 240 rpm on a
reciprocal shaker. After 20 h, 3 ml of seed culture was
inoculated into 500 ml Erlenmeyer flasks containing
50 ml of the medium consisting of 8% L-sorbose or 8%
.
D-sorbitol, 0.25% MgSO4 7H2 O, 3.0% corn steep
liquor, 5.0% baker’s yeast, 0.05% glycerol, 0.5% urea,
1.5% CaCO3 , and 0.15% antifoam. Cultivation was
carried out at 30
C at 180 rpm on a rotary shaker. L-AA
contents in the culture broth was periodically measured
at reaction times of 20 and 45 h by high-performance
liquid chromatography (HPLC). All L-AA values shown
in this report pertain to its free form. L-AA was
measured at a wavelength of 264 nm by HPLC com-
posed of a UV detector (TOSOH UV8000, Tosoh,
Tokyo), a dualpump (TOSOH CCPE, Tosoh, Tokyo), an
integrator (Shimadzu C-R6A, Shimadzu, Tokyo), and a
660
column (YMC-Pack Polyamine-II, 4.6 mm i.d.  15 cm,
YMC, CA). The mobile phase was 50 mM NH4 H2 PO4 –
CH3 CN (3:7, volume/volume) with a flow rate at 2 ml
per min.
As for the result, L-AA was not detectable at 20 h, but
0.12 g per liter of L-AA was produced at 45 h from D-
sorbitol, and 0.41 and 0.42 g per liter of L-AA was
produced from L-sorbose at 20 and 45 h respectively. As
mentioned above, K. vulgare DSM 4025 can produce
2KGA from L-sorbose, and in line with expectations, 40
and 70 g per liter of 2KGA was produced from L-sorbose
at 20 h and 40 h respectively. The 2KGA produced from
D-sorbitol was 10 and 15 g per liter at the 20th hour and
the 40th hour respectively.
Furthermore, L-AA production was studied under
resting cell conditions using D-sorbitol, L-sorbose, L-
gulose, and L-sorbosone as substrates. K. vulgare DSM
4025 cultivated on agar medium consisting of 8.0% L-
.
sorbose, 0.25% MgSO4 7H2 O, 1.75% corn steep liquor,
5.0% baker’s yeast, 0.5% urea, 0.5% CaCO3 , and 2.0%
agar at 27
C for 4 d was transferred into 50 mM
potassium phosphate buffer (pH 7.0), washed twice
with the same buffer, and then resuspended in that
buffer. The cell suspension contained wet cells in the
range of 0.057 to 0.064 g per ml. The reaction mixtures
consisted of 0.5 ml of the above cell suspension and
4.5 ml of 50 mM potassium phosphate buffer (pH 7.0)
containing 8% D-sorbitol, 8% L-sorbose, 1% L-gulose, or
0.5% L-sorbosone (generously supplied by Hoffmann-La
Roche Inc., Nutley, NJ). The sodium salt of 2KGA
(0.5%) was also applied to this reaction. The reaction
was started by inoculating the cell suspension, and it was
carried out at 30
C and 240 rpm on a reciprocal shaker.
L-AA contents in the reaction mixture were periodically
measured at reaction times of 4, 20, and 24 hours by
HPLC as well. Table 1 shows the time course of L-AA
accumulation in reaction mixtures containing each
substrate concentration as described above. The sub-
strates L-sorbosone and L-gulose, which can be regarded
as structurally closer precursors to L-AA formation than
D-sorbitol or L-sorbose, gave higher titers of L-AA. As
the best result from the viewpoint of L-AA productivity
in this study, K. vulgare DSM 4025 produced 1.37 g per
liter of L-AA from 5 g per liter of L-sorbosone within 4 h
reaction time when resting cells corresponding to 5.7 g
Table 1. L-Ascorbic Acid Production from D-Sorbitol, L-Sorbose, L-Sorbosone, and L-Gulose by Resting Cells of Ketogulonicigenium vulgare DSM
4025
Optical densitya
Substrate
(at 600 nm)
8% D-Sorbitol 2.54
8% L-Sorbose 2.62
0.5% L-Sorbosone 2.82
1% L-Gulose 3.49
0.5% Na-2-keto-L-gulonic acid 3.55
None 2.90
a
Optical density was measured before the start of the reaction, and each reaction mixture contained 5.70 to 6.40 g per liter of wet cells.
T. S UGISAWA et al.
per liter of wet cells were applied to the reaction. L-
Sorbosone was also converted to 2KGA, and 0.50, 1.5 to
2.0, and 2.0 to 2.5 g per liter of 2KGA were detected at
4, 20, and 24 h respectively, as estimated by thin layer
chromatography (TLC) analysis.9) Prolonged incubation
with L-sorbosone resulted in a slight decrease in the L-
AA content of the reaction mixtures, indicating L-AA
degradation, because an increase of unidentified com-
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pounds, possibly L-AA degradation products, were
detected by TLC. No L-AA production from 2KGA
was observed, which excludes the possibility that L-AA
was produced from L-sorbosone via 2KGA, and there
was no consumption of 2KGA when sodium 2KGA was
provided as a substrate.
The results indicate that K. vulgare DSM 4025 can
convert D-sorbitol, L-sorbose, L-gulose, and L-sorbosone
to L-AA. The previously described enzymes L-sorbose/
L-sorbosone dehydrogenase and L-gulono-
-lactone de-
hydrogenase isolated from K. vulgare DSM 4025 by our
group catalyzed exclusively the oxidation reactions of L-
sorbosone to 2KGA and of L-gulono-
-lactone to L-AA
in vitro, respectively. Therefore we assume that this
strain has different kinds of enzyme systems responsible
for L-AA production from L-sorbosone. Regarding the L-
gulose to L-AA conversion, it was assumed that L-AA
was produced via the L-gulono-
-lactone pathway,4) but
it was also assumed that this strain expresses an
additional enzyme responsible for L-AA production
from L-sorbosone. In fact, we have confirmed by activity
staining of native polyacrylamide gel electrophoresis
(PAGE) with L-sorbosone and nitrobluetetrazolium
(NBT) the presence of so far undescribed L-sorbosone
dehydrogenases in a cell free lysate of K. vulgare DSM
4025 (manuscript in preparation). The activity staining
was carried out as follows: The native PAGE performed
with a slab gel (10 cm  10 cm, pH 9.4) containing 10%
(weight/volume) polyacrylamide was immersed in a
staining solution of 100 ml consisting of 100 mM
potassium phosphate buffer (pH 7.0), 4 mg of NBT,
14 mg of phenazine methosulfate, and 200 mg of L-
sorbosone as the substrate for the enzymes, and
incubated with gentle shaking at room temperature for
20 min. The reaction was stopped with 7% (volume/
volume) acetic acid solution, and then dark-purple
colored bands corresponding to enzymes having oxida-
L-Ascorbic acid produced (gram per liter)
4th h 20th h 24th h
0.0 0.062 0.090
0.64 0.908 0.874
1.37 1.12 1.04
0.49 1.36 1.67
0.0 0.0 0.0
0.0 0.0 0.0
 Microbial Production of L-Ascorbic Acid 661

A Oxidation product from L-sorbosone estimated B L-Sorbosone formation and their products estimated
by Fischer`s projection

COOH COOH CHO CH2OH

(certain equilibrium constant
C O HO C O OH in aqueous solution) 4
HO O
HO C H O C OH
HO C H OH CHO

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H C OH H C OH C O
or
CHO HO O
HO C H HO C H HO OH HO C H
C O L-Sorbosone pyranose form L-Sorbosone furanose form
CH2OH H2C H C OH
HO C H 1 HO C H
2KGA Hemi-acetal form
H C OH 5 CH2OH 6
of 2KGA
HO C H (L-Sorbosone)
COOH
CH2OH 3 2 2 CH2OH
O OH
HO O
L-Sorbosone O C OH C O
HO C O
HO C HO OH HO OH
H C Hemi-acetal form of 2KGA L-AA
HO C H
L-AA
CH2OH

Fig. 1. Proposal as to Bioconversion Pathway from L-Sorbosone to L-Ascorbic Acid by Ketogulonicigenium vulgare DSM 4025.
(A) Oxidation product from L-sorbosone based on the Fisher projection. Pathway 1: 2KGA is produced by the oxidation of –CHO residue.
Pathway 2: Ketogulonicigenium vulgare DSM 4025 has no activity. Pathway 3: Two steps of reaction, oxidation and lactonization, are required.
(B) L-Sorbosone formations and their products estimated. Formula 4: Hypothesis of L-sorbosone formation with a certain equilibrium constant in
aqueous solution. Pathway 5: 2KGA formation from pyranose-form L-sorbosone. Pathway 6: Pathway proposed to convert L-sorbosone to L-AA
by Ketogulonicigenium vulgare DSM 4025 described in this report.

tion activity of L-sorbosone appeared on the gel.
As described in Fig. 1-A, pathway 1 is well-known,
but it was unclear to us how to explain the direct
formation of L-AA from the structure of L-sorbosone
expressed by the Fischer projection. In addition, Loewus
et al.8) presented as a suitable explanation that the
conversion of L-sorbosone to L-AA by partially purified
NADP-dependent dehydrogenase from spinach leaf
involves two steps, oxidation and lactonization (path-
way 3). But L-AA formation was undetectable from
2KGA (pathway 2) which was assumed to be an
oxidation product of L-sorbosone in our study.
To explain in the best possible way, we propose L-
sorbosone conformations (pathway 4) and the oxidation
pathway to 2KGA (pathway 5) and L-AA (pathway 6)
from L-sorbosone, as shown in Fig. 1-B. As for the
conversion of L-sorbosone to L-AA, described above, we
envision that the reaction starts from the putative
(1 ! 4)-furanose form of L-sorbosone, in which the
hydroxyl group at C-1 is oxidized. The reaction would
be completed by a keto-enol tautomerization forming
the final product, L-AA. Generation of 2KGA could start
either from the open form of L-sorbosone or from the
putative (2 ! 6)-pyranose form. Oxidation of the C-1
carbonyl group of the open form or the exo-carbonyl
group of the putative (2 ! 6)-pyranose form would then
result in the open or the hemi-acetal form of 2KGA
respectively. Dehydrogenases such as L-sorbose/L-sor-
bosone dehydrogenase, producing 2KGA from L-sorbo-
sone, might be specific for the open form or the pyranose
form of L-sorbosone as substrate. Dehydrogenases
catalyzing the conversion of L-sorbosone to L-AA might
be limited to the 5-ring furanose form as substrate. This
model requires that both of these conformational
isomers of L-sorbosone are present in the reaction
mixture at concentrations allowing significant reaction
velocities at the given Km values of the enzyme
involved. Structural analysis of L-sorbosone in aqueous
solution should provide insight into the rather unusual
reaction mechanism where the conformation determines
by which of two alternative enzymes present in an in
vitro reaction mixture or within an intact cell a substrate
is converted to different reaction products.
Acknowledgment
The authors would like to thank Dr. Markus Goese
and Dr. Hans-Peter Hohmann of DSM Nutritional
Products Ltd., Biotechnology R&D for discussion of
the chemical structures of L-sorbosone and 2-keto-L-
gulonic acid.
662 T. S UGISAWA et al.
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