Biochemical Engineering Journal 29 (2006) 35–40

Microbial synthesis of cis,cis-muconic acid from benzoate bySphingobacterium sp. mutants

Chun-Ming Wu a, Chang-Chan Wu a, Chien-Chou Su a, Sung-Nung Lee a,∗,Yung-An Lee b, Jiumn-Yih Wu c

a Department of Chemistry, Fu Jen Catholic University, Hsinchuang, Taipei, Taiwan 242, ROCb Department of Life Science, Fu Jen Catholic University, Hsinchuang, Taipei, Taiwan 242, ROCc Department of Chemical Engineering, I-Shou University, Ta-Hsu, Kaohsiung, Taiwan 840, ROC

Received 19 October 2004; accepted 16 February 2005

Abstract

The mutants used in this study, Sphingobacterium sp. M4113/M4115, were originated from a wild-type strain GCG, which was isolatedfrom the sewage of a styrene monomer-manufacturing factory. The culturing medium contains appropriate amounts of K2HPO4, (NH4)2SO4,Miiyt©

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1

ppbdioawtd

mch

1d

gSO4·7H2O, Na-succinate, yeast extract and EDTA–FeCl3 complex. The highest conversion yield was obtained with mutant strain M4115,n which more than 0.560 g of cis,cis-muconic acid was accumulated in 28 h from a Na-benzoate solution of 2.0 g/l (∼28% yield). Growthnhibition appeared as the concentration of Na-benzoate exceeded 3.0 g/l. In addition, an increase of 17% and 6% of cis,cis-muconic acid inield were observed for M4115 and M4113, respectively, as EDTA–FeCl3 complex was employed. It’s believed that this complex can enhancehe yield of catechol 1,2-dioxygenase, and therefore, the yield of cis,cis-muconic acid according to �-ketoadipate biosynthesis pathway.

2005 Elsevier B.V. All rights reserved.

eywords: Aerobic process; Biosynthesis; Bioprocess design; Global environment; cis,cis-Muconic acid; Sphingobacterium sp

. Introduction

Adipic acid is an important industrial chemical, whoseroduction is necessary for the manufacture of nylon 66,olyurethane, acidulant in baking powder, insecticides andactericides [1]. The worldwide industrial adipic acid pro-uction is up to 2.3 million metric tons per year [2]. Mostndustrial processes of adipic acid production use nitric acidxidation of cyclohexanol, cyclohexanone or both, which isccessible from benzene [3,4]. An inevitable stoichiometricaste (N2O), which is emitted from the nitric oxidation reac-

ion is commonly thought to cause global warming and ozoneepletion as well as acid rain and smog.

On the other hand, cis,cis-muconic acid, an potential rawaterial for new functional resin, pharmaceuticals and agri-

ultural chemicals, can be easily converted to adipic acid byydrogenation at 50 psi for 3 h at room temperature. Thus, the

∗ Corresponding author. Tel.: +886 2 2905 2475; fax: +886 2 2791 8687.E-mail address: chem1000@mails.fju.edu.tw (S.-N. Lee).

development of a benzene-free route for adipic acid synthesishas been pursued with great enthusiasm. Several microbialstrains were reported for producing cis,cis-muconic acid fromaromatic compounds such as benzene, benzoate, toluene orcatechol [5–7]. A mutant strain of Arthrobacter sp. accu-mulated 44 g/l of cis,cis-muconic acid in 2 days culture wasreported by Mizuno and Yoshikawa [7]. Other microorgan-isms like Pseudomonas sp., Mycobacterium sp., Acinetobac-ter sp. and Micrococcus sp. were also attempted to use forproduction of cis,cis-muconic acid [8,9]. A mutant strainof Pseudomonas sp. was employed to synthesize cis,cis-muconic acid from toluene, reported by Maxwell [10]. Anbenzene-free synthesis route has recently been establishedby frost, who used a microbial catalyst (i.e. a mutant ofEscherichia coli) enzymatically converted d-glucose into cis,cis-muconic acid (ca. 24% yield) [11].

In our previous study [12], a Sphingobacterium sp. wasisolated from the sewage of a styrene monomer manufactur-ing factory located in Taiwan. The bacterium can convertbenzoic acid to cis,cis-muconic acid and gave a yield of

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

oi:10.1016/j.bej.2005.02.034

36 C.-M. Wu et al. / Biochemical Engineering Journal 29 (2006) 35–40

100 mg/l with a medium contain 2.4 g/l of benzoic acid. In thisstudy, two mutant strains generated from the original wild-type GCG strain, Sphingobacterium sp. M4113 and M4115were obtained from treatment of the original GCG strain witha mutagenesis agent, N-methyl-N′-nitrosoguanidine (NTG).The effect of the culture conditions such as types of carbonsources, medium compositions and the involvement of metalcomplex on the yield of cis,cis-muconic acid were examined.

2. Materials and methods

2.1. Materials

Sodium succinate and catechol were obtained from SigmaChemical Co. (USA). Yeast extract powder, beef extract pow-der and pure agar were obtained from Difco (Detroit, Michi-gan, USA). cis,cis-Muconic acid standard for HPLC and N-methyl-N′-nitrosoguanidine for mutagenesis were obtainedfrom Fluka (Switzerland). All other chemicals were ofreagent grade and were purchased from J.T. Baker andRiedel-deHaen (Germany). The compositions of variousmediums are shown in Table 1. The BY series representthe mediums that contain sodium benzoate as the carbonsource and ammonium sulfate as the nitrogen source andyeast extract. The SBY series are mediums that containsclrdi

2

sgsfI

a

TC

A

NNKM(YEFp

the general technique of Adelberg et al. [13] and physicalmutagenesis using short wave UV irradiation. The mutagen-esis procedure is outlined in Fig. 1. After culturing the cell tothe middle logarithmic growth phase for NTG mutagenesis,the cell suspensions contained a DCW (dry cell weight) valueof 250 (mg/l) were incubated for 45 min at 30 ◦C and shakenwith 500 mg/l of NTG. The cells were recovered by centrifu-gation at 5000 × g for 20 min and were used to inoculate theenriched culture experiments. Mutagenesis derived by UVirradiation was performed by placing 10 ml of microbial sus-pension on a plastic plate, which was exposed to a germicidalUV lamp (λ ∼ 260 nm) at a distance of 30 cm for 15 min. Theaction of NTG/UV mutagenesis can cause 99.99% death ofthe cell population, as determined by CFU (colony formingunit) on the beef extract nutrient (BP) medium plates main-tained at 30 ◦C for 24 h before and after mutagenesis. Thenthe mutagenized cells were re-inoculated to SBY3 mediumshaking flask, and cultured at 30 ◦C for 24 h.

2.3. Screen mutants

After culturing in SBY3 medium for 24 h, the mutage-nized cells were harvested and washed twice with physio-logical saline, and then incubated with BY medium for 1 h.The PC (penicillin-G/d-cycloserine) agent [14] was addedto this BY medium, and shaken under 30 ◦C for 6 h. ThecwceScalwftGH

2

mrta1ciwatcim

odium benzoate and sodium succinate acid as the complexarbon source. In the SBY3(Fe) mediums, the EDTA (ethy-ene diamine tetraacetic acid)–FeCl3 complex was added toender Fe(III) as a cofactor to catA enzyme (catechol 1,2ioxygenase). All solutions were prepared in distilled, deion-zed water.

.2. Strain and mutagenesis

The microbial used in this study was Sphingobacteriump.-GCG [12], this bacterium identified to be a strain of Sphin-obacterium sp. based on the physiological tests, 16S rDNAequence analysis, and fatty acid profile analysis method per-ormed by FIRDI (Food Industry Research and Developmentnstitute) in Taiwan.

The mutants were derived by N-methyl-N′-nitrosogu-nidine (NTG) chemical mutagenesis of GCG according to

able 1ompositions of culture mediuma

A SBY3 SBY3(1-3) BY3 SY3 SBY3(Fe)

a-benzoate 1 1/2/3 1 – 1a-succinate 2 2 – 2 2

2HPO4 1 1 1 1 1gSO4·7H2O 1 1 1 1 1

NH4)2SO4 1.2 1.2 1.2 1.2 1.2east extract 0.5 0.5 0.5 0.5 0.5DTA – – – – 0.1eCl3 – – – – 0.1H 7.1–7.5a unit: g/l.

ells were harvested, washed and re-suspended in distilledater. Repeat the above procedures five times. The treated

ells were diluted and transferred to the BP agar plate. Selectach single colony from BP agar plate and replicate on BY,Y3 and SBY3 agar plates, respectively. Then pick up smallolony on BY agar plate but large colony on SY and SBY3gar plates. The cells picked were placing on the SBY3itmus agar plate. After 24 h, incubate the colonies, whichere assumed blue on litmus plate to tube slant array (TSA)

or culturing at SBY3 medium for 2 days. By centrifuga-ion at 5000 × g for 20 min and filtrating through 0.22 �mHP membrane, the cultured broths were analyzed byPLC.

.4. Bacterial cultures and growth conditions

The cells were cultured in 1 l conical flasks with 200 mledium. The shaker was set at 250 rpm and operated at

oom temperature (approximately 27 ◦C) or 30 ◦C. The cul-ure medium was prepared according to that listed in Table 1nd an initial pH value was adjusted to 7.1–7.5 by using2N NaOH solution. The concentrations of Na-benzoate andis,cis-muconate were monitored for every 4 h by HPLC dur-ng the fermentations. TSA screen method was performedith the 10 ml tube, which contained 3 ml SBY3 medium

nd was slanted in a holder on the shaker. Each colony wasransferred to a tube from the SBY3 litmus agar for pre-ulture. After 24 h, 10% (v/v) of the pre-culture broth wasncubated to three SBY3 medium main culture tubes to screen

utants.

C.-M. Wu et al. / Biochemical Engineering Journal 29 (2006) 35–40 37

Fig. 1. Outline of the NTG/UV mutagenesis and screening of mutants.

2.5. HPLC analysis

The HPLC system used consists of Lab Alliance Model500 Virable UV/VIS Detector and SISC computer integratorsoftware of Version 3.02. Vercopak Inertsil 6 ODS-3 typeC18 column (4.6 mm × 250 mm) was used. The mobile phaseconsists of a water/methanol mixture (7:3, v/v) and a 0.1 MKH2PO4 solution, and its pH value was adjusted to 7.80 byadding phosphoric acid. The flow rate was 1.0 ml/min.

3. Results and discussion

All the screening methods were based on the �-ketoadipate pathway (Fig. 2). The resetting cells fromSY3/BY3/SBY3 nutrition screening could generate cis,cis-muconate cycloisomerase (catB) regulated (or knock-out)mutants, which might accumulate more cis,cis-muconic acidthan the original wild-type strain GCG did. Based on ourprevious experiences with the GCG’s fermentation reactions[12], we note that higher cis,cis-muconate production rate

could be obtained with increasing pH value. This result isattributed to the reduction of benzoic acid and the increasingof metabolites such as cis,cis-muconate and nitrides. Accord-ing to this pH effect, a litmus agar could be designed as a highcis,cis-muconate accumulating strain detecting agent. ThisTSA method seems efficient to screen suitable mutants from alarge number of the possible resetting cells. With the purposeof enhancing the yield of cis,cis-muconic acid, we choosethe mutant strain which can produce more than 100 mg/l ofthe product; as compared to the yield of the original GCGstrain. By using the TSA screen method, we can found themutants M4113 and M4115 can produce more than 100 mg/lof cis,cis-muconic acid as shown in Table 2. The yields were112 and 264 mg/l, respectively.

Fig. 3 shows the batch fermentation results obtained fromthe SBY3 series medium. The time course of cells growthcurves indicates that the medium with a Na-benzoate con-centration of 1.0 g/l exhibits the highest growth rate both inGCG, M4113 and M4115 in the first 16-h period, while themedium with a Na-benzoate concentration of 2.0 g/l gave thebest growth performance at the end of the cultivation (i.e.,

38 C.-M. Wu et al. / Biochemical Engineering Journal 29 (2006) 35–40

Fig. 2. The relationship with compounds and genes in �-ketoadipate path-way [15].

Table 2Conversion yield data of various mutant strains

Strain DCW (g/l) pH MA (mg/l)

GCG 0.571 7.8 58M4110 0.45384 7.7 44M4112 0.34449 8.0 58M4113 0.31169 8.3 112M4115 0.38823 8.5 264M4151 0.47936 8.1 73

Fig. 3. The growth curves of GCG, M4113 and M4115 in SBY3 (1-3)medium.

36 h), since there are more carbon source supply in the lat-ter case. The growth curves shown in Fig. 3 revealed thatthe log-phase located approximately at the period of 5–20 h,and the stationary phase was found after reacted for 25 h.The result also indicates that the growth of the GCG, M4113and M4115 were inhibited as the Na-benzoate concentrationreaches 3.0 g/l. These results also showed that the originalGCG exhibited the highest growth rate. It seems the GCGutilized the Na-benzoate to be a growth substrate when Na-succinate was completely consumed. Owing to this reason,the mutants M4113 and M4115 can utilize more benzoate toproduce cis,cis-muconic acid than GCG does.

Figs. 4 and 5 depicted the consumption of Na-benzoateand the formation of cis,cis-muconic acid during the fermen-tation process, respectively. Results from both Figs. 4 and 5were comparable, since the forming of cis,cis-muconic acidwas accompanied with the usage of Na-benzoate. As shownin Fig. 5, the highest conversion yield was obtained withmutant strain M4115, in which more than 560 mg/l of cis,cis-muconic acid was accumulated in 28 h from a Na-benzoate

FS

ig. 4. The Na-benzoate usage curves by GCG, M4113 and M4115 inBY3(1-3) medium.

C.-M. Wu et al. / Biochemical Engineering Journal 29 (2006) 35–40 39

Fig. 5. The production of cis,cis-muconic acid by GCG, M4113 and M4115in SBY3 (1-3) medium.

solution of 2.0 g/l (∼28% yield). This result is nearly nine-fold increase in yield comparing to the original strain at thesame reaction condition. In addition, the high muconic acidconversion rate exhibited by M4115 samples further provedthe TSA screen results mentioned above.

Cosper et al. [15] reported the catM-regulated genesinclude catA (Catechol 1,2 dioxygenase), which encodesa dioxygenase to produce cis,cis-muconate from catechol,and the catBCIJFD genes, which are needed to generate tri-carboxylic acid cycle intermediates from cis,cis-muconate.According to the �-ketoadipate pathway, catA is an oxy-genase necessary for converting catechol to cis,cis-muconicacid. The enzyme structure of catA (E.C.1.13.11.1) consistsof two Fe(III) ions as the cofactor. In order to improvingthe yield of catA and therefore the yield of cis,cis-muconate,FeCl3 was added to the SBY3 medium to form a SBY3(Fe)medium. For dissolving of FeCl3, EDTA was employed toprepare a EDTA–FeCl3 complex solution. Fig. 6 presents

FM

the effect of EDTA–FeCl3 complex on the culture processproceeded in the mediums of SBY3(1). It revealed that anincrease of 43%, 17% and 6% of cis,cis-muconic acid inyield were observed for GCG, M4115 and M4113, respec-tively, as EDTA–FeCl3 complex was used. In spite of the factthat the SBY3(Fe) medium increase the yield of the originalGCG much high than that of the original GCG much higherthan that of the mutants, the overall yield of cis,cis-muconicacid was still much higher for the mutant strains, M4113 andM4115, as shown in Fig. 6.

These results indicate that the NTG/UV mutagenesis andscreening methods (such as TSA method) used in this studyare technically feasible. While these results are encourag-ing, they must be extended further research are neededin order to elucidate the fermentation mechanism and toraise the yield of cis,cis-muconic acid suitable for industrialproduction.

Acknowledgements

We are grateful to the support for this work provided bythe National Science Council of ROC and Fu Jen CatholicUniversity.

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ig. 6. The comparison of the accumulate of cis,cis-muconate by GCG,4113 and M4115 in SBY3(1) and SBY3(Fe) medium.

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