Appl Microbiol Biotechnol (1991)34:436-440 017575989100002B

App//ed

Micob/obgy Biotechnology © Springer-Verlag 1991

Biotransformation of 2-acetylthiophene by micromycetes Fran~oise Seigle-Murandi ~, Serge Krivobok m, R~gine Steiman ~, Georges-Andr~ Thiault 2, and Jean-Louis Benoit-Guyod a i Laboratoire de Botanique, Cryptogamie, Biologie Cellulaire et Gbn~tique, GEDEXE, UFR de Pharmacie, Universit+ J. Fourier, Grenoble I, BP 138, F-38243 Meylan C6dex, France 2 Produits Chimiques Auxiliaires et de Synth6se (PCAS), 23 Rue Bossuet, F-91160 Longjumeau, France 3 Laboratoire de Toxicologic et Ecotoxicologie, GEDEXE, UFR de Pharmacie, Universit6 J. Fourier, Grenoble I, BP 138, F-38243 Meylan C6dex, France Received 13 March 1990/Accepted 12 September 1990

Summary. The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. A m o n g them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. O f these, 106 strains were able to completely deplete 2acetylthiophene with or without production of intermediary metabolites, 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. A m o n g them, eight strains have been selected for further optimization of this bioconversion.

cals (Trophires, Thiopheol, Atrikan, Difluorex). In previous articles, we have reported the specific 5-hydroxylation of benzimidazole by micromycetes (Chapelle et al. 1986; Seigle-Murandi et al. 1986a). The aim of this work was to consider the possibility of using microbial enzyme systems as a safe and effective process for producing 2-thienylacetic acid. The screening p r o g r a m m e described in this p a p e r was intended to determine which microorganisms, if any, could perform this bioconversion. Such microorganisms could then be studied further in order to understand the transformation process and try to optimize the production of each metabolite.

Introduction 2-Thienylacetic acid is an important intermediate for industrial synthesis and especially for production of some cephalosporins. It is possible to obtain this comp o u n d by classic synthesis (Fig. 1A) from thiophene (Blicke and Zienty 1941; Blicke and Burckhalter 1942). However, chloromethylation (first step) gives 2-chloromethylthiophene with a low yield and furthermore, this c o m p o u n d is carcinogenic. In the second step, the use of sodium cyanide is unsuitable because of its toxicity and the difficulty of destroying the waste products obtained. More recently, a new synthesis of 2-thienylacetic acid has been described (Fig. 1B), but it is no more effective (Thiault and Le G u e n 1977). The drawbacks of the available processes (toxicity or p o o r yield) led us to find another method for preparation, using micromycetes to transform 2-acetylthiophene into 2-thienylglyoxylic acid (Fig. 1C). Further reduction of 2-thienylglyoxylic acid into 2-thienylacetic acid can be achieved by chemical reduction (Fig. 1C). We have also paid attention to other possible metabolites from the biotransformation of 2-acetylthiophene, especially the production of 2-thiophenecarboxylic acid (Fig. 1C), which is also an industrially interesting intermediate used for the synthesis of several pharmaceuti-

Offprint requests to: F. Seigle-Murandi

Materials and methods Microorganisms. About 800 strains of fungi were taken from the collection of our Institute (CMPG: Collection Mycology Pharmacy Grenoble). Most of them were fungi imperfecti isolated from different substrates (Seigle-Murandi et al. 1980a, 1981; de Hoog et al. 1985). Phanerochaete chrysosporium was a gift of Prof. Dr. K.-E. Eriksson from the Swedish Forest Products Research Laboratory, Stockholm, Sweden. They were maintained on solid malt extract agar medium (1.5%) at 4 ° C. Cultures conditions. Strains were selected after cultivation on malt extract medium and the synthetic medium of Galzy and Slonimski (1957) without glucose. Agar was added at 1.5% and the media were sterilized at 120° C for 20 rain. For the first experiments (50 strains), the final concentrations of 2-acetylthiophene were 0.10, 0.30, 0.50, 0.75, and 1.00 g/l. The results allowed us to select a final concentration of 0.50 g/l for further cultivation of all the strains. Malt extract medium was used to check the toxicity of 2-acetylthiophene against microorganisms. Cultivation on synthetic medium was a test of consumption or metabolism. Synthetic medium was also used without 2-acetylthiophene in order to estimate any nutrient effect of agarose, which is not an inert substrate (Seigle-Murandi et al. 1980b). Cultivation was made for a 30-day period at 24° C. To obtain sufficient inoculum for liquid media cultures, the selected fungi were grown for 1-2 weeks on solid malt extract medium with 2-acetylthiophene (0.01 g/l) for induction. Five different liquid media were used for preliminary experiments: Galzy and Slonimski (1957), Czapek Dox medium (Pitt 1979), soya medium (Smith and Rosazza 1974), cornstarch medium (Golbeck et al. 1983) and tartaric acid medium, a modification of dimethylsuc-

437

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NaCN

~ -CH~CN

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2- Cyanomethylthiophene

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Fig, 1. A Classic synthesis of 2-thienylacetic acid. B Synthesis of 2-thienylacetic acid following Thiault and Le Guen (1977). C Possible synthesis of 2-thienylacetic acid and of 2-thienylcarboxylic acid

cinic acid medium (Ander et al. 1984). Four preliminary studies were made under shaking conditions (180 rpm) at 24°C with 2acetylthiophene (0.50 g/l). The glucose concentration was 0, 5, 10 or 20 g/l and the pH was 4.6, 6.0 or 7.0. In a first series of experiments, malt extract medium or Galzy and Slonimski medium (with or without 10 g/1 glucose) were used. The addition of 2-acetylthiophene was made either at the beginning of the culture or 48 h after inoculation. The inoculum was obtained from one Petri dish (90 mm) for 100 ml of medium when 2-AT was added 48 h after inoculation and from four Petri dishes when 2-AT was added at the beginning of the culture. Massive inoculation was used in this case to overcome the toxicity of 2-AT that was the only carbon source. Cultures were also made in two steps, whereby mycelium grown in Galzy and Slonimski medium including 20 g/l glucose was transferred after 4 days to medium without glucose but including 2-acetylthiophene. Samples

were taken either every day or after 8 days of growth with 2-acetylthiophene. In a second series of experiments, we compared strains that presented good growth or no growth on solid media. Cultures were made in 1-1 erlenmeyer flasks containing 150 ml Galzy and Slonimski medium (glucose 10 g/l) pH 4.6. 2-Acetylthiophene was added after 4 days of growth. Samples were taken after 8 days of culture. In a third series of experiments, four different culture media were used with glucose (5 or 10 g/l) or saccharose (5 or 10 g/l), pH 6.0 or 7.0, as follows: malt extract medium (15 g/l); soya medium (glucose 5 and 10 g/l), pH 6.0 and 7.0; Galzy and Slonimski medium (glucose 5 and 10 g/l), pH 6.0 and 7.0; Czapek Dox medium (saccharose 5 and 10 g/l), pH 6.0 and 7.0. Cultures were grown in 250-ml erlenmeyer flasks containing 50 ml medium. 2Acetylthiophene was added after 3 days of growth and samples were taken after 7 days of culture. In a last series of experiments, results obtained with Galzy and Slonimski medium were compared with results obtained with cornstarch medium and tartaric acid medium (glucose 5 g/l, pH 6.0). Addition of 2-acetylthiophene and sampling were made as in the third series of experiments. Finally, fungi selected on solid media (460 strains) were cultivated in 250-ml erlenmeyer flasks containing 50 ml Galzy and Slonimski medium with glucose (5 g/l), pH 6.0, under shaking conditions (180 rpm) at 24 ° C. 2-Acetylthiophene was added after 3 days of culture and sampling carried out after 7 days of growth.

Extraction of metabolites. Each culture medium was filtered off, and the aqueous phase was acidified to pH 2.0 with 6 M HC1 and extracted with bidistilled ethyl acetate (3 vol). The combined extracts were dried over anhydrous Na2SO4 and evaporated to dryness at 40°C under vacuum. The residue was dissolved in ethyl acetate for TLC analysis.

Thin-layer chromatography. TLC was carried out on silica gel 60 F 254 plates (Merck, Darmstadt, FRG). Different solvent systems were used: (1) chloroform/ethyl acetate/acetic acid (5:4:1), (2) benzene/chloroform (9/1), (3) cyclohexane/ethyl formate/formic acid (15:10:1), (4) cyclohexane/ethyl acetate/acetic acid (15:10:1), (5) cyclohexane/ethyl acetate/formic acid (15:10:1) and (6) toluene/ethyl acetate/formic acid (10:9:1). Finally, solvent systems (3) and (2) were used successively for proper separation (2-acetylthiophene, Re: 0.62; 2-thienylglyoxylic acid, Re: 0.17; 2-thiophenecarboxylic acid, Re: 0.41 ; 2-thenaldehyde, Re: 0.71 ; 2thenyl alcohol, Re: 0.47). These metabolites were visible in UV light at 254 nm. Their maximum absorbance in UV light was: 2acetylthiophene (260 nm), 2-thienylglyoxylic acid (290 nm), 2thiophenecarboxylic acid (248 nm), 2-thenaldehyde (260 nm), 2thenyl alcohol (240 nm). Gas chromatography (GC) - mass spectrometry (MS). GC was carfled out with a Girdel instrument (model 32; Argenteuil, France) equipped with a capillary column (SE-30, 25 m by 0.32 mm). The products were well separated using a programme with a 130°C initial temperature and thereafter a rise of 6 °C/rain to 270 ° C. The carrier gas was He at a flow rate of 1.5 ml/min. MS was performed with a Nermag (model R 10-10 C; Argenteuil, France) mass spectrometer. The compounds, dissolved in dry pyridine, were silylated with bis (trimethylsilyl) trifluoroacetamide for 2 h at room temperature. Identification of products was based on comparison with authentic reference compounds (from PCAS, Longjumeau, France).

Results Selection o f strains on solid media P r e l i m i n a r y s t u d i e s w e r e m a d e w i t h 50 s t r a i n s i n c l u d ing representative genera of our culture collection and

438 with concentration of 2-acetylthiophene from 0.1 to 1 g/1. Toxicity assays on malt extract medium showed that 2-acetylthiophene was not highly toxic since all the strains could grow except Aphanocladium album, Acremonium murorum and Verticillium lecanii, which were, however, only inhibited at the concentration of 1 g/1. When the concentration of 2-acetylthiophene increased, growth diminished with the appearance of sterile mycelia and sometimes discolouration of the colonies. Some exceptions were found: for Aspergillus niger, growth increased as a function of the added 2-acetylthiophene concentration: for Botrytis cinerea and Fusaium moniliforme, the best growth was observed at 0.5 g/l; the zonation of Trichoderma increased and disappeared as a function of the concentration. Growth on Galzy and Slonimski medium was less than on malt extract medium: the growth of four strains was inhibited

(Absidia spinosa, Cunninghamella blakesleeana, Scopulariopsis brevicaulis, Syncephalastrum racemosum) and the growth of two strains diminished (Acremonium murorum, Candida pseudotropicalis). For several strains, 0.5 g/l was the optimum concentration both for growth and fructification (Fig. 2). The preliminary results allowed us to use a concentration of 2-acetylthiophene of 0.5 g/l.

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Toxicity assays on malt extract medium (2-acetylthiophene, 0.5 g/l) allowed to divide fungi into two groups (Table 1). For 92% of the strains, 2-acetylthiophene was not toxic; basidiomycetes (22 strains) and the genus Penicillium showed particularly good growth. For 8% of the strains, 2-acetylthiophene was a toxic substrate: reduction or inhibition of growth was observed. The toxicity of the molecule was not specific for a particular genus since 33 different genera were inhibited. Some of them were particularly sensitive, including Acremonium, dermatophytes and yeasts (Kloeckera, Lipomyces, Schizosaccharomyces). Finally, 597 strains were selected for metabolic assays. After cultivation on Galzy and Slonimski synthetic medium (without glucose, 2-acetylthiophene 0.5 g/l), the fungi were distributed as follows. In a first group (137 strains), growth was inhibited (Table 1). Sixty different genera could not utilize 2-acetylthiophene as a whole, although some species did. The only genus to be completely inhibited was Mortierella. Except for the genus Mucor, the Mucorales gave poor results since 21 strains out of 37 were inhibited. The sensitivity of Acremonium and dermatophytes to 2-acetylthiophene was confirmed. In the second group (460 strains), growth was normal or increased. The best represented genera were Mucor, Trichoderma and the Basidiomycetes group (10 strains out of 22).

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First experiments were made to determine the use of solid media cultures, the culture medium to be used, pH, glucose concentration, time of addition of 2-acetylthiophene to the culture and time of sampling. The first series of experiments was made using two strains with very good growth on solid media: Paecilomyces variotii and Trichoderma viride. The highest growth was in malt extract medium and in Galzy and Slonimski medium containing 20 g/l glucose for 4 days followed by medium without glucose for 8 days. Using a large inoculum in Galzy and Slonimski medium without glucose did not give good results. However, the best consumption of 2-acetylthiophene was obtained with condition 2 when the substrate was added after 48 h of growth in Galzy and Slonimski medium. In order to test the value of strain selection on solid media, one group of fungi that grew on solid medium and another that did not grow on solid medium were cultivated in liquid medium. The first group was able to grow in liquid media, and also the second group (Absidia spinosa, Cunninoha-

mella blakesleeana, Cunninghamella echinulata, Helieostylum syriforme and Scopulariopsis brevicaulis). The Table 1. Toxicityof 2-acetylthiophene(2-AT) for fungi cultivated on two different solid media Medium

Number of strains

Strains inhibited

Malt extract Galzy and Slonimski

800 597

8% 23%

third series of experiments confirmed that the highest metabolism of 2-acetylthiophene was obtained with Galzy and Slonimski medium when the glucose concentration was 5 g/1 and pH 6.0. The comparison of results obtained with Galzy and Slonimski medium with those obtained with cornstarch medium and tartaric acid medium (glucose 5 g/l, pH 6.0) is given in Table 2.

439 Table 2. Metabolism of 2-AT by fungi in three different liquid media Medium

Metabolism of 2-AT (number of strains, %)

Galzy and Slonimski Tartaric acid Cornstarch

90 79 36

Table 3. Production of 2-thiophenecarboxylicacid in Galzy and

Slonimski medium Fungus"

Productionb

Aureobasidium pullulans CMPG4 Basidiomyceteno. 7 CMPG17 Penicilliurnfellutanum CMPG7 P. fellutanum CMPG15 P. fellutanum CMPG19 P. italicum CMPG12 Ophiostoma piceae CMPG433 Phoma hibernica CMPG434

+++ ++ ++ +++ +++ ++ ++ ++

a CMPG: Collection MycologiePharmacie Grenoble b + + +, very good production; + +, good production

According to these results, 460 strains were cultivated in Galzy and Slonimski liquid medium (glucose 5 g/l, pH 6.0) under shaking conditions at 24°C for 7 days (2-acetylthiophene added on day 3). The total consumption of 2-acetylthiophene was observed for 106 strains: two basidiomycetes, six yeasts, six Mucorales, 21 Penicillium, four Asperoillus, and 67 other fungi imperfecti. We did not find 2-thienylglyoxylic acid among the metabolites, but some strains metabolized 2-acetylthiophene into 2-thenaldehyde. 2-Thiophenecarboxylic acid was produced by 72 strains, eight of them giving a high production yield (Table 3). The mass spectrum of this compound was the same when compared with the authentic compound (from PCAS).

Discussion

Microorganisms have been utilized extensively for the biotransformation of drugs or their precursors (Charney and Herzog 1980; K_ieslich 1984). A significant advantage is that preparative quantities of otherwise difficult-to-synthesize metabolites may be obtained by scale-up of the fermentation process. The aim of this work was to find strains competent in the transformation of 2-acetylthiophene into 2-thienylglyoxylic acid or (and) 2-thiophenecarboxylic acid. Biological oxidation may be a detoxification process for fungi and we had first to determine for all the strains the possible toxicity of 2-acetylthiophene on solid malt extract medium, then its eventual biotransformation by selected fungi on synthetic medium, as we had done for phenolic compounds (Steiman and Seigle-Murandi 1984; Seigle-Murandi et al. 1986b; Rahouti et al. 1989) and benzimida-

zole (Chapelle et al. 1986). Malt extract medium allowed the growth of all the fungi tested and was convenient for a toxicity assay. Consumption or metabolism of a substrate must be carried out with a synthetic medium where the only carbon source is the substrate and Galzy and Slonimski (1957) medium was chosen from the results obtained. Studies on solid media with various concentration of 2-acetylthiophene showed that this compound was toxic only at 1 g/l for a few strains. If weak inhibition could be seen as a function of the concentration, it was also observed that some strains were stimulated at 0.5 g/1. The toxicity assay confirmed that 2-acetylthiophene was not toxic for most of the strains (92%) and the consumption assay selected 460 strains able to metabolize this substrate. It was also found that some fungi that did not grow on solid medium containing 2acetylthiophene were able to metabolize it in liquid medium. Some strains may have different behaviour depending on the culture conditions. For example, Aureobasidium pullulans did not grow on solid media containing crude oil whereas it produced an emulsifier in liquid media (Seigle-Murandi et al. 1978). In the same way, Embellisia annulata was unable to grow on solid media in the presence of phenolic compounds whereas it metabolized them in liquid media (de Hoog et al. 1985). Much work has dealt with the microbial oxidation of organic compounds, mostly with bacteria (Kieslich 1976). Hydroxylation of various substrates by fungi have been described: naphthalene (Ferris et al. 1973; Cerniglia and Gibson 1977; Cerniglia et al. 1978), benzene (Smith and Rosazza 1974), biphenyl (Smith et al. 1980), benzimidazole (Seigle-Murandi et al. 1986a), biphenyl oxide (Seigle-Murandi et al. 1990). Fungi serve as effective models for mammalian metabolism, since the cytochrome P-450-1inked monooxygenase enzymes are often similar to those found in mammalian liver (Ferris et al. 1973; Smith and Rosazza 1974; Cerniglia et al. 1978). We did not find any work concerning the biotransformation of 2-acetylthiophene. Thiophenes, benzothiophenes and dibenzothiophenes are found in petroleum and the metabolism of condensed thiophenes has been reviewed by Ensley (1984). Most research has involved dibenzothiophene metabolism because of its presence in conventional crude oils (Kodama et al. 1970; Laborde and Gibson 1977; Monticello et al. 1985). More recently microbial degradation of substituted thiophenes and tetrahydrothiophenes has been reported, essentially by bacteria (Kanagawa and Kelly 1987; Fedorak et al. 1988). Cultivation in liquid media has demonstrated the difficulties in the choice of the best conditions for the biotransformation of a compound by many strains. Complex media allowed good growth for most microorganisms and have been used for the hydroxylation of naphthalene (Smith and Rosazza 1974) or of biphenyl (Golbeck et al. 1983), but the extraction of metabolites was easier in synthetic media. As previously reported for the hydroxylation of benzimidazole (Seigle-Murandi et al. 1986a), we found that better growth did not

440 imply better biotransformation. More recently, the hydroxylation of biphenyl oxide to 4-hydroxybiphenyl oxide was obtained in tartaric acid medium when the mycelial weight was minimum. The lowest yield was obtained in cornstarch medium in which the fungal growth was the highest (Seigle-Murandi et al. 1990). Tartaric acid medium is N-limited (2.6 mM) compared with Galzy and Slonimski medium (82.5 mM) and Nlimitation of the medium was shown to be a very important factor in some metabolic reactions, particularly of lignocellulosic c o m p o u n d s (Ander et al. 1984). Maybe this medium should be used for further optimization. In conclusion, the cultivation of 460 strains in liquid synthetic medium showed that 23% were able to completely deplete 2-acetylthiophene (0.5 g/l) with or without the production of intermediary metabolites. The lack of 2-thienylglyoxylic acid and the presence of 2-thiophene-carboxylic acid and 2-thienaldehyde may be explained by the fact that 2-thienylglyoxylic acid is produced rapidly and immediately transformed into 2thiophenecarboxylic acid and 2-thenaldehyde. A m o n g the strains transforming 2-acetylthiophene into 2-thiophenecarboxylic acid, eight fungi have been selected.

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