3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway.

Article abstract of DOI:10.1271/bbb.67.2124

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline qui

Full text of DOI:10.1271/bbb.67.2124

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3-Dehydroquinate Production by Oxidative
Fermentation and Further Conversion of 3-
Dehydroquinate to the Intermediates in the
Shikimate Pathway
Osao ADACHIa, Somboon TANASUPAWATb, Nozomi YOSHIHARAa, Hirohide TOYAMAa &
Kazunobu MATSUSHITAa
a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi
UniversityYamaguchi 753-8515, Japan
b Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn
UniversityBangkok 10330, Thailand
Published online: 22 May 2014.
To cite this article: Osao ADACHI, Somboon TANASUPAWAT, Nozomi YOSHIHARA, Hirohide TOYAMA & Kazunobu MATSUSHITA
(2003) 3-Dehydroquinate Production by Oxidative Fermentation and Further Conversion of 3-Dehydroquinate to the
Intermediates in the Shikimate Pathway, Bioscience, Biotechnology, and Biochemistry, 67:10, 2124-2131, DOI: 10.1271/
bbb.67.2124
To link to this article: http://dx.doi.org/10.1271/bbb.67.2124
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Biosci. Biotechnol. Biochem., 67 (10), 2124–2131, 2003
3-Dehydroquinate Production by Oxidative Fermentation and Further
Conversion of 3-Dehydroquinate to the Intermediates in the
Shikimate Pathway
† Somboon TANASUPAWAT,2 Nozomi YOSHIHARA,1 Hirohide TOYAMA
,
1,
1
Osao ADACHI
,
1
and Kazunobu MATSUSHITA
1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330, Thailand
Received April 7, 2003; Accepted July 24, 2003
3-Dehydroquinate production from quinate by oxida-
tive fermentation with Gluconobacter strains of acetic
acid bacteria was analyzed for the ˆrst time. In the
bacterial membrane, quinate dehydrogenase, a typical
quinoprotein containing pyrroloquinoline quinone
(PQQ) as the coenzyme, functions as the primary en-
zyme in quinate oxidation. Quinate was oxidized to
biodegrading herbcides and pesticides are originated
from the shikimate pathway. Besides shikimate,
3-dehydroquinate (DQA) and 3-dehydroshikimate
(DSA) are also common intermediates in these path-
ways.2,3) In spite of the importance of the shikimate
pathway to a variety of applications, little informa-
tion has been accumulated about the shikimate
pathway regarding interconversion of intermediates.
One reason can be that the shikimate pathway is very
remote from the initial substrate, glucose, and it is
di‹cult to control the pathway by means of mo-
3-dehydroquinate with the ˆnal yield of almost 100
% in
earlier growth phase. Resting cells, dried cells, and
immobilized cells or an immobilized membrane fraction
of Gluconobacter strains were found to be useful
biocatalysts for quinate oxidation. 3-Dehydroquinate
was further converted to 3-dehydroshikimate with a
reasonable yield by growing cells and also immobilized
cells. Strong enzyme activities of 3-dehydroquinate
dehydratase and NADP-dependent shikimate de-
hydrogenase were detected in the soluble fraction of the
same organism and partially fractionated from each
other. Since the shikimate pathway is remote from
glucose in the metabolic pathway, the entrance into the
shikimate pathway from quinate to 3-dehydroquinate
looks advantageous to produce metabolic intermediates
in the shikimate pathway.
lecular biology. 3-Deoxy-7-phospho- -arabinohep-
D
tulosonate, a key intermediate to shikimate, is pro-
duced by merging two diŠerent metabolic pathways,
phosphoenolpyruvate from the glycolytic pathway,
and erythrose-4-phosphate from the pentose phos-
phate pathway. Thus, many steps of enzymatic
reactions are required before getting shikimate from
glucose (Fig. 1).
Quinate utilization by microorganisms is known as
an important index in the systematic determinative
bacteriology for Pseudomonas, and many other aer-
obic bacteria. Quinate is used as a growth substrate
by fungi as well as by bacteria and is metabolized via
3-dehydroquinate (DQA) and 3-dehydroshikimate
(DSA). DQA dehydratase (synonymous to 5-de-
Key words: acetic acid bacteria; 3-dehydroquinate; 3-
dehydroshikimate; quinate oxidation;
shikimate pathway
hydroquinase, EC 4.2.1.10) (DQD) was isolated
4)
from cell extract of Escherichia coli
.
The presence
Shikimate is a versatile intermediate in the shiki-
mate pathway of aromatic compounds in animals
and plants, and even in microorganisms.1) The shiki-
mate pathway is important in pharmaceutical and
industrial signiˆcance, because three aromatic amino
acids, more than ten diŠerent antibiotics, and many
of quinate 5-dehydrogenase (synonymous to
NAD(P)-dependent quinate dehydrogenase, EC
1.1.1.24) and shikimate 5-dehydrogenase (synony-
mous to 5-dehydroshikimate reductase, EC 1.1.1.25)
(SKDH) were found in Aerobacter aerogenes5) and in
6)
E. coli
.
These enzymes were also indicated from
†
To whom correspondence should be addressed. Tel:
＋
81-839-33-5857; Fax:
＋
81-839-33-5820; E-mail: osao agr.yamaguchi-u.ac.jp
＠
Abbreviations: DQA, 3-dehydroquinate; DQD, 3-dehydroquinate dehydratase (EC 4.2.1.10); DSA, 3-dehydroshikimate; PQQ, pyrrolo-
quinoline quinone; QDH, quinoprotein quinate dehydrogenase (EC 1.1.99.25); SKDH, NADP-dependent quinate dehydrogenase (EC
1.1.1.25)

Formation of 3-Dehydroquinate and Other Intermediates in Shikimate Pathway
2125
Fig. 1. Metabolic Map of Shikimate Pathway.
Entrance to the shikimate pathway from quinate is indicated by a fat arrow.
plant origins in a series of aromatic biosyntheses.7–10)
The ˆrst report of a quinate-oxidizing enzyme in
acetic acid bacteria was done by Whiting and Cog-
gins.11) They pointed out quinate oxidation to DQA
by NAD(P)-independent quinate dehydrogenase
(QDH) (EC 1.1.99.25) and shikimate oxidation to
DSA by an enzyme associated to the particulate
enzyme in the cytoplasmic membrane. van Kleef and
Duine12) indicated the occurrence of QDH in the
periplasm of Acinetobacter calcoaceticus LMD 79.41
and suggested that QDH is a quinoprotein in which
pyrroloquinoline quinone (PQQ) is involved. Else-
more and Ornston did genetic analysis of pro-
tocatechuate catabolism13) and claimed that a con-
secutive action of three genes, quiA encoding QDH,
quiB for DQD, and quiC for DSA dehydratase, are
required in the catabolism of quinate to pro-
tocatechuate.14)
Materials and Methods
Chemicals. NADP, NADPH, and yeast extract
were kind gifts from Oriental Yeast Co. (Tokyo,
Japan). Other chemicals used were from commercial
sources of guaranteed grade unless otherwise stated.
DQA and DSA were prepared as described in the
text.
Microorganisms
and
culture
conditions.
Gluconobacter oxydans IFO 3244, G. oxydans IFO
3292, and G. melanogenus IFO 3294 were kind
donations from the Institute for Fermentation,
Osaka (IFO). The basal medium consisted of
glycerol, 3 g of yeast extract, and 1 g of polypepton
in 1 liter of tap water. Glycerol concentrations in the
culture medium were varied up to 10 g per liter
according to the conditions. If it was necessary to
induce QDH in the bacterial cells in advance, 2 g of
quinate was added to the culture medium and the pH
of the medium was adjusted to 7.0. Microorganisms
were grown in 100 ml of the medium in a 500-ml side-
As reported elsewhere, DQA production from
quinate is catalyzed by a membrane-bound QDH,
and NAD-dependent quinate dehydrogenase in the
cytoplasmic fraction makes no contribution to
quinate oxidation.15) Since DQA is produced rapidly
under various conditions by acetic acid bacteria,
bioconversion of quinate to DQA by immobilized
cells is examined. In this paper, microbial production
of DQA by oxidative fermentation with acetic acid
bacteria and further enzymatic conversion of DQA
to DSA is brie‰y done.
armed Erlenmeyer ‰ask at 309C with shaking. The
bacterial growth was measured by a Klett-Summer-
son photoelectric colorimeter with a red ˆlter. For
the cell-mass preparation, a seed culture in 100 ml of
the medium in a 500-ml Erlenmeyer ‰ask was done
overnight and transferred it to ˆve liters of a fresh
medium in a 10-L table top fermentor (MD500-10L,

2126
O. ADACHI et al.
Marubishi Bioengineering, Co., Tokyo, Japan), and
cultivated for another 12 hr under vigorous aeration.
If necessary, the ˆve liters of culture was used as
another seed culture for 40 liters of medium in a 50-L
fermentor (MSJ U2-50L, Marubishi Bioengineering,
Co., Tokyo, Japan). The harvested cells were sus-
ically in 0.1
total volume of 1 ml of a reaction mixture in the
presence of 0.2 mol of NADPH and arbitrary units
M
potassium phosphate, pH 6.0, in the
m
of puriˆed SKDH. Reading the decrease in ab-
srobance at 340 nm in the rate assay showed a good
proportionality to the concentration of DSA.
Shikimate. Shikimate was measured enzymatically
pended in cold 5 m
M
potassium phosphate, pH 6.5,
and centrifugation was repeated with a conventional
centrifuge to remove materials coming from the
culture medium. The precipitated cells were stored
without freezing before use. Freshly harvested cells
were usually used for every experiment where the
intact cells were required.
in 0.1
of 1 ml of a reaction mixture in the presence of
0.2 mol of NADP and arbitrary units of puriˆed
M
glycine-NaOH, pH 10.0, in the total volume
m
SKDH. Reading the increase in absorbance at 340 nm
in the rate assay showed a good proportionality to
the concentration of shikimate.
Protocatechuate. A qualitative detection of pro-
tocatechuate was done by reacting DSA with DSA
dehydratase. Protocatechuate formation allowed a
color change of the reaction mixture to orange
having lmax at 460 nm. It was only observed when
DSA dehydratase was added to a reaction mixture
containing enzyme reaction mixtures yielding DSA.
Preparation of membrane fraction and cytoplas-
mic fraction. Cell suspension was made by
homogenizing freshly harvested cells at a ratio of
about 10 g wet cells per 10 ml of 5 m
M
potassium
phosphate, pH 6.5. Trace amounts of RNase and
DNase were added before cell disruption. The cell
suspension was treated by passing it twice through a
French pressure cell press (SIM AMINCO, Spectron-
ic Instruments, Inc., Rochester, NY, USA) at 16,000
Detection of quinate metabolites by paper chro-
matography. Paper chromatography and detection
of intermediates in the shikimate pathway. Paper was
developed with a solvent containing benzylalcohol:2-
lb in2. After removal of intact cells by a conventional
W
low speed centrifuge, the crude cell-free extract was
×
＝
butanol:2-propanol:water 3:1:1:1 (w v) containing
W
further centrifuged at 68,000
g
for 90 min to
separate the membrane fraction from the cytoplas-
mic soluble fraction. The membrane fraction was
washed by homogenizing the precipitate in a glass
homogenizer with the same buŠer and by repeating
ultracentrifugation.
2
z
formic acid. After this was dried up in a draft
chamber, freshly prepared sodium metaperiodate
(160 mg) in a mixture of each 12.5 ml of 1 acetic
acid and 1 Na-acetate was sprayed over the paper.
About 20 min later when the paper was still wet, the
second coloring agent, 3 (v v) aniline in alcohol,
M
M
z
W
Enzymatic measurements of quinate, DQA, DSA,
and shikimate.
was sprayed over the paper according to the method
described by Yoshida and Hasegawa.17) In most
Quinate. Quinate in a culture medium or in an
enzymatic reaction mixture was measured enzymati-
cally using a puriˆed QDH. Puriˆed QDH was
obtained as reported previously.15) Composition of
the reaction mixture was essentially the same as de-
scribed previously and either potassium ferricyanide
or a combination of phenazine-methosulfate (PMS)
and 2,6-dichlorophenolindophenol (DCIP) were used
as the electron acceptors.16)
cases, quinate of which the
spot of pale pink, DQA (
R
R
f value of 0.23 gave a
＝
f
0.28) gave yellow,
＝
＝
0.54)
shikimate (
Rf
0.43) gave red, and DSA (
R
f
gave yellow, as shown in the text. Due to the dual
sprayings by diŠerent coloring agents while the paper
was still wet, it was di‹cult to show a clear picture.
Immobilization of acetic acid bacteria. Conditions
for cell immobilization of acetic acid bacteria were
examined according to the method described by
Saeki.18) The bacterial culture of mid-exponential
3-Dehydroquinate. DQA was measured by reading
decreasing intensity at 340 nm spectrophotometrical-
ly. First, DQA was converted to DSA by a puriˆed
growth (1 g wet cells 10 ml of 0.1
M
Tris-acetate, pH
W
DQD. DSA was then measured with SKDH in 0.1
Tris-HCl, pH 8.0, by the addition of 0.2 mol of
M
6.5) was mixed with 3
in 0.1 Tris-acetate buŠer, pH 6.5. The optimum of
the cell suspension and Na-alginate solution was
2.5 ml 7.5 ml. The mixture was dropped into a
z Na-alginate solution (w v)
W
m
M
NADPH in the total volume of 1 ml of the reaction
mixture. After already existing DSA in the samples
had been eliminated by SKDH and NADPH, reac-
tion was started by the addition of arbitrary units of
DQD. After it was left standing for 20 min to ensure
W
chilled solution of 8
z
CaCl (w v). Gentle stirring
W
2
was continued for 5 hr to conˆrm the embedding of
the cells in the Ca-alginate. Beads were collected,
washed with the same buŠer to remove excess CaCl2,
and stored in 0.1
C until use. The immobilized cells were mixed with
completion of the reaction at 309C, the intensity of
the reaction mixture was read and the absorbance
from a control run was subtracted to give an appar-
ent estimation of DQA.
M
Tris-acetate buŠer, pH 6.5, at
4
9
quinate in various ratios and the reaction was done
by gentle stirring at room temperature. In order to
3-Dehydroshikimate. DSA was measured enzymat-

Formation of 3-Dehydroquinate and Other Intermediates in Shikimate Pathway
2127
check the optimum immobilization, the concentra-
tion of Na-alginate, cell density, CaCl2 concentra-
tion, and gel bead size were examined. As the opti-
Results and Discussion
mum immobilization conditions, 1.2
z
Na-alginate,
Quinate oxidation by acetic acid bacteria
3
z
CaCl2, 1.5 mm beads size, of which initial viable
When several strains of Gluconobacter were cul-
5
×
cells were set to 6 10 ml gel were prepared. During
tured in the basal medium containing 0.1
z glycerol
W
quinate oxidation, the viable cell numbers were
and 0.2 quinate, quinate rapidly disappeared from
z
increased by three orders to 108–9 ml of the gel.
the culture medium and stoichiometric amount of
DQA was accumulated instead before the culture
came to the mid exponential growth phase. This is
the ˆrst stoichiometric observation of DQA forma-
tion by means of oxidative fermentation. DQA ac-
cumulation was strongly catalyzed by Gluconobacter
strains like G. oxydans IFO 3244, G. oxydans IFO
3292, G. melanogenus IFO 3294, G. liquefaciens IFO
12388, and several isolated Gluconobacter strains.
G. oxydans IFO 3244 was grown on the basal medi-
um to the mid exponential growth phase (after 12 hr)
of which Klett intensity was 40, 120, and 150 when
W
Polyacrylamide gel was also useful for cell im-
mobilization according the method described by Das
19)
et al
24
.
The cell suspension (10 ml) was mixed with
z
acrylamide stock solution, 10
z
ammonium
-tetramethyl-
l). The mixture was poured to
persulfate (135
enediamine (35
ml), and N,N, N?, N?
m
solidiˆy into a shallow glass container. After the gel
was solidiˆed, the gel plate was cut into small cubes
before use. Cell immobilization using k-carrageenan
gave unfavorable results due to unsuccessful gel for-
mation. Dried cells of acetic acid bacteria were rather
useful and the results in quinate oxidation was almost
comparable to quinate oxidation with Ca-alginate
immobilized cells, although the free cells must be
removed after the reaction before analysis. In order
to prepare dried cells, wet cells were spread over on a
ˆlter paper (Advantec-Toyo No. 2) and kept air-dried
glycerol was added to 0
tively. When quinate was added to the culture medi-
um to 0.2 (w v), quinate contents in the culture
z z z
, 0.1 , and 1 , respec-
z
W
media dropped rapidly to zero after three hr of incu-
bation. When quinate oxidation was done with the
resting cells, the cell concentration was adjusted to
0.75 by measuring turbidity at 600 nm. Judging from
the results with resting cells, rapid quinate oxidation
occurred with quinate grown cells, while a 90-min lag
time was observed with the cells which had been
grown in the absence of quinate. These results indi-
cate that induction of quinate oxidase system occurs
soon after addition of quinate.
9
at 30 C until the wet cells were completely dried. The
air-dried cells were further dried over P2O5 under
reduced pressure in a desiccator.
Preparation of 3-dehydroquinate and 3-de-
hydroshikimate. Since DQA and DSA are not
available commercially, standard solutions of both
compounds were prepared and used for further
experiments. Freshly harvested cells were mixed with
Quinate oxidation by immobilized cells of acetic
acid bacteria
9
sodium quinate, the mixture was kept at 30 C under
vigorous shaking, and remaining quinate in the reac-
tion mixture was checked periodically. Immediately
after the quinate reaction disappeared in the reaction
mixture, the cells were separated by a conventional
centrifuge. The supernatant was lyophilized and
stored until use. The dried materials were dissolved in
a minimal volume of water and the insoluble materi-
als were removed by centrifugation. The supernatant
Immobilized cells were also useful catalysts in
quinate oxidation. When examined with Ca-alginate
immobilized cells and polyacrylamide gel immobi-
lized cells of G. oxydans IFO 3244, quinate oxidation
was completed within 12 hr and 20 hr, respectively,
under the conditions used as shown in Fig. 2. Almost
the same results as above were obtained with other
strains, G. oxydans IFO 3292 and G. melanogenus
IFO 3294. The Ca-alginate immobilized cells showed
a faster rate of quinate oxidation than poly-
acrylamide-gel immobilized cells. The diŠerence may
come from the diŠerences of availability of the sur-
face area between two carriers used for cell immobili-
zation. It was obvious that the other factors such
as the matrix size of the gel and hydrodynamic
parameters must aŠect substrate diŠusion rate
through the gels. Taking all these factors into con-
sideration, the optimized immobilization of acetic
acid bacteria must be obtained to give the highest
quinate oxidation to DQA.
×
was put on a column (1.5 70 cm) of Ca-form of
Dowex 50 and eluted with water. When fractionation
was done every 5 ml, unreacted quinate appeared at
the fraction number of 30 and DQA came out at the
fraction number of 55. The fractions containing
DQA were combined and lyophilized and stored be-
fore use in a dark desiccator under reduced pressure.
DSA was prepared by the reaction of DQD puriˆed
from G. oxydans IFO 3244. An alternative method
of DSA formation was done with dried cells. The
dried cells (1 mg ml) were put into 1
z
DQA solution
W
(3 ml) in a test tube, shaken at 30
9C (200 rpm), and a
sample of the reaction mixture was picked up period-
ically.

2128
O. ADACHI et al.
Fig. 4. 3-Dehydroshikimate Production by Dried Cells and Wet
Cells.
Dried cells and freshly harvested wet cells of G. oxydans IFO
3244 were reacted with 30 mg quinate in 3 ml with shaking at
30 C for the period indicated. Samples of the reaction mixture
Fig. 2. Quinate Oxidation by Ca-Alginate Immobilized Cells and
Polyacrylamide Gel Immobilized Cells.
Freshly harvested cells of G. oxydans IFO 3244 were used for
immobilization. Squares and circles mean Ca-alginate immobi-
lized cells and polyacrylamide gel immobilized cells, respec-
9
were taken periodically for the measurement of DSA.
tively. Immobilized cells were mixed with 10 mgWml of quinate
and gently shaken (90 rpm) for the period indicated. Quinate in
the reaction mixture was measured periodically under the stan-
dard assay conditions.
the substrate (data not shown). The unexpected low
yield of DSA might come from the diverse metabolic
pathways of the product, some of the DSA produced
may be converted without accumulating in the reac-
tion mixture. This was suggested by the following
experiments. When dried cells and freshly harvested
wet cells were tested for DSA production, DSA was
clearly detected in the reaction mixture including
dried cells, while a scarce amount of DSA was detect-
ed in the reaction mixture examined with freshly har-
vested cells (Fig. 4). EŠectiveness and usefulness of
dried cells for the production of metabolic intermedi-
ate by means of bioconversion has been described in
other cases.20) Permeability of substrate and product
through the cytoplasmic membranes becomes im-
portant in the experiment where whole cells were
used as biocatalyst. Anyway, it was something far
from a stoichiometric account when compared to
DQA production by oxidative fermenation, and DSA
may be controlled by the cytoplasmic metabolism.
The DSA produced may not stay stable and may be
further converted to shikimate and also pro-
tocatechuate. In fact, in the reaction mixture in
which dried cells or immobilized cells were reacted
with DQA, shikimate and protocatechuate were
always detected more or less in addition to DSA.
Fig. 3. 3-Dehydroquinate Oxidation by Immobilized Cells of
Acetic Acid Bacteria.
Ca-Alginate immobilized cells of Gluconobacter strains were
incubated with 30 mg DQA in 3 ml with shaking at 309C for the
period as indicated. The numbers shown in the ˆgure cor-
respond to the strain: 3244, G. oxydans IFO 3244; 3292, G.
oxydans IFO 3292; 3294, G. melanogenus IFO 3294. Samples of
the reaction mixture were taken periodically for the measure-
ment of DSA.
Chromatographic detection of oxidation products
To avoid heavy tailing in paper development, rest-
ing cells were prepared from freshly harvested cells
and directly used for quinate oxidation at the ˆnal
cell density of 0.75 at 600 nm. As shown in Fig. 5,
3-Dehydroshikimate production from quinate and
3-dehydroquinate
DSA production by Ca-alginate immobilized cells
of G. oxydans IFO 3244, G. oxydans IFO 3292, and
G. melanogenus IFO 3294 proceeded with 11, 10,
quinate initially loaded at 10 mg ml was converted to
W
and 8.4
z
of ˆnal yield, respectively, when DQA was
DQA within 12 hr of reaction. After the same situa-
tion was maintained for another 12 hr until quinate
was completely converted to DQA, DQA was grad-
ually converted to DSA in the next 24 hr. After 72 hr
used as the initial substrate (Fig. 3). When DSA
production was done with quinate, the total yield of
DSA was lower than those examined with DQA as

Formation of 3-Dehydroquinate and Other Intermediates in Shikimate Pathway
2129
KCl concentration between 500 ml of BuŠer A con-
taining 0.03 KCl and 500 ml of BuŠer A containing
0.11 KCl. Judging from the elution pattern (not
shown), SKDH came out at about 0.075 KCl
M
M
M
concentration. The partially puriˆed SKDH at this
stage contained no enzyme activities of DQD and
DSA dehydratase, though several minor protein
bands were still appeared in polyacrylamide gel elec-
trophoresis (data not shown). In further puriˆcation
of DQD, the impurity was then removed by a
×
hydroxyapatite column (5 10 cm) equilibrated with
BuŠer A, to which DQD was adsorbed while the
major impurity was not adsorbed under these condi-
tions. DQD was eluted from the column with BuŠer
A of which potassium phosphate was increased to
50 m . The dialyzed DQD was put on a small
M
×
DEAE-Sephadex A-50 column (1 10 cm), which
had been equilibrated with the same BuŠer A used
for enzyme dialysis. After the column was washed
Fig. 5. Chromatographic Detection of Oxidation Products of
Quinate.
Dried cells of G. oxydans IFO 3244 (1 mg
with 10 mg ml of quinate and shaken at 30 C for the period in-
Wml) were mixed
with BuŠer A containing 0.175
ˆnally eluted from the column with BuŠer A contain-
ing 0.2 KCl. During DQD puriˆcation, the enzyme
M
KCl, DQD was
W
9
dicated. Samples of the reaction mixture were taken periodically
and spotted on a paper. Other chromatographic conditions are
given in Materials and Methods.
M
activity of DSA dehydratase was separated from
DQD at the ˆnal step of chromatography. DQD was
eluted from the column under the conditions above,
whereas DSA dehydratase was eluted from the
column only when KCl concentration in BuŠer A was
KCl. A manuscript on the
overall puriˆcation of SKDH and DQD is now being
prepared.
of incubation, almost equal amounts of DQA and
DSA were detected in the reaction mixture. Since
acetic acid bacteria catalyze a one-step incomplete
oxidation reaction in the oxidative fermentation, it is
advantageous to collect the oxidation product by
controlling the reaction conditions. Thus, QDA
production by acetic acid bacteria was shown here
for the ˆrst time by criteria using growing cells, im-
mobilized cells, and dried cells. If the cells were
removed from the reaction mixture after quinate
depletion, DQA can be collected with the highest
yield. Other oxidative bacteria like Pseudomonas
strains or Acinetobacter strains also contain QDH
oxidizing quinate to DQA. However, since these
aerobic bacteria contain strong assimilating enzymes
like DSA dehydratase which leads DSA catabolism to
succinate and acetyl-CoA via protocatechuate; such
bacteria do not accumulate DQA and also DSA as
high as acetic acid bacteria.
increased to 0.225
M
Preliminary production of shikimate by a single-
cellular system
As mentioned above, characterization of the en-
zymes relating to enzymatic conversion of quinate to
shikimate was done in this study. In the case of dried
cell experiments, a positive SKDH reaction was de-
tected by increased absorbance at 340 nm with a
sample of the reaction mixture when shikimate
production was measured with SKDH in the presence
of NADP. This strongly indicates that shikimate
production by the single-cellular system of acetic acid
bacteria would be possible, although the reaction
conditions or water contents in the dried cells must be
optimized until the data become reproducible and
convincing. To conˆrm shikimate formation by the
single-cellular system of acetic acid bacteria, quinate
was oxidized ˆrst to DQA, which must be coupled
with DQD and then with SKDH in the presence of
NADPH at pH 7.0. Dried cells (3 mg) were poured
into a 3-ml reaction mixture containing 30 mg DQA
Mutual separation of SKDH, DQD, and SDA de-
hydratase
The cytoplasmic fraction of G. oxydans IFO 3244
was prepared from 90 g of wet cells and put on a
×
DEAE-cellulose column (2 25 cm), which had been
equilibrated with 2 m potassium phosphate, pH
6.5, containing 5 m -mercaptoethanol (BuŠer A).
M
M
b
After the column was washed with BuŠer A, SKDH
was eluted from the column with BuŠer A containing
and shaken on a rotary shaker (200 rpm) at 309C.
Samples were taken periodically for the SKDH reac-
tion. Measurement of shikimate produced was done
with SKDH and NADP in glycine-NAOH, pH 10.0,
and shikimate production was almost proportional to
reaction time and DQA concentration. A similar
system was reconstructed in vitro in the presence of
0.15
BuŠer A containing 0.25
puriˆed by a DEAE-Sephadex A-50 column (1.2
M
KCl and DQD came out from the column with
M
KCl. SKDH was further
×
20 cm), which had been equilibrated with buŠer A.
Elution of SKDH was done by a linear gradient of

2130
O. ADACHI et al.
References
1) Whipp, M. J., Camakaris, H., and Piottard, A. J.,
Cloning and analysis of the shiA gene, which ecodes
the shikimate transport system of Escherichia coli
K-12. Gene, 209, 185–192 (1998).
2) Euverink, G. J. W., Hessels, G. I., Vrijbloed, J. W.,
Coggin, J. R., and Dijkhuizen, L., Puriˆcation and
characterization of a dual function of 3-dehydro-
quinate dehydratase from Amycolatopsis methanoli-
ca. J. Gen. Microbiol., 138, 2449–2457 (1992).
3) Jude, D. A., Ewer, C. D. C., Thalin, J. K. L.,
Davies, G. M., and Nichols, W. W., Transport of the
antibacterial agent (6
S)-6-‰uoroshikimate and other
Fig. 6. Schematic Diagram of Shikimate Production from
Quinate by a Single-cellular System.
The results discussed in this paper are linked and a series of
reaction from quinate to shikimate is schematically drawn.
shikimate analogues by the shikimate transport
system of Escherichia coli
1279, 125–129 (1996).
. Biochim. Biophys. Acta,
4) Mitsuhashi, S., and Davis, B. D., Aromatic biosyn-
thesis. XII. Conversion of 5-dehydroquinic acid to 5-
dehydroshikimic acid by 5-dehydroquinase. Biochim.
Biophys. Acta, 15, 54–61 (1954).
DQD and SKDH, where DQA was given as the start-
ing substrate. DQA was the preferential substrate
to quinate in any experiments. In addition to
NADP, additions of NADP-dependent glucose
5) Mitsuhashi, S., and Davis, B. D., Aromatic biosyn-
thesis. XIII. Conversion of quinic acid to 5-de-
hydroquinic acid by quinic dehydrogenase. Biochim.
Biophys. Acta, 15, 268–281 (1954).
dehydrogenase21) and excess glucose (100
m
mol) con-
ˆrmed shikimate formation as schematically shown
in Fig. 6. Any enzyme yielding NADPH, like
glucose-6-phosphate dehydrogenase22) or 6-phospho-
-gluconate dehydrogenase23) from Gluconobacter
strains can be coupled as NADPH generating en-
zyme. -Glucose dehydrogenase must be suitable for
6) Yaniv, H., and Gilvarg, C., Aromatic biosynthesis.
XIV. 5-Dehydroshikimic reductase. J. Biol. Chem.
,
D
-
213, 787–795 (1955).
7) Balinsky, D., and Davies, D. D., Aromatic biosyn-
thesis in higher plants. 1. Preparation and properties
D
of dehydroshikimic reductase. Biochem. J.
292–296 (1961).
, 80,
D
routine purposes because of the expenses of glucose.
As written above, DSA dehydratase was separated
from DQD. One strategy to pull this system to shiki-
mate production is genetic deletion or inactivation of
DSA dehydratase, which may be the most straight-
forward, because most Gluconobacter strains can
grow using the pentose phosphate pathway, even
though the protocatechuate pathway is inherently
closed. Only a few enzymes in the shikimate pathway
are disclosed in this study. Encouraged with the
results discussed here, it would be possible to op-
timize shikimate production and other intermediates,
like chorismate and prephenate, in the further study.
8) Balinsky, D., and Davies, D. D., Aromatic biosyn-
thesis in higher plants. 2. Mode of attachment of
shikimic acid and dehydroshikimic acid to de-
hydroshikimic reductases. Biochem. J., 80, 296–300
(1961).
9) Balinsky, D., and Davies, D. D., Aromatic biosyn-
thesis in higher plants. 2. Preparation and properties
of dehydroquinase. Biochem. J., 80, 300–303 (1961).
10) Gamborg, O., Aromatic metabolism in plants. III.
Quinate dehydrogenase from mung bean cell suspen-
sion cultures. Biochim. Biophys. Acta, 128, 483–491
(1966).
11) Whiting, G. C., and Coggins, R. A., The oxidation of
D
-quinate and related acids by Acetomonas oxydans.
Biochem. J., 102, 283–293 (1967).
Acknowledgments
12) van Kleef, M. A. G., and Duine, J. A., Bacterial
NAD(P)-independent quinate dehydrogenase is a
quinoprotein. Arch. Microbiol., 150, 32–36 (1988).
A part of this work was done under collaboration
in the Core University Program between Yamaguchi
University and Kasetsart University supported by the
Scientiˆc Cooperation Program from the Japan
Society for the Promotion of Science (JSPS) and the
National Research Council of Thailand (NRCT).
Technical contributions by Mr. Tomonori Funahashi
are highly acknowledged. Grant support to O.A.
from the Uehara Life Science Foundation for 2002
was also grateful.
13) Elsemore, D. A., and Ornston, L. N., The pca-pob
supraoperonic cluster of Acinetobacter calcoaceticus
contains quiA, the structural gene for quinate-shiki-
mate dehydrogenase. J. Bacteriol., 176, 7659–7666
(1994).
14) Elsemore, D. A., and Ornston, L. N., Unusual
ancestry of dehydratases associated with quinate
catabolism in Acinetobacter calcoaceticus
teriol., 177, 5971–5978 (1995).
. J. Bac-
15) Tanasupawat, S., Adachi, O., Yoshihara, N.,
Toyama, H., and Matsushita, K., Puriˆcation
and characterization of quinoprotein quinate de-

Formation of 3-Dehydroquinate and Other Intermediates in Shikimate Pathway
2131
hydrogenase, in the proceedings of the 2nd Joint
Seminar of JSPS-NRCT on Thermotolerant Microbi-
al Resources, Nov. 21–25, p. 36 (2000).
beads. Biotechnol. Appl. Biochem., 27, 25–29 (1998).
20) Tochikura, T., Kawai, H., Kawaguchi, K.,
Mugibayashi, Y., and Ogata, K., Sugar nucleotide
fermentation by yeasts. In ``Fermentation Technolo-
gy Today'' Proc. IVth Intl. Ferment. Symp., ed.
Terui, G., Soc. Ferment. Technol., Japan, pp.
463–471 (1972).
16) Adachi, O., Fujii, Y., Ano, Y., Moonmangmee, D.,
Toyama, H., Shinagawa, E., Theeragool, G.,
Lotong, N., and Matsushita, K., Membrane-bound
sugar alcohol dehydrogenase in acetic acid bacteria
catalyzes
L
-ribulose formation and NAD-dependent
21) Adachi, O., Matsushita, K., Shinagawa, E., and
Ameyama, M., Crystallization and characterization
ribitol dehydrogenase is independent of the oxidative
fermentation. Biosci. Biotechnol. Biochem.
115–125 (2001).
,
65,
of NADP-dependent -glucose dehydrogenase from
D
Gluconobacter suboxydans. Agric. Biol. Chem., 44,
17) Yoshida, S., and Hasegawa, M., Microcolorimetric
method for the determination of shikimic acid. Arch.
Biochem. Biophys., 70, 377–381 (1957).
18) Saeki, A., Vinegar production using immobilized
Acetobacter aceti cells entrapped in calcium alginate
gel beads. J. Jpn. Soc. Food Sci. Technol., 37,
191–198 (1990).
19) Das, K., Kayastha, A. M., and Malhotra, O. P.,
Immobilization of urease from pigeonpea (Cajanus
cajan L.) in polyacrylamide gels and calcium alginate
301–308 (1980).
22) Adachi, O., Matsushita, K., Shinagawa, E., and
Matsushita, K., Occurrence of old yellow enzyme in
Gluconobacter suboxydans, and the cyclic regenera-
tion of NADP. J. Biochem., 86, 699–709 (1979).
23) Adachi, O., Osada, K., Matsushita, K., Shinagawa,
E., and Ameyama, M., Puriˆcation, crystallization
and properties of 6-phosphogluconate dehydrogenase
from Gluconobacter suboxydans
Chem., 46, 391–398 (1982).
.
Agric. Biol.