INTRODUCTION

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Ionones and their derivatives are important intermediates in the metabolism of terpenoids, e.g., in carotenoid biosynthesis, and have been isolated from many sources (1a, 11). Compounds with a trimethylcyclohexane building block constitute essential aroma elements in many plant oils and thus have attracted the attention of the flavor and fragrance industry (3). Further, ionone derivatives, e.g., 3-hydroxy--ionone, could prove valuable intermediates for the chemoenzymatic synthesis of carotenoids, e.g., for astaxanthin and zeaxanthin (5).

Microbial transformation of - and/or -ionone to a number of hydroxy and oxo derivatives has been reported for several fungal strains (2, 4, 8, 9, 18), mainly of the genus Aspergillus, but not for bacterial strains. 3-Hydroxy--ionone was observed, among other metabolites, when Cunninghamella blakesleeana ATCC 8688 (2) or Aspergillus niger JTS 191 (18) was used.

Many species of the order Actinomycetes are known to catalyze a broad spectrum of xenobiotic transformations. Several cytochrome P-450-dependent monooxygenases from Streptomyces strains, which catalyze the hydroxylation of a wide range of substrates, have been investigated on the molecular level (12) and thus provide an interesting potential as biocatalysts for specific hydroxylation reactions by recombinant techniques.

As a first step in this direction, we now report the screening of 215 Streptomyces strains for their capacity to hydroxylate - and/or -ionone to the respective 3-hydroxy derivatives in a regio- and stereoselective manner. The structure and stereochemistry of the main biotransformation product were characterized unequivocally by nuclear magnetic resonance (NMR) spectroscopy.

	MATERIALS AND METHODS

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Materials. Substrates for the biotransformation reactions were purchased from Merck (racemic -ionone) and Fluka (-ionone), respectively. BASF AG (Ludwigshafen, Germany) kindly provided 3- and 4-hydroxy--ionone as references. Soybean meal, yeast extract, and nutrient broth were obtained from Difco.

Strains. For the first screening round, 215 Streptomyces strains were selected from the collection of the Institute of Microbiology/Biotechnology, University of Tübingen, Tübingen, Germany. Streptomyces griseus ATCC 13273 was purchased from the American Type Culture Collection. Streptomyces hygroscopicus Lu 1537 was kindly provided by the BASF AG.

Media. Three complex media and one synthetic medium were investigated for strain cultivation and bioconversion of ionones (all amounts given per liter): medium A (20 g of soybean meal, 20 g of mannitol [pH 7.5]), medium B (8 g of nutrient broth, 10 g of yeast extract, 5 g of glucose [pH 7.0]), medium C [5 g of (NH₄)₂SO₄, 0.5 g of MgSO₄ · 7H₂O, 0.05 g of MnSO₄ · H₂O, 3.6 g of K₂HPO₄, 1.5 g of KH₂PO₄, 2 g of glucose, 0.2 g of FeSO₄ · H₂O, 10 mg of ZnSO₄ · 7H₂O, 3 mg of MnCl₂ · 4H₂O, 30 mg of H₃BO₃, 20 mg of CaCl₂ · 6H₂O, 1 mg of CuCl₂ · 2H₂O, 2 mg of NiCl₂ · 6H₂O, 3 mg of Na₂MoO₄ · 2H₂O, 500 mg of Titriplex III (pH 7.0)], and medium D (15 g of glucose, 15 g of soybean meal, 5 g of corn steep liquor, 5 g of NaCl, 2 g of CaCl₂ [pH 7.0]).

Initial screening for -ionone bioconversion. The first screening round for an overall ionone hydroxylation potential of the individual Streptomyces strains was performed with -ionone as the substrate. Precultures (5 ml of medium A) were inoculated from slant agar stocks and incubated for 2 days at 28 to 30°C. Medium A (100 ml) was inoculated with 3 ml of the precultures and incubated at 28 to 30°C with shaking (150 rpm). -Ionone (0.1% [wt/vol]) was added to each of the cultures after 2 days, and cultivation continued for 5 days. Cells were then separated by filtration. A 2-ml aliquot of the culture supernatant was extracted with ethyl acetate-hexane (3:2) or diethyl ether, and the extract was analyzed by thin-layer chromatography (TLC). Control cultivations were carried out analogously but without addition of -ionone. The stability of -ionone in medium A was tested under incubation conditions identical to those as employed for strain cultivation.

Bioconversion of - and -ionone. The strains Streptomyces griseus ATCC 13273 and S. hygroscopicus Lu 1537, selected during the initial screening round for their ability to convert -ionone, were employed for a second screening round under the same cultivation conditions. A 1-ml aliquot was withdrawn every other day, and the culture supernatant was extracted as described above and analyzed for conversion products by TLC and gas liquid chromatography (GLC). After 10 to 12 days of cultivation, the cultures were filtered, and the supernatant was subjected to the final product analysis. Bioconversion of -ionone was performed as described above for -ionone.

Extraction of conversion products from culture supernatant. Larger volumes of culture supernatant (e.g., 100 ml) were extracted twice with 50 ml each of diethyl ether, and the extracts were washed with a saturated aqueous NaCl solution, dried with MgSO₄ or Na₂SO₄, and then evaporated to yield the unused substrate and conversion products.

Analytical methods. Samples for TLC analysis were spotted onto TLC plates (silica gel layer thickness, 0.2 mm; Silica Gel 60 F₂₅₄; Merck), and the plates were developed with hexane-ethyl acetate (3:2). Product spots were visualized first by fluorescence quenching at 254 nm and then by spraying with a 2.5% (wt/vol) vanillin solution in 95% ethanol-H₂SO₄ and subsequent heating. Authentic - and -ionone and 3- (R_f, 0.37) and 4-hydroxy--ionone (R_f, 0.33) were employed as standards.

	RESULTS AND DISCUSSION

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Hydroxylation of -ionone. In the first screening round, 215 different Streptomyces strains (from the strain collection of the Institute of Microbiology/Biotechnology, University of Tübingen) were tested for their potential to hydroxylate -ionone in position 3, i.e., for their ability to transform -ionone to 3-hydroxy--ionone. Twenty Streptomyces strains were selected at random and cultivated on a small scale (50 ml) in four different media (medium A to medium D [see Materials and Methods for medium composition]) in the presence of -ionone. Medium A proved best for both strain cultivation and -ionone biotransformation and was therefore selected for all further cultivation studies. TLC analysis showed bioconversion of -ionone to more-polar products, e.g., hydroxylated or oxygenated products, for 13 of the 215 Streptomyces strains.

Bioconversion of -ionone. Our screening results suggest that selective enzymatic hydroxylation of -ionone at C-3 is difficult if not impossiblea finding which is in line with all earlier reports on the microbial conversion of -ionone. While stereochemical reasoning implies that the two methyl groups at C-1 should direct any oxidative attack toward C-3 rather than C-2, it is the electronic activation of the allylic hydrogens at C-4 by the C-5==C-6 double bond which governs the regiochemistry of -ionone hydroxylation (Fig. 1). This view is supported by the fact that the main products of -ionone oxidation found in cultures of Aspergillus were 2- and 4-hydroxy--ionone. In the isomeric -ionone, however, it is C-3 which is in allylic position to the double bond and thus should be most susceptible to oxidative attack. In fact, among the 13 most promising strains from our primary screening capable of -ionone hydroxylation, 5 hydroxylated -ionone at C-3 with a much higher hydroxylation activity (Table 1). S. fradiae, for instance, converted 75% of the -ionone added to the culture medium; i.e., its activity is 19 times higher for the -isomer than for the -isomer. Good conversion of both ionone isomers was found for S. arenae.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Products of oxidative transformation of -ionone (A) and -ionone (B).

View this table: [in this window] [in a new window]   TABLE 1.   Biotransformation of - and -ionone by various Streptomyces strains

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Hydroxylation of (6R)- and (6S)--ionone with Streptomyces strains.

Structural characterization of (3R,6R)- and (3S,6S)-hydroxy--ionone. One transformation (S. fradiae [Table 1]), where the GLC trace showed sufficiently high turnover of the substrate, racemic -ionone, was worked up as described above, and the ether extract was dried and evaporated. The oily residue was taken up in CDCl₃ (1 ml), the solution was dried again on a molecular sieve, and the extract was filtered carefully and used for the individual ¹H and ¹³C NMR experiments.

View this table: [in this window] [in a new window]   TABLE 2.   NMR data for (3R,6R)- and (3S,6S)-hydroxy--ionone