High shikimate production from quinate with two enzymatic systems of acetic acid bacteria

Article abstract of DOI:10.1271/bbb.60259

3-Dehydroshikimate was formed with a yield of 57-77% from quinate via 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. 3-Dehydroshikimate was then reduced to shikimate (SKA) with NADP-dependent SKA dehydrogenase (SKDH) from the same organism. When SKDH was coupled with NADP-dependent D-glucose dehydrogenase (GDH) in the presence of excess D-glucose as an NADPH regenerating system, SKDH continued to produce SKA until 3-dehydroshikimate added initially in the reaction mixture was completely converted to SKA. Based on the data presented, a strategy for high SKA production was proposed.

Full text of DOI:10.1271/bbb.60259

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
High Shikimate Production from Quinate with Two
Enzymatic Systems of Acetic Acid Bacteria
Osao ADACHI, Yoshitaka ANO, Hirohide TOYAMA & Kazunobu MATSUSHITA
To cite this article: Osao ADACHI, Yoshitaka ANO, Hirohide TOYAMA & Kazunobu MATSUSHITA
(2006) High Shikimate Production from Quinate with Two Enzymatic Systems of Acetic Acid
Bacteria, Bioscience, Biotechnology, and Biochemistry, 70:10, 2579-2582, DOI: 10.1271/
bbb.60259
To link to this article: http://dx.doi.org/10.1271/bbb.60259
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Date: 04 June 2016, At: 14:49

Biosci. Biotechnol. Biochem., 70 (10), 2579–2582, 2006
Communication
High Shikimate Production from Quinate with Two Enzymatic Systems
of Acetic Acid Bacteria
Osao ADACHI,y Yoshitaka ANO, Hirohide TOYAMA, and Kazunobu MATSUSHITA
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
Received May 10, 2006; Accepted September 4, 2006; Online Publication, October 7, 2006
[doi:10.1271/bbb.60259]
3-Dehydroshikimate was formed with a yield of 57–
77% from quinate via 3-dehydroquinate by two succes-
sive enzyme reactions, quinoprotein quinate dehydro-
genase (QDH) and 3-dehydroquinate dehydratase, in
the cytoplasmic membranes of acetic acid bacteria. 3-
Dehydroshikimate was then reduced to shikimate (SKA)
with NADP-dependent SKA dehydrogenase (SKDH)
from the same organism. When SKDH was coupled with
NADP-dependent D-glucose dehydrogenase (GDH) in
the presence of excess D-glucose as an NADPH re-
generating system, SKDH continued to produce SKA
until 3-dehydroshikimate added initially in the reaction
mixture was completely converted to SKA. Based on the
data presented, a strategy for high SKA production was
proposed.
effective and convenient SKA production contributing
to oseltamivir synthesis.
QDH and 3-dehydroquinate dehydratase are localized
predominantly on the outer surface of the cytoplasmic
membranes of some species of Gluconobacter strains,
and quinate is oxidized to 3-dehydroshikimate via 3-
dehydroquinate in a sequential manner.4–6) In the
cytoplasm, SKDH (EC 1.1.1.25) catalyzes a reversible
reaction of SKA oxidation to 3-dehydroshikimate and
3-dehydroshikimate reduction to SKA. In the previous
papers, we proposed SKA production by a single
cellular system of acetic acid bacteria,4,5,7,8) but many
times of trial and error combining the two separately
localized enzymatic systems taking place outside and
inside the cells together have given insufficient produc-
tion of SKA. As a positive response to global needs for
oseltamivir, we have developed a better strategy for high
SKA production allowing the two separately localized
enzymatic systems to work sequentially. Dried cells or
the membrane fraction involving QDH and 3-dehydro-
quinate dehydratase can be used for 3-dehydroshikimate
production in the first reaction (enzymatic system 1 in
Fig. 1). The second reaction is a coupling reaction
composed of two cytosolic enzymes, SKDH and GDH
(EC 1.1.1.47) as an NADPH re-generating enzyme
(enzymatic system 2 in Fig. 1). The coupling reaction
of the two cytosolic enzymes in the presence of excess
D-glucose worked well, as expected, until 3-dehydro-
shikimate added initially was converted completely to
SKA. The overall reaction carried out for SKA produc-
tion by the two different enzymatic systems is depicted
in Fig. 1.
Key words: Gluconobacter oxydans; NADP-shikimate
dehydrogenase; oxidative fermentation; qui-
nate oxidation; shikimate production
SKA is a key intermediate for aromatic amino acids
as well as for large numbers of antibiotics, alkaloids, and
herbicides. Recently, another immediate impact has
been given to SKA as a precursor for oseltamivir
synthesis protecting people from pandemic flu infection.
In spite of global warnings from WHO about pandemic
flu, including avian influenza, there are insufficient
measures for oseltamivir over the world. One reason is
the technical difficulties in preparing SKA, because
two different metabolic pathways, the glycolysis and
the pentose phosphate pathway, must be combined in
forming 3-deoxy-arabino-heputulosonate 7-phosphate
before reaching SKA. Furthermore, the metabolic
location of SKA is far from D-glucose and it is difficult
to lead the metabolic flow to SKA production by classic
fermentation technology or by modern molecular bio-
technology.1,2) Although resources are limited, SKA is
also produced by extraction from plants like Illicium
anisatum or I. verum.3) Total synthesis of SKA by
organic chemistry has not been practical. Nevertheless,
it is necessary to develop a novel method for more
Dried cells and membrane fraction were prepared
from Gluconobacter oxydans IFO 3244, as described
previously.4) As will be explained elsewhere, a highly
purified SKDH having 60 units/mg of SKA oxidation
activity at pH 10.0 and 25 units/mg of 3-dehydro-
shikimate reduction activity at pH 6–7 was prepared.
According to the previous paper,9) GDH having 120
units/mg at pH 9 was purified from the same organisms.
For enzymatic determination of SKA and 3-dehydro-
y
To whom correspondence should be addressed. Tel: +81-83-933-5857; Fax: +81-83-933-5859; E-mail: osao@yamaguchi-u.ac.jp

2580
O. ADACHI et al.
System 1: Oxidative fermentation for 3-dehydroshikimate formation from quinate
QDH
3-Dehydroquinate dehydratase
COOH
HO COOH
HO
OH
HO COOH
O
OH
O
OH
OH
OH
OH
3-Dehydroquinate
Quinate
3-Dehydroshikimate
System 2: Conversion of 3-dehydroshikimate to shikimate by SKDH coupled with GDH
COOH
COOH
SKDH
HO
OH
O
OH
NADPH
OH
OH
3-Dehydroshikimate
Shikimate
NADP
Glucono-δ-lactone
Glucose
GDH
Fig. 1. Schematic Drawings of Overall Reaction for SKA Production.
shikimate, SKDH has been used extensively because it
is highly reactive only with SKA and 3-dehydroshiki-
mate. On the other hand, quinate, 3-dehydroquinate, 3-
dehydroshikimate, and SKA were detected or identified
by paper chromatography, as described by Yoshida and
Hasegawa.10) Since 3-dehydroquinate and 3-dehydro-
shikimate are not commercially available, they were
prepared after chromatographic separation from the
reaction products of quinate oxidation by dried cells, as
described previously.4)
DSA
DQA
QA
QA DQA
DSA
For enzymatic system 1, the dried cells or the
membrane fraction were incubated with quinate under
different pHs (pH 3–10), and the 3-dehydroshikimate
formed was measured using the deproteinized super-
natants. In most cases, 1 g of quinate was incubated with
2.5 g of dried cells or 5 g of dried membrane fraction
in 100 ml of McIlvaine buffer, pH 3.0–8.0, in a 500-ml
Erlenmeyer flask. For the reactions at pH 9.0 and 10.0,
50 mM glycine–NaOH buffers were used. Under the
conditions above, the initial quinate concentration
corresponded to 52 mM. Incubation was carried out at
30 ꢀC under shaking. Under these conditions, QDH
oxidized quinate transferring electrons generated to
ubiquinone-10 in the cytoplasmic membrane and then
to the terminal oxidase functioning as ubiquinol oxidase,
in which molecular oxygen is reduced to water. The
cells or the membrane fraction was removed by
centrifugation after the addition of trichloroacetic acid
(TCA) to 5.5%. After 50 times dilution of the super-
natant with 0.1 M acetate buffer, pH 6.0, 3-dehydro-
shikimate reduction activity was measured at 25 ꢀC with
SKDH by incubating an aliquot of the diluted super-
natant with 0.25 mmol of NADPH and the enzyme
(1 unit of 3-dehydroshikimate reduction activity) in
0.1 M acetate buffer, pH 6.0, in a total volume of 1.0 ml.
Since SKDH was highly specific to 3-dehydroshikimate
reduction and SKA oxidation, either the rate assay or the
Fig. 2. Paper Chromatogram of 3-Dehydroshikimate Production.
Dried cells or dried membrane preparation was incubated with
quinate (0.1 M) under various pHs. Reaction mixtures obtained after
20 h incubation were developed by paper chromatography. Standard
quinate, 3-dehydroquinate, and 3-dehydroshikimate were also
developed, as indicated. QA, DQA, and DSA mean quinate, 3-
dehydroquinate, and 3-dehydroshikimate respectively.
end point measurement was valid to estimate the
concentration of the two substrates by reading the
absorbance change at 340 nm. 3-Dehydroshikimate was
detected as a brilliant yellow spot on a paper chromato-
gram, although the efficiency and reaction rate for 3-
dehydroshikimate production varied under the condi-
tions employed. Under the conditions above, incubation
at pH 7.0–9.0 for 20 h gave the fastest conversion rate
of quinate to 3-dehydroshikimate (Fig. 2). The apparent
concentration of 3-dehydroshikimate reached 30–40 mM,
indicating 57–77% conversion efficiency from quinate.
At pH 6.0, about 50% of quinate oxidation appeared,
while no positive 3-dehydroshikimate production was
observed at pH 4.0 or 5.0, although the quinate initially
added was almost completely converted to 3-dehydro-
quinate, judging from the developed color spots. Quinate
and 3-dehydroquinate gave almost the same Rf -values on

High Shikimate Production from Quinate
2581
the paper chromatogram under the conditions employed,
but they were identified with each other because of
different colors developed. Quinate gave a pink spot
while 3-dehydroquinate was colored yellow, as noted
in the previous paper.4) Under prolonged incubation
with dried cells at pH 7.0 to 9.0 for over 50 h, the 3-
dehydroshikimate produced was readily converted to
protocatechuate, an unfavorable byproduct for 3-dehy-
droshikimate production. More stable 3-dehydroshiki-
mate production was observed when quinate oxidation
was conducted for 2 d at rather acidic pH, pH 4 to 6
(data not shown). With more minute control of quinate
oxidation, higher yields of 3-dehydroshikimate are
expected in future. The fractions containing 3-dehy-
droshikimate from Dowex column chromatography
were neutralized with alkali and freeze-dried to concen-
trate the compound. The dried material was dissolved in
a small volume of water and insoluble materials were
removed by centrifugation. The 3-dehydroshikimate
thus prepared was determined to be 2.1 M by assay of
3-dehydroshikimate reduction activity, and was used for
SKA production.
0
15 30 60 90 120 240 DSA
SKA
125
100
75
50
25
0
The reaction mixture for enzymatic system 2 con-
sisted of 250 mg of 3-dehydroshikimate (about 1.4
mmol), NADP (5 mmol), SKDH (20 units of 3-dehy-
droshikimate reduction activity), GDH (1,000 units), and
excess D-glucose (5 mmol). The total volume was
adjusted to 20 ml with 30 mM potassium phosphate
buffer, pH 7.0, containing 5 mM ꢀ-mercaptoethanol and
5 mM EDTA. Incubation was carried out at 25 ꢀC by
standing the reaction mixture. Part of the reaction
mixture (0.1 ml) was taken out periodically and mixed
with 10 ml of 60% TCA to terminate the reaction. After
removal of precipitate, an aliquot of the reaction
mixtures (5 ml) was developed by paper chromatogra-
phy, and two different coloring reagents were sprayed
under the conditions employed previously.4) A clear red
spot of SKA appeared, as shown in the upper frame of
Fig. 3. Under the conditions above, the reduction of 3-
dehydroshikimate to SKA was complete within 75 min.
As expected, the spot of 3-dehydroshikimate almost
disappeared from the reaction mixture before 90 min
incubation. The SKA formed was also measured with an
aliquot of the reaction mixture by end point measure-
ment with SKDH (1 oxidation unit) in 50 mM glycine–
NaOH, pH 10.0, in the presence of 0.25 mmol of NADP.
The conversion rate of 3-dehydroshikimate to SKA by
the coupled enzyme reaction was almost 100% (lower
frame in Fig. 3) and showed a good coincidence with the
results from paper chromatography.
0
50
100
150
200
250
Incubation time (min)
Fig. 3. Conversion of 3-Dehydroshikimate to SKA by SKDH
Coupled with GDH.
Upper frame: The coupled enzymatic reaction by two NADP-
dependent enzymes was conducted in the presence of 3-dehydro-
shikimate and NADP as described in the text. Part of the reaction
mixture was taken out periodically, as indicated, and mixed with
TCA to terminate the reaction. An aliquot of the reaction mixture
(5 ml) was spotted and developed by paper chromatography. Lower
frame: An aliquot of the terminated reaction mixture (25 ml) was
diluted 50 times with 50 mM of glycine–NaOH, pH 10.0. SKDH
activity was measured in 50 mM of glycine–NaOH, pH 10.0, with
30 ml of the diluted samples by the end point measurement of SKA
oxidation. After 1 h incubation, the optical density was measured at
340 nm and the conversion rate of 3-dehydroshikimate to SKA was
determined as plotted.
tion should continue until the given 3-dehydroshikimate
is totally converted to SKA in the same way as
exemplified in this study. There should be no negative
factors against SKA production when the two different
enzymatic systems are employed in a sequential manner.
Thus other forms of SKDH and GDH such as immo-
bilized enzyme catalysts should also be successful for
SKA production, as suggested in the previous report.4)
In order to make these results practical for industrial
applications like other oxidative fermentation, we must
confirm that the system developed here is valid even
in increased substrate concentrations. Since abundant
quinate can be supplied readily from industrial process-
ing of coffee wastes,11–13) scaling up of this enzymatic
system for higher SKA production should be possible to
provide the direct precursor for oseltamivir, contributing
to prevent unexpected pandemic flu.
Since SKDH showed strong reactivity with 3-dehy-
droshikimate even in highly diluted solution, the
reaction system for SKA production can be scaled up
successfully with some increased SKDH and GDH. It
may not be necessary to increase NADP or NADPH if
NADPH re-generation is guaranteed with excess D-
glucose. The above experiment allowed us to finish
within the expected reaction period. Thus SKA produc-

2582
O. ADACHI et al.
membrane-bound quinoprotein quinate dehydrogenase.
Biosci. Biotechnol. Biochem., 67, 2115–2123 (2003).
Acknowledgment
6) Vangnai, A. S., Toyama, H., De-ekanamkul, W.,
Yoshihara, N., Adachi, O., and Matsushita, K., Quinate
oxidation in Gluconobacter oxydans IFO 3244: purifi-
cation and characterization of quinoprotein quinate
dehydrogenase. FEMS Microbiol. Lett., 241, 157–162
(2004).
The quinate used in this work was generously
provided by Fuji Chemical Industry. Co., Ltd.
(Wakayama, Japan). Part of this work was conducted
under the 2005–2006 Venture Business Laboratory
Program of Yamaguchi University.
7) Adachi, O., Moonmangmee, D., Shinagawa, E., Toyama,
H., Yamada, M., and Matsushita, K., New quinoproteins
in oxidative fermentation. Biochim. Biophys. Acta, 1647,
10–17 (2003).
8) Adachi, O., Moonmangmee, D., Toyama, H., Yamada,
M., Shinagawa, E., and Matsushita, K., New develop-
ments in oxidative fermentation. Appl. Microbiol. Bio-
technol., 60, 643–653 (2003).
9) Adachi, O., Matsushita, K., Shinagawa, E., and
Ameyama, M., Crystallization and chracterization of
NADP-dependent D-glucose dehydrogenase from Glu-
conobacter suboxydans. Agric. Biol. Chem., 44, 301–308
(1980).
10) Yoshida, S., and Hasegawa, M., Microcolorimetric
method for the determination of shikimic acid. Arch.
Biochem. Biophys., 70, 377–381 (1957).
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