Reductive transformation of benzoate by Nocardia asteroides and Hormoconis resinae

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 69, No. 4, 220-223. 1990

Reductive Transformation of Benzoate by Nocardia asteroides and Hormoconis resinae N O B U O K A T O , l* H I S A T O S H I K O N I S H I , 2 M U N E T O M A S U D A , 1 E U N - H O J O U N G , l M A S A Y U K I S H I M A O , 1 AND C H I K A H I R 0 S A K A Z A W A *

Departments of Biotechnology I and Materials Science, 2 Faculty of Engineering, Tottori University, Tottori 680, Japan Received 29 November 1989/Accepted 30 January 1990 A variety of substituted aromatic acids were reduced to the corresponding alcohols by Nocardia asteroides JCM3016 under aerobic conditions. An isolated mold, Hormoconis resinae F328, could also reductively transform benzoate, the erythro isomer of (1R, 2S)-l-phenyl-l,2-propanediol being yielded as well as benzyl alcohol. C-1 of the diol was found to be derived from the a-carbon of benzoate by 13C-NMR analysis. The acyloin condensation between pyruvate and benzaldehyde formed from benzoate is assumed to participate in the diol formation. An ATP-dependent and NADPH-linked benzoate reductase (EC 1.2.1.30) and an NADPH-iinked benzaldehyde reductase (EC 1.1.1.91) were demonstrated to participate in the benzoate reduction in both N. asteroides JCM 3016 and H. resinae F328.

The microbial d e g r a d a t i o n of benzoate is generally recognized to be initiated by the oxidation to catechol or p r o t o c a t e c h u a t e under aerobic conditions (1), and to occur through ring hydrogenation to cyclohexane carboxylic acid under anaerobic conditions (2). Besides such degradation routes involving ring cleavage, several basidiomycetes and molds are known to reduce aromatic carboxylic acids to aldehydes and alcohols (3). In the course of a screening p r o g r a m for the microbial t r a n s f o r m a t i o n of benzoate, we found that an actinomycete, Nocardia asteroides J C M 3016, and several m o l d strains had the ability to reduce aromatic carboxylic acids under aerobic conditions (4). In general, reduction of carboxyl group is an strong endergonic reaction, which is an inconvenient biochemical process in aerobic conditions. Benzoate reduction, however, is found in a variety o f microorganisms. A metabolic p a t h w a y involving the reduction is assumed to be significant in the microbial conversion o f aromatic compounds in nature. Such reduction also seems to be applicable to the microbial p r o d u c t i o n o f useful c o m p o u n d s from benzoate, which is now rather a cheep substrate for microbial conversion. Further investigation demonstrated that an optically pure isomer o f 1-phenyl-l,2-propanediol was the main p r o d u c t in a reductive t r a n s f o r m a t i o n o f benzoate by an isolated mold. Such an optically active c o m p o u n d is a promising starting substrate for an asymmetric synthesis. In this paper, we report the reduction o f substituted benzoates by resting cells o f N. asteroides and the f o r m a t i o n o f an optically active diol from benzoate by an isolated mold. The enzymes related to the reductive t r a n s f o r m a t i o n are also discussed.

asteroides J C M 3016, showing the highest benzoate reduction activity, was selected. A m o n g 376 mold strains, Rhizopus oryzae IFO 5440 and I F O 4706, Mucorfragilis I F O 6449, Mucor javanicus IFO 4569, and isolate F328 reduced benzoate. Isolate F328 was identified as Hormoconis resinae by the identification service o f the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Three stock cultures of H. resinae, IFO 31770, 31771, and 31772, were used for comparison with the isolate. Cultivation and resting-cell reaction The actinomycetes and molds were cultivated at 30°C for 48 and 72 h, respectively, in test tubes (18 × 180 mm) each containing 5 ml of a medium under reciprocal shaking. The composition o f the medium for actinomycetes was (per 100 ml): 2 g of glucose, 1 g o f sucrose, 1 g o f meat extract, 0.5 g o f peptone, 0.2 g of yeast extract, 0.3 g of NaC1, 0.4 g o f KzHPO 4, 0.2 g of KH2PO4, 0.1 g o f MgSO4' 7H20, and 0.1 g o f corn steep liquor, p H 7.0. The molds were grown in a m e d i u m composed of 5 g of glucose and 5 g o f corn steep liquor in 100 ml H20 (pH 6.0). The cells o f the actinomycete and the molds were harvested by centrifugation and by filtration on filter paper, respectively, and then washed with 0.9%o NaC1 three times. The resting-cell reactions with N. asteroicles J C M 3016 and H. resinae F328 were done in the following reaction mixture: 50 mg (as dry weight) of the washed cells, 25 mg o f sodium benzoate, and 2 0 0 m g o f glucose in 5 ml of 50 m M potassium phosphate buffer (pH 7.0). The reaction mixture in a test tube (18 × 180mm) was shaken at 30°C for an a p p r o p r i a t e period. To prepare the reaction product, 100 ml o f medium in a 300-ml Erlenmeyer flask was inoculated and shaken on a rotary shaker at 30°C for 48h. After the addition of sodium benzoate ( 0 . 5 ~ , w/v) and glucose (4.0%), the shaking was continued until the productivity reached the m a x i m u m level (48 to 72 h). Analysis of the reaction product The clear supernatant o f the reaction mixture was acidified to p H 2.0 with conc. HC1, extracted with ethyl acetate, and dried under reduced pressure. The resulting oily material or powder

MATERIALS AND METHODS Microorganisms Sixty-eight strains o f n o c a r d i o f o r m actinomycetes, including ones from the genera Nocardia, Rhodococcus, Nocardioides, and Pseudonocardia, were screened for benzoate transforming activity, and N. * Corresponding author.

220

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MICROBIAL REDUCTION OF BENZOATE

was dissolved in a m i n i m u m volume o f 1,4-dioxane. The trimethylsilyl (TMS) derivative o f the p r o d u c t was assayed by gas c h r o m a t o g r a p h y under the following conditions: glass column (2.6 m m × 2 m), 5% silicon OV-1 (Uniport H P 80/100mesh), 200°C. N 2 gas ( 6 0 m l / m i n ) , F I D . The retention times o f TMS-derivatives o f benzyl alcohol, benz o a t e / b e n z a l d e h y d e , and 1-phenyl-l,2-propanediol were 0.47, 0.58, and 1.0 min, respectively. Benzaldehyde was separately assayed using yeast aldehyde dehydrogenase (5). N M R analyses 1H and 13C N M R spectra were recorded on a J E O L GX-270 spectrometer, in CDC13, operating at 270 and 67.9 M H z with tetramethylsilane as an internal standard, Preparation o f authentic 1-phenyl-l,2-propanediol

v

7 T

o, .-z 4

6

\ E "~ 3 az o o

E n z y m e assays with a cell-free extract Cells o f N. a s t e r o i d e s J C M 3016 and H . resinae F328 were disrupted

0

Aromatic carboxylic acid reduction by N. a s t e r o i d e s We have reported that iV. a s t e r o i d e s J C M 3016 converted benzoate to benzyl alcohol under aerobic culture conditions, where the carboxyl group underwent direct reduction to alcohol (4). In this work, the conversion reaction was done using resting cells to facilitate the analyses of the reaction process. The conversion o f the acid to the alcohol was dependent on the addition of glucose: the m a x i m u m conversion yield to substrate a d d e d (600/00 w / w ) was obtained in a reaction mixture containing 0.5% (w/v) benzoate and 4.0O//oo(w/v) glucose. The conversion yield in the resting-cell reaction was almost the same as that under the growing culture conditions (4). The conversion reaction only occurred under aerobic conditions, i.e., not under N2 gas. The course of the reaction is shown in Fig. 1. Unlike

t

t)°

-6 T,, ¢.'~ 2 _o

RESULTS

8

5

One hundred milligrams o f 1-phenyl-l,2-propanedione ( W a k o Pure Chemical Ind. Ltd., Osaka) in 5 ml o f 50% ethanol was reduced to racemic (1RS, 2 S R ) - l - p h e n y l - l , 2 p r o p a n e d i o l by the a d d i t i o n o f sodium b o r o h y d r i d e powder. The isomer (1R, 2S) o f the diol was prepared through a fermentative reaction with b a k e r ' s yeast from benzaldehyde and glucose, by the m e t h o d o f O h t a et al. (6). The diol was extracted with dichloromethane and then purified by preparative thin layer c h r o m a t o g r a p h y (SiO2, 60F254, 20 × 20 cm, Merck, benzene : ether : acetic acid = 40:10:5). with a sonic oscillator and a Braun MSK homogenizer, respectively, and then a clear cell-free extract was obtained in each case by centrifugation at 40,000g for 2 h with a Hitachi preparative centrifuge, model 70P-72. Protein was measured by Bio-Rad Protein Assay with bovine serum albumin as a standard. Benzaldehyde reductase (aryl-alcohol dehydrogenase, EC 1.1.1.91) (7) was assayed in a reaction mixture containing 3 3 r a M potassium p h o s p h a t e (pH 7.0), 0 . 1 5 m M N A D P H , 1 0 m M benzaldehyde, and an a p p r o p r i a t e amount of cell-free extract, in a total volume o f 3.0 ml. The activity o f benzoate reductase (aryl-aldehyde dehydrogenase, EC 1.2.1.30) (3,8) was measured in a reaction mixture containing 3 3 m M Tris-HC1 (pH 7.5), 0 . 1 5 m M N A D P H , 2 . 0 m M A T P , 1 0 m M MgC12, 1 0 m M sodium benzoate, and an a p p r o p r i a t e a m o u n t of cell-free extract, in a total volume of 3 . 0 m l . These enzyme activities were measured as the oxidation o f N A D P H , which was followed at 340 nm at 30°C. Blank tests were done in reaction mixtures without benzaldehyde and benzoate, respectively.

221

-'--" t) o

I

._ o

N

c cn

0

I

I

I

J

24

48

72

96

]

Reaclion p e r i o d (h) FIG. 1. Course of the conversion of benzoic acid to benzyl alcohol in the resting-cell reaction with N. asteroides JCM 3016. Glucose was measured by a colorimetric method with glucose oxidase and peroxidase (9). Symbols: e , benzoate; m, glucose; ~, benzyl alcohol; :~, pH. the preceding report (4), benzoate and glucose did not completely disappear in the resting-cell reaction, in which the yield to consumed benzoic acid was 94%o. No accumulation o f benzaldehyde was observed. A variety of benzoates substituted with hydroxyl, halogen, nitro or methyl groups were transformed to the corresponding alcohols by the actinomycete (Table 1). Exceptionally, no reaction occurred with o-hydroxybenzoate. No relationships between the reactivity and the substitution position were indicated by these data. Several aliphatic acids substituted with a phenyl group were also good substrates for the reductive t r a n s f o r m a t i o n (Table 2). On the other hand, no reduction was observed in the TABLE 1. Reduction of substituted benzoates by resting cells of N. asteroides JCM 3102 Substrate, benzoate substituted by o-OH m-OH

p-OH m-F p-F o-Cl m-Cl p-Cl

Yield" (%) 0 72 63 66 78 62 43 24

Substrate, benzoate substituted by o-Br nl-Br p-Br m-NO, p-NO2 o-CH3 m-CH3 p-CH3

Yield (O/oo) 52 47 32 10 20 46 55 70

The substrates and products were measured by gas chromatography as described under Materials and Methods. No aldehyde was accumulated from any substrate. ~' Ratio of alcohol formed to acid added.

222

KATO ET AL.

J. FERMENT. BIOENG.,

TABLE 2. Reduction of various aromatic acids by resting cells of N. asteroides JCM 3016 Substrate

Yield" (%)

Phenylacetic acid 2-Phenylpropionic acid 3-Phenylpropionic acid 2-Phenylbutyric acid 3-Phenylbutyric acid 4-Phenylbutyric acid Phenoxyacetic acid D-Mandelic acid L-Mandelic acid Phthalic acid

68 52 58 44b 52 58 89 38 30 0

Conversions were assayed by gas chromatography as described under Materials and Methods. Ratio of alcohol formed to acid added. b The corresponding aldehyde was also produced. cell-reaction with simple aliphatic acids with C~ to C8, or phthalic acid. Reaction product from benzoate with H. resinae F328 To 100 ml o f a 48-h culture o f the m o l d was a d d e d 0.5 g o f sodium benzoate and then the shaking was continued for a further 72 h at 30°C. F r o m the filtrate o f the reaction mixture, the main p r o d u c t was isolated (73 rag) by the same procedures as used for p r e p a r a t i o n o f authentic samples, as described under Materials and Methods. The IH N M R spectrum (CDC13) o f the p r o d u c t [5 1.100 (d, J = 6 . 4 , 3H, CH3), 3.6 (br.s. 2H, OH), 4.023 (dq, J = 4 . 5 , 6.4, 1H, CHCH3), 4.693 (d, J = 4 . 6 , 1H P h C H ) , 7 . 3 - 7 . 4 (m, 5H,

m

E c~ 4

E O "10

--" 3 0 tO (3

N

r-Q

(D fl[:l

2

0

0

24

48

72

96

R e a c t i o n p e r i o d (h) FIG. 2. Course of the conversion of benzoic acid in the restingcell reaction with H. resinae F328. Symbols: O, benzoate; i , glucose; •, benzyl alcohol; A, 1-phenyl-l,2-propanediol.

TABLE 3. Enzyme activities related to benzoate reduction in N. asteroides JCM 3016 and H. resinae F328

Specific activity (nmol rain ~mg ~) Enzyme

N. asteroides

H. resinae

Aa

Bb

Benzoate reductase

35

nd c

A 8

Benzaldehyde reductase

11

19

199

B nd 146

a Induced cells were obtained after incubation for 48 h with benzoate (0.5%), which was added to a 48-h culture. Non-induced cells were obtained after 96 h of cultivation. ~ Not detected. aromatic)] agreed exactly with that o f the authentic sample of 1-phenyl-l,2-propanediol prepared with b a k e r ' s yeast. The assignment of peaks of the ~3C N M R spectrum (CDCI3) o f the product was as follows; ~ 17.4 (q, CH3), 71.3 (d, C2), 77.6 (d, C-I), 126.6 (d, o-C of Ph), 127.9 (d, p- C of Ph), 128.4 (d, m-C of Ph), 140.3 (ipso-C of Ph). The stereochemistry o f the diol was identified by comparison of the ~H N M R spectra o f the product and authentic samples o f a racemic diol and a (1R, 2S) isomer. The N M R spectra were measured in the presence o f a chiral shift reagent, (+)tris(di(perfluoro-2-propoxyprophyl)methanato) europium ( l i d (in 30O/oo w / v Freon 113, Daiichi Pure Chem. Co., Tokyo). The ortho protons in the phenyl ring of the racemic diol appeared as a pair of doublets. When the authentic (1R, 2S) isomer was added to the racemic sample, the strength o f the doublet in the lower magnetic field was enhanced. The doublet in the lower magnetic field was assigned to the signal of the (1R, 2S) isomer. Since a similar spectral change was observed for the product, its configuration was identified a (1R, 2S). The optical purity was also shown to be very high within the limits of NMR analysis. The microbial t r a n s f o r m a t i o n was done using [~-13C] benzoate (10% a t o m % ~3C) and the isolated product was analyzed by I~C-NMR. On comparing the spectrum with that o f the product from natural benzoate (1% atom % ~3C), the peak intensity ratio of C-1 (5 77.6) to C-2 (5 71.6) o f the ~3C-labeled sample was found to be about 10 times higher than that o f the non-labeled one. This indicates that C-1 o f 1-phenyl-l,2-propanediol is directly derived from the ~-carbon o f benzoate. Course of the reaction with H. resinae As shown in Fig. 2, the aerobic shaking reaction of benzoate with H. resinae F328 gave 1-phenyl-l,2-propanediol in a good yield as to added benzoic acid (55%), benzyl alcohol also being accumulated. Other strains of H. resinae, IFO 31770, 31771, and 31772, also accumulated the diol (the conversion yields ranging from 10 to 25o/00) and a detectable a m o u n t o f benzyl alcohol. The reductive transformation required the addition o f glucose and only occurred under aerobic conditions. Enzyme activities related to benzoate reduction The activities o f benzoate and benzaldehyde reductases were found in the cell-free extracts o f both N. asteroides J C M 3016 and H. resinae F328 (Table 3). The benzoate reductases o f both organisms were inducibly formed in response to benzoate in the culture medium, and their N A D P H linked activities were absolutely dependent on A T P and Mg 2-. On the other hand, N A D P H - l i n k e d benzaldehyde reductase was constitutively formed in both strains.

VOL. 69, 1990

MICROBIAL REDUCTION OF BENZOATE

HO H ~P ~ C'c'CH3 ~ • & H OH

ATP, NAD(P)H .CH3 ---~ .CO COOH

Glucose ---D

ucoo

.uc.o I

tion, and A T P and N A D P H for the benzoate reduction. The unique coupling reactions of benzoate reduction and the condensation leads to the formation of the optically pure isomer (1R, 2S)-l-phenyl-l,2-propanediol (Fig. 3). In N . asteroides JCM 3016, the consumed benzoate was almost completely converted to benzyl alcohol and no accumulation of the diol was observed. This may be due to a lower rate of the acyloin-condensation reaction compared with the rate of benzaldehyde reduction in vivo.

. 2

FIG. 3. Reductive transformation of benzoate by H. resinae F328. Enzymes: 1, benzoate reductase; 2, benzaldehyde reductase; 3, pyruvate decarboxylase; 4, alcohol dehydrogenase.

223

ACKNOWLEDGMENT The authors are grateful to Prof. H. Ohta, Faculty of Science and Technology, Keio University, for the valuable suggestions as to the identification of 1-phenyl-l,2-propanediol. REFERENCES

DISCUSSION Although several species of basidiomycetes and molds are k n o w n to reduce aromatic carboxylic acids to the corresponding aldehydes and alcohols, the reductive transform a t i o n of benzoates by N . asteroides is the first example by a procaryotic organism (4). A n A T P - d e p e n d e n t and N A D P - l i n k e d benzoate reductase and an N A D P H - l i n k e d benzaldehyde reductase were demonstrated to participate in the direct reduction of carboxyl groups to alcohols. Both enzymes showed broad substrate specificities (data not shown), indicating that the conversion of a variety of aromatic acids to alcohols was the result of identical enzyme reactions. That the conversion was dependent on glucose and oxygen can be explained because the oxidative metabolism of glucose furnishes N A D P H and A T P for the reductive reaction. 1-Arylpropane-l,2-diol was reported to have been produced from substituted benzaldehyde with baker's yeast (6, 10). This t r a n s f o r m a t i o n is supposed to involve acyloin condensation with pyruvate decarboxylase between benzaldehyde and pyruvate to give 1-phenyl-l-hydroxy-2-propanone, which is reduced to the diol by alcohol dehydrogenase. In H . resinae F328, C-1 of the diol was demonstrated to be derived from the a - c a r b o n of benzoate on ~3C N M R analysis, the intermediate, benzaldehyde, being formed from benzoate through the action of the A T P dependent reductase. The acyloin condensation followed by the reduction is considered to occur in the same way as in baker's yeast. The reductive t r a n s f o r m a t i o n by H. resinae F328 was linked with the aerobic metabolism of glucose, which provides pyruvate for the acyloin condensa-

1. Fewson, C.A.: Biodegradation of aromatics with industrial relevance, p.141-179. In Leisinger, T., Cook, A. M., Hutter, R., and Nuesche, J. (ed.), Microbial degradation of xenobiotics and recalcitrant compounds. Academic Press Inc., London (1981). 2. Young, L. Y.: Anaerobic degradation of aromatic compounds, p.487- 523. In Gibson, D.T. (ed.), Microbial degradation of organic compounds. Marcel Dekker, Inc., New York and Basel (1984). 3. Gross, G. G. and Zenk, M. H.: Reduktion Aromatischen Sauren zu Aldehyden und Alkoholen im Zellfreien System. I. Reinigung und Eigenschaften von Aryl-Aldehyd: NADP-Oxidoreductase aus Neurospora crassa. Eur. J. Biochem., 8, 413-419 (1969). 4. Kato, N., Konishi, H., Uda, K., Shimao, M., and Sakazawa, C.:

5.

6.

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8. 9.

10.

Microbial reduction of benzoate to benzyl alcohol. Agric. Biol. Chem., 52, 1885-1886 (1988). Lundquist, F.: Determination with aldehyde dehydrogenase, p. 1509-1513. In Bergmeyer, H.U. (ed.), Methods of enzymatic analysis, 2nd ed., vol. 3. Verlag Chemie GmbH, Weinheim (1974). Ohta, H., Ozaki, K., Konishi, J., and Tsuehihashi, G.: Reductive C2-homologation of substituted benzaldehydes by fermenting baker's yeast. Agric. Biol. Chem., 50, 1261-1266 (1986). Gross, G. G. and Zenk, M. H.: Reduktion Aromatisher Sauren zu Aldehyden und Alkoholen in Zellfreien System. II. Reinigung und Eigenschaften yon Aryl-Allkohol: NADP-Oxidoreductase aus Neurospora crassa. Eur. J. Biochem., 8, 420-425 (1969). Gross, G.G.: Formation and reduction of intermediate acyladenylate by aryl-aldehyde NADP oxidoreductase from Neurospora crassa. Eur. J. Biochem., 31, 585-592 (1972). Kunst, A., Draeger, B., and Ziegenhorn, J.: Colorimetric methods with glucose oxidase and peroxidase. In Bergmeyer, H.U. (ed.), Methods of enzymatic analysis, 3rd ed., vol. 6. Verlag Chemie GmbH, Weinheim (1984). Voets, J. P., Vandammer, E.J., and Vleriek, C.: Some aspects of phenylacetylcarbinol biosynthesis by Saccharomyces cerev&iae. Allg. Mikrobiol., 13, 355-366 (1973).