High-yield production of cis,cis-muconic acid from catechol in aqueous solution by biocatalyst

Article abstract of DOI:10.1246/cl.2011.381

A fed-batch process was used to produce cis,cis-muconic acid from catechol by recombinant Escherichia coli cells expressing the catA gene, which encodes the Pseudomonas putida mt-2 catechol 1,2-dioxygenase responsible for catalyzing ortho-cleavage of catechol, as biocatalysts. We succeeded in producing 415mM (59.0 g L^-1) cis,cis-muconic acid in aqueous solution without generation of by-products in 12 h under the optimal conditions with successive addition of 10mM catechol. The molar conversion yield based on the amount of consumed catechol was the theoretical value of 100% (mol mol^-1).

Full text of DOI:10.1246/cl.2011.381

doi:10.1246/cl.2011.381
Published on the web March 12, 2011
381
High-yield Production of cis,cis-Muconic Acid from Catechol
in Aqueous Solution by Biocatalyst
Aya Kaneko, Yoshitaka Ishii, and Kohtaro Kirimura*
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University,
3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555
(
Received January 7, 2011; CL-110022; E-mail: kkohtaro@waseda.jp)
A fed-batch process was used to produce cis,cis-muconic
acid from catechol by recombinant Escherichia coli cells
expressing the catA gene, which encodes the Pseudomonas
putida mt-2 catechol 1,2-dioxygenase responsible for catalyzing
ortho-cleavage of catechol, as biocatalysts. We succeeded in
OH
OH
COOH
COOH
+
O
2
Catechol
1
,2-dioxygenase
Catechol
CA)
cis,cis-Muconic acid
(
(ccMA)
¹
1
producing 415 mM (59.0 g L ) cis,cis-muconic acid in aqueous
solution without generation of by-products in 12 h under the
optimal conditions with successive addition of 10 mM catechol.
The molar conversion yield based on the amount of consumed
Figure 1. Enzymatic production of ccMA from CA by
catechol 1,2-dioxygenase (CatA; EC1.13.11.1) from P. putida
mt-2.
¹
1
catechol was the theoretical value of 100% (mol mol ).
rapid production of ccMA by a biocatalyst in aqueous solution
under environmentally benign conditions.
cis,cis-Muconic acid (ccMA, 1,3-butadiene-1,4-dicarbox-
ylic acid) has a dicarboxylic acid structure with conjugated
double bonds (Figure 1). This compound is expected to gain
widespread use as a raw material for new functional resins,
As prerequisites for ccMA production, we examined the
enzymatic properties of the recombinant His-tagged CatA in
comparison to those of native CatA reported previously. The
9
purified His-tagged CatA yields a single 34-kDa band in
SDS­polyacrylamide gel electrophoresis (data not shown).
The specific activity of the purified His-tagged CatA was 25.7
1
pharmaceuticals, and agrochemicals. For example, ccMA can
be easily converted to adipic acid, which is used as a commodity
chemical for production of nylon-6,6 by hydrogenation at 50 psi
¹
1
¹1
¯mol min mg -protein. This specific activity is the same level
2
¹1
¹1
for 3 h at room temperature. Furthermore, highly stereoregular
as that (31.6 ¯mol min mg -protein) of a purified native CatA
9
polymers can be produced through topochemical polymerization
previously reported. The optimal temperature of the purified
3
of muconic acid esters. These polymers are useful as functional
His-tagged CatA was 35 °C although the optimal temperature of
the purified native CatA has not yet been reported. The optimal
pH of His-tagged CatA was 7.5 and is identical to that of the
resins. Verrucarin is an antibiotic that can be synthesized from
ccMA by organic synthesis.4
9
ccMA had been reported to be synthesized from phenol by
purified native CatA.
5
peracetic acid oxidation, but the reaction also generates by-
We confirmed that whole cells of recombinant E. coli
BL21(DE3) expressing catA, instead of the purified His-tagged
CatA or a cell-free extract of the recombinant E. coli cells, can
be directly used to produce ccMA from CA. This whole-cell
reaction is advantageous for practical production of ccMA
because no additional pretreatment, such as disruption of
recombinant E. coli cells or purification of the recombinant
His-tagged CatA, is necessary for the reaction. We confirmed
that E. coli BL21(DE3) cells without pEcatA show no activity
toward ccMA or CA, and that they did not consume ccMA or
CA in whole-cell reactions.
products. Therefore, biocatalytic methods have been recently
developed based on site-specific oxidation of aromatic com-
pounds such as benzoate and toluene using enzymes and
6­8
microbial cells, because they enable selective production of
ccMA under environmentally benign conditions. For example,
Mizuno et al. reported that a mutant strain of Arthrobacter sp.,
which lacks muconate-lactonizing enzyme, could produce
¹
1
4
4.1 g L in 48 h in a 30-1 jar fermenter by successive feeding
6
of benzoate. However, biocatalytic methods have not yet been
applied in industrial production of ccMA, probably due to low
yields, formation of by-products, and/or long reaction periods.
As shown in Figure 1, ccMA production by catechol 1,2-
dioxygenase (CatA; EC 1.13.11.1), which catalyzes the ortho-
cleavage of CA, is a direct 1-step conversion, and this enzymatic
reaction is efficient and advantageous for recovery and purifi-
cation of ccMA in practical processes because it does not
generate any by-product. However, there has been no report
concerning high-yield production of ccMA using CA as a
starting substrate by enzymatic or whole-cell reactions using
CatA.
To optimize conditions for ccMA production from CA in
whole-cell reaction, we examined the effects of several
1
0
parameters. Because the optimum temperature of the purified
His-tagged CatA is 35 °C, the whole-cell reaction was first
performed at 35 °C. Effects of CA concentration on the whole-
cell reaction by E. coli cells BL21 (DE3) with pEcatA were
examined at 35 °C. As shown in Figure 2, below CA concen-
trations of 40 mM, CA was stoichiometrically converted to
ccMA, and the pH was decreased from 7.5 to 6.2 with
accompanying production of ccMA. At 50 mM, CA was not
completely degraded to ccMA. Above CA concentration of
60 mM, very little CA was degraded, and ccMA production was
essentially stopped. These effects may be due to the toxic effects
In this report, we describe the production of ccMA without
by-product through whole-cell reaction of recombinant Esche-
richia coli cells highly expressing the gene (catA) encoding
CatA. To our knowledge, this is the first report of high-yield and
11
of CA against the enzymes and microbial cells. Thus, under
Chem. Lett. 2011, 40, 381­383
© 2011 The Chemical Society of Japan www.csj.jp/journals/chem-lett/

3
82
1
00
8
6
4
2
0
80
60
40
20
0
10
8
(A)
8
6
4
2
0
0
0
0
0
6
4
2
0
0
20
40
60
80
100
0
50
100
Initial CA/mM
Time/min
5
4
3
2
1
00
00
00
00
00
0
14
12
Figure 2. Effects of initial CA concentration on ccMA
production by whole-cell reaction using E. coli BL21 (DE3)/
pEcatA cells. ccMA production was performed under the
following conditions. The reaction mixture contained CA at
each concentration, 10­100 mM, and E. coli cells (O.D.600 = 10)
in 500 mM Tris-acetate buffer (pH 7.5) in 18 mmº test tube
(B)
10
8
6
4
2
(total volume of 0.6 mL). Each reaction was performed at 35 °C
with shaking at 160 rpm for 2 h. Symbols; square, ccMA; circle,
CA; triangle, pH.
0
2
4
6
8
10 12
the conditions tested, feeding of CA at concentrations less than
Time/h
40 mM was found to be effective for high-yield production of
ccMA. Effects of shaking speed in relation to oxygen supply on
the rate of ccMA production were also examined, and we
confirmed that shaking speeds above 180 rpm were necessary for
Figure 3. ccMA production through fed-batch process by
whole-cell reaction using recombinant E. coli BL21 (DE3)/
pEcatA cells under the optimal conditions with 0.1 mM FeCl3.
¹
1
rates of ccMA production higher than 1.5 mM min (Support-
(
1
A) Initial stage (0­90 min) of the reaction. (B) The reaction for
2 h. Both of the reactions, (A) and (B), were performed at 20 °C
10
ing Information; SI, Figure S1 ). Effects of temperature on
ccMA production were examined, and we confirmed that the
ccMA production rates were almost constant within a temper-
with 0.1 mM FeCl3 with successive feeding of CA. The fed-
batch process was initiated by addition of 10 mM CA and
continued with successive additions of 0.25 mL of 4 M CA (final
concn: 10 mM) as the substrates and 0.15 mL of 12.7 M NaOH
solution for adjustment of the pH at 15-min intervals. A 0.4 mL
portion of the reaction mixture was collected at 15-min intervals
for measurement of CA and ccMA. Symbols; square, ccMA;
circle, CA; triangle, pH.
10
ature range of 10­30 °C (SI, Figure S2 ).
Effects of temperature on continuous CA degradation
through the fed-batch process with successive additions of CA
1
0
were therefore examined in the range of 10­30 °C. The
terminal point of successive CA addition for the fed-batch
process was determined as the point at which CA degradation is
no longer observed and the concentration of CA exceeds 5 mM
in the reaction mixture. The terminal points of the fed-batch
process with successive additions of CA at 10, 20, and 30 °C
were 31, 32, and 24 additions of CA (7.75, 8.00, and 6.00 h),
terminal point of successive CA addition was prolonged for
more than 1 h relative to reaction carried out without the
additive. The effects of the concentration of FeCl3 on continuous
CA degradation through the fed-batch process with successive
additions of CA were the same in the range of 0.1­5.0 mM (data
not shown). Based on these results, further ccMA production
through the fed-batch process was performed under the
conditions with the addition of 0.1 mM FeCl3.
10
respectively (SI, Table S1 ). Although ccMA production rates
were almost constant over the temperature range of 10­30 °C
1
0
(
Figure S2 ), the maintenance of CA degradation in the fed-
batch process was affected by temperatures in the range of 10­
0 °C, and the optimum temperature was determined to be 20 °C.
3
Based on these results, further ccMA production was performed
at 20 °C with rotary shaking at 180 rpm and successive additions
of 10 mM CA.
The fed-batch process using whole cells with successive
additions of CA was performed at 20 °C with addition of 0.1 mM
FeCl . Solutions of 10 mM CA and NaOH were added at 15-min
3
CatA contains nonheme iron(III) as a cofactor in its active
center.12 Wu et al. reported that addition of Fe(III)­EDTA
complex was effective to increase ccMA production from
benzoate by Sphingobacterium sp. strain GCG.13 Effects of the
addition of Fe(II) and Fe(III) on continuous CA degradation
through the fed-batch process were examined with successive
additions of CA. We used 1 mM FeCl , 1 mM FeCl ­EDTA
intervals. The accumulation of undegraded CA was not detected
until 8 h, and ccMA production reached 300 mM (42.6 g L ) at
¹1
8 h. After 48 additions of CA over 12 h, the final concentration
¹1
of ccMA reached 415 mM (59.0 g L ) without by-product as
shown in Figure 3. The molar conversion yields based on the
amount of consumed CA were almost the theoretical value of
¹
1
100% (mol mol ), until 12 h.
3
3
complex, 1 mM FeSO4­10 mM ascorbic acid, and 10 mM
ascorbic acid as additives. Among the tested compounds
In conclusion, we have demonstrated that high-yield and
rapid ccMA production can be achieved using whole cells of
E. coli expressing catA as a biocatalyst in aqueous solution
under environmentally benign conditions. Through the direct 1-
containing Fe(II) or Fe(III), addition of FeCl showed a clear
3
effect on prolongation of continuous CA degradation. The
Chem. Lett. 2011, 40, 381­383
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

3
83
step conversion from CA, we succeeded in producing 415 mM
2
3
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¹1
(59.0 g L ) ccMA without generation of by-product in 12 h. The
molar conversion yields based on the amount of consumed CA
¹
1
were almost the theoretical value of 100% (mol mol ) during
2 h of the whole period of reactions. These results demonstrate
4
5
6
1
that this biocatalytic method enables us to produce ccMA with
high yield in a short time. In comparison to other bioprocesses
for ccMA production,6 the fed-batch process for ccMA
production from CA described in this report is advantageous
from the viewpoints of practical production, since ccMA
produced without by-product in the reaction mixture is easily
separated from CA as a substrate through an ion-exchange
column. Therefore, the method described in this report repre-
sents a significant model process for the biocatalytic production
of ccMA from CA by a whole-cell reaction.
­8
7
8
9
10 Supporting Information is available electronically on the
CSJ-Journal Web site, http://www.csj.jp/journals/chem-lett/
index.html.
This work was partly supported by the Global-COE program
Practical Chemical Wisdom” from MEXT, Japan.
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“
References and Notes
1
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13 C.-M. Wu, T.-H. Lee, S.-N. Lee, Y.-A. Lee, J.-Y. Wu,
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Chem. Lett. 2011, 40, 381­383
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/