Introduction

The process of bioconversion, which is also referred to as biotransformation, involves the modification of natural compounds by microorganisms. For example, the anti-hyperlipidemia drug pravastatin is produced on an industrial scale using Streptomyces carbophilus to hydroxylate the compound ML-236B.1 In addition, the precursor of anti-fungal drug micafungin is produced via the microbial deacylation of an acyl chain in the lipopeptide FR901379.2 As microorganisms are also capable of hydroxylating a wide range of compounds,3, 4, 5, 6, 7 bioconversion is considered to be a powerful tool for the modification of useful natural products with complicated structures.

Cyclosporin A (CsA) is a cyclic peptide that inhibits cellular cyclophilins and was developed for clinical use as an immunosuppressant critical for organ transplants. The inhibitory activity of CsA is dependent on its binding to cyclophilin, as the resulting complexes inhibit the function of calcineurin in T cells.8 Recently, it was reported that CsA suppresses replication of the hepatitis C virus (HCV) genome, and that the inhibition of cyclophilins are critical for HCV replication.9, 10 As CsA treatment in combination with interferon was effective against HCV,11 a number of CsA derivatives, including DEBIO-025, SCY-635 and NIM-811,12 are currently being evaluated in clinical trials as anti-HCV drugs.

Compound FR901459 is a member of the cyclosporin family and was first identified in the fermentation broth of Stachybotrys chartarum No. 19392.13 This compound exerts immunosuppressive activity upon binding to cyclophilin and also has potent neuroprotective effects by inhibiting mitochondrial damage,14 but is structurally distinct from CsA (Figure 1). This compound is considered to have an attractive backbone structure for the development of an anti-HCV drug, but lacks active moieties, which are essential for synthesizing derivatives of this compound. In the present work, we describe the biotransformation, isolation and physicochemical characterization of seven bioconversion products (1–7) of FR901459 that were derived using an actinomycete strain, designated 7887, which was revealed as a species of Lentzea based on phylogenetic analysis.

Figure 1
figure 1

Structures of FR901459 and cyclosporin A. Bmt, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine; Me, methyl.

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Results

Isolation of bioconversion products (compounds 1–7) from strain 7887

A screening for actinomycete strains with bioconversion activity was performed with 30 strains isolated from soil samples. We identified one strain, designated 7887, that converted FR901459 to a number of derivatized products. To isolate the bioconversion products, strain 7887 was incubated in the presence of 0.5 mg ml−1 FR901459 for 13 h, and the reaction mixture was extracted with an equal volume of acetone at room temperature. The mixture was filtered with an aid of diatomaceous earth and the diluted filtrate was passed through a 14-l Diaion HP-20 column (Mitsubishi Chemical, Tokyo, Japan). The column was washed with 25% aqueous acetone, eluted with methanol and the active fraction was passed through an 8-l Daisogel SP-120-ODS-B column (15/30 μm, Daiso, Osaka, Japan). After eluting the column with 50 and 60% aqueous acetonitrile, the following three fractions containing a total of seven compounds were obtained: fractions I (compound 1, 60% aqueous acetonitrile), II (compounds 2–4, 60% aqueous acetonitrile) and III (compounds 5 and 7, 50% acetonitrile).

To isolate the bioconversion products from fraction I, the sample was diluted with an equal volume of water and then passed through a 2-l Daisogel SP-120-ODS-B column. The column was eluted with 60% aqueous acetonitrile and the eluate was rechromatographed as above. The active fraction was again passed through a 2-l Daisogel SP-120-ODS-B column and eluted with 70% aqueous acetonitrile containing 0.1% trifluoroacetic acid. The active fraction was diluted with an equal volume of water, passed through a 2-l Daisogel SP-120-ODS-B column and eluted with ethyl acetate. The solvent fraction was concentrated to dryness in vacuo, dissolved in CHCl3 and applied to silica gel column (Silica Gel 60 N, spherical, neutral, 40–100 μm, Kanto Chemical Co., Inc., Tokyo, Japan). The column was washed with 2% MeOH in CHCl3, eluted with 3 and 5% MeOH in CHCl3, and the active fraction was concentrated to dryness in vacuo and then precipitated with n-hexane to give 3.7 g of compound 1 as a white powder.

A similar approach was used to isolate the bioconversion products of FR901459 in fractions II and III. The water-diluted fraction II was passed through an 8-l Daisogel SP-120-ODS-B column, which was then eluted with 60% aqueous acetonitrile. The active fraction was diluted with an equal volume of water and passed through a 2-l Daisogel SP-120-ODS-B column. After washing the column with water and adding ethyl acetate, the eluted solvent fraction was concentrated to dryness in vacuo and precipitated by n-hexane to give 32.8 g of compound 2 as a white powder. The first elution of the ODS column with 60% aqueous acetonitrile gave a subfraction that contained compounds 3 and 4. To separate compounds 3 and 4, this diluted subfraction was passed through a 2-l Daisogel SP-120-ODS-B column, and was then eluted with 60% aqueous acetonitrile. The active fraction of compound 3 was diluted with an equal volume of water, passed through a 1-l Daisogel SP-120-ODS-B column and then eluted with 60% aqueous acetonitrile. The active fraction was concentrated to dryness in vacuo to give 322 mg of compound 3 as a pale yellow powder. Using the same purification method as described above for compound 3, 740 mg of compound 4 as a pale yellow powder was obtained.

Similarly, fraction III was diluted with an equal volume of water and passed through an 8-l Daisogel SP-120-ODS-B column. After eluting the column with 50% aqueous acetonitrile, the active fractions containing compound 5 or 7 were concentrated in vacuo and then extracted with ethyl acetate. To purify compound 5, the solvent extract was concentrated in vacuo, dissolved in CHCl3 and applied to a silica gel column, which was then washed with 3% MeOH in CHCl3 and eluted with 4 and 5% MeOH in CHCl3. The active fraction was concentrated in vacuo, dissolved in MeOH and then loaded onto a preparative HPLC column (Mightysil RP-18 GP 250 mm × 20 mm I.D., 5 μm; Kanto Chemical Co., Inc.). The column was eluted with 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 10 ml min−1. The diluted active fraction was passed through a 19-ml Daisogel SP-120-ODS-B column. After the column was washed with water and eluted with ethyl acetate, the solvent fraction was concentrated to dryness in vacuo and precipitated with n-hexane to give 205 mg of compound 5 as a white powder. Using the same purification method as described above for compound 5, 227 mg of compound 7 as a white powder was obtained.

To purify compound 6, a part of the filtrate from the reaction mixture was diluted with an equal volume of water, passed through a 2-l Daisogel SP-120-ODS-B column and eluted with 50% aqueous acetonitrile. The active fraction was diluted with an equal volume of water, passed through a 2-l Daisogel SP-120-ODS-B column and eluted with 45% aqueous acetonitrile containing 0.1% trifluoroacetic acid. The active fraction was diluted with an equal volume of water, passed through a 2-l Daisogel SP-120-ODS-B column and eluted with ethyl acetate after the column was washed with water. The solvent fraction was concentrated to dryness in vacuo and precipitated with n-hexane to give 1.3 g of compound 6 as a pale yellow powder. The isolation procedures for compounds 1–7 are summarized in Figure 2.

Figure 2
figure 2

Isolation and purification of compounds 1–7.

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Structure elucidation

The physicochemical properties of the isolated bioconversion products are summarized in Table 1. All compounds were soluble in CHCl3, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), pyridine and MeOH, and were slightly soluble in water, but were insoluble in n-hexane. The ESI-TOF (time-of-flight) MS analysis (Table 1) showed each molecular formula for compounds 1–4 to be C62H111N11O14, and C62H111N11O15 for compounds 5–7, indicating that compounds 1–4 were mono-hydoxylated forms of FR901459, whereas compounds 5–7 were bis-hydoxylated.

Table 1 Physicochemical properties of compounds 1–7
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For the NMR study of compounds 1–7, pyridine-d5 was used as a solvent because it resulted in the best separation of all tested solvents. At least two conformers were observed for all compounds as in the case of FR901459.13 By observing 13C NMR and HSQC spectra, the existence of 62 carbons was confirmed, including 11 carbonyl carbons, 11 Cα carbons and six N-methyl carbons for all compounds. Five NH protons were indicated by correlation with each adjacent Cα proton in the COSY spectra. The structure of each amino-acid residue was elucidated by the combination of NMR spectra, COSY, TOCSY and HMBC analyses. The peptide sequences were determined to be unchanged from FR901459 by analyzing multiple correlations in the HMBC spectra from Cα, NH and N-methyl protons to carbonyl carbons.

NMR assignments are shown in the Table 2. Hydroxylated positions were clearly detected by chemical shifts of the terminal methylene (δH 4.35 p.p.m. and δC 63.1p.p.m.) of 1-MeBmt for compound 1, the terminal methylene of 9-MeLeu (δH 3.77 and 3.71p.p.m., and δC 67.9 p.p.m.) for compound 2, the quaternary carbon of 9-MeLeu (δC 69.0 p.p.m.) for compound 3 and the terminal methylene of 9-MeLeu (δH 3.91 and 3.79 p.p.m., and δC 66.1 p.p.m.) for compound 4. Oxygenated positions of compounds 6, 5 and 7 were concluded to be combinations of compounds 2, 3 and 4 with compound 1 as they have both hydroxylated carbons resonating at 63.1 p.p.m. of 1-MeBmt, and 67.9, 69.0 and 66.1p.p.m. of 9-MeLeu, respectively (Table 2).

Table 2 NMR assignments of compounds 1–7 and FR901459
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During the attempted derivatization of these bioconverted products, we obtained FR310363, a tert-butyldimethylsilyl-protected compound in 2-Thr, which was determined to be a crystalline derivative of compound 2. A single crystal of FR310363 was obtained from a solution of ethyl acetate, and X-ray crystallography analysis allowed assignment of the absolute configuration of 4-MeLeu to be (S). The X-ray structure analysis data have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 1048671. An ORTEP drawing is shown in Figure 3. We speculated that the absolute configuration of the same position of compound 6 to be (S), and those of compounds 4 and 7 to be (R) based on their 13C NMR similarity. The proposed gross structures of compounds 1–7 are shown in Figure 4.

Figure 3
figure 3

ORTEP drawing of FR310363. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Figure 4
figure 4

Structures of bioconversion products (compounds 1–7) derived from FR901459.

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Biological activity

Compounds 1–4 inhibited the replication of HCV subgenomic replicons in #50-1 cells with IC50 values of 1.5, 2.6, 0.47 and 1.0 μM, respectively. FR901459 also exerted an inhibitory effect with an IC50 value of 0.14 μM. In contrast, no obvious inhibitory effects were observed for compounds 5–7 at 10 μM.

Taxonomic characterization of strain 7887

This strain formed compact colonies on the agar surface and had well developed and irregularly branched substrate mycelia that penetrated the agar surface. The cultural characteristics of strain 7887 are summarized in Table 3. Aerial mycelia were moderately developed on 1/5 yeast extract–starch agar, glycerol-asparagine agar (ISP-5), tyrosine agar (ISP-7), Czapek agar, yeast-starch agar and glucose-asparagine agar, and were fragmented into rod-shaped elements. Sclerotic granules, sporangia and motile spores or fragments were not observed on any of the examined media.

Table 3 Cultural characteristics of strain 7887
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The reverse side of colonies appeared reddish brown to pale orange. No melanoid pigments were produced in tryptone-yeast extract broth or peptone-yeast extract–iron agar (ISP-6). Light orange to brown soluble pigments were produced by colonies cultured on yeast extract–malt extract agar (ISP-2), oatmeal agar (ISP-3), inorganic salts–starch agar (ISP-4), glycerol-asparagine agar (ISP-5), tyrosine agar (ISP-7), Bennett’s agar, Czapek agar, yeast-starch agar, glucose-asparagine agar and sucrose-nitrate agar. The color of mycelia and soluble pigments were not pH sensitive.

The physiological characteristics of strain 7887 are summarized in Table 4. L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol, D-mannitol, cellobiose, dextrin, D-galactose, glycerol, D-mannose, maltose, D-melibiose, soluble starch and trehalose were all utilized as carbon sources, but L-rhamnose, raffinose, adonitol, dextran, dulcitol, inulin, lactose, D-melezitose, sorbitol, L-sorbose and xylitol were not utilized. Strain 7887 was able to grow in the temperature range of 10.5–32.5 °C and exhibited optimal growth at 31.0 °C. Meso-diaminopimelic acid was detected in whole-cell hydrolysates.

Table 4 Physiological characteristics of strain 7887
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Comparison of 16S ribosomal DNA (rDNA) sequences showed that strain 7887 had the highest similarity with Lentzea violacea LM 036T (99.2%). The similarity values of 16S rDNA sequences between strain 7887 and members of the genus Lentzea were between 97.1% (L. californiensis) and 99.2% (L. violacea). These species comprised a single cluster on the phylogenetic tree (Figure 5).

Figure 5
figure 5

Phylogenetic tree based on near full-length 16S rDNA sequences (~1400 nucleotides) showing the position of strain 7887. The numbers on the branches are confidence limits (expressed as percentages) estimated from a bootstrap analysis with 1000 replicates (limits >50% are indicated). Scale bar, 0.01 substitutions per nucleotide position.

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Based on the morphological and chemical characteristics of strain 7887, and the results of the phylogenetic analysis, we concluded that strain 7887 belonged to the genus Lentzea.15, 16, 17 Therefore, this strain was designated as Lentzea sp. 7887.

Evaluation of members of the genus Lentzea for bioconversion capability

To evaluate the bioconversion activity of strain 7887, the bioconversion products generated by six other strains of Lentzea were analyzed as described above for the reaction products of strain 7887. HPLC analysis revealed that all of the examined strains converted FR901459 to derivatives 1–7 (Table 5). Compound 3 was the major conversion compound produced by four of the Lentzea strains. In contrast, strain 7887 only produced relatively low levels of compound 3, but formed compounds 2 and 6 at markedly higher rates than all of the other strains. Taken together, these results suggested that several members of the genus Lentzea are suitable for metabolizing FR901459, and that strain 7887 has a unique profile for the bioconversion of FR901459 to potentially useful compounds.

Table 5 Bioconversion capabilities of members of the genus Lentzea
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Discussion

The CsA analog FR901459 is a useful chemical template for the development and design of new classes of anti-HCV drugs; however, this compound is resistant to chemical modification. Because bioconversion has the potential to efficiently hydroxylate chemically resistant compounds; here, we screened for actinomycete strains capable of bioconverting FR901459, and included actinomycete strains that reportedly catalyze the conversion other compounds, such as AS1387392.18 We identified a novel isolate, strain 7887, which was capable of converting FR901459 to several bioconversion products (1–7). Isolation and structural elucidation of the derivatization products revealed that this strain was able to efficiently hydroxylate two positions of FR901459: the methyl-Bmt group at position 1, generating compound 1; the N-methyl-Leu group at position 9, generating compounds 2–4; and both positions 1 and 9, generating compounds 5–7. These compounds might serve as useful chemical templates that can be further modified to produce novel anti-HCV drugs.

Compound 2 was generated from FR901459 by strain 7887 at a reasonably high rate. It was previously reported that compounds chemically derived from compound 2 had potent anti-HCV effects, but reduced immunosuppressive activity, and that further derivatization of the three-position generated compounds with powerful anti-HCV activity and excellent pharmacokinetic profiles.19 Thus, strain 7887 may be useful for the large-scale production of compound 2 as a template for the development of highly effective and safe anti-HCV agents.

Morphological and chemical characterization and phylogenetic analysis of 16S rDNA indicated that strain 7887 belongs to the genus Lentzea.15, 16, 17 Strain 7887 could be distinguished from the most closely related strain, L. violacea LM036T based on phenotypic characteristics, including the temperature range for growth, substrate mycelium color and utilization of L-rhamnose, adonitol and lactose (data from Lee et al.).20 We therefore suggest that strain 7887 belongs to a new species of the genus Lentzea, but further study will be needed.

Nearly, all of the Lentzea strains examined in the present study were able to convert FR901459 to derivative compounds. In a preliminary study, several members of the closely related genera Saccharothirix, Actinokinespora, Lechevalieria and Actinosynnema only poorly metabolized FR901459, as demonstrated by the low amount of compound 1 produced by these strains. These results indicate that Lentzea strains have superior FR901459 bioconversion activity. However, within the genus Lentzea, widely varying bioconversion rates and products were observed, at least under the present culture conditions. In view of the high productivity of compound 2, strain 7887 is considered to be the most promising strain among those examined in the present study for the synthesis of bioactive compounds from FR901459.

In conclusion, we successfully identified a new strain Lentzea sp. 7887 that is capable of bioconverting the CsA derivative FR901459 into a chemically accessible template that is amenable for further modification. We anticipate that the findings from this study will lead to the targeted development of more effective and safer anti-HCV drugs.

Methods

Materials

FR901459 was isolated from the fermentation broth of S. chartarum No. 19392, as previously reported.13 The actinomycete Lentzea sp. 7887 was isolated from a soil sample collected in Ibaraki Prefecture, Japan. All other Lentzea strains used in this study were purchased from the Japan Collection of Microorganisms.

Culture media

Medium A consisted of 1% corn flour, 6% MS#3600 (Nihon Shokuhin Kako Co., Ltd, Tokyo, Japan), 1.2% Pharmamedia (Archer Daniels Midland Company, Lubbock, IL, USA), 0.8% dried yeast, 0.3% KH2PO4, 0.3% MgSO4·7H2O and 0.02% FeSO4·7H2O (pH 6.5). Medium B consisted of 0.5% glucose, 0.5% sucrose, 0.5% oatmeal, 0.2% yeast extract, 0.5% peptone, 0.5% peanut powder, 0.01% humic acid, 0.1% Tween 80 and 0.5% CaCO3 (pH 7.0).

Bioconversion of FR901459 using Lentzea sp. 7887

Aqueous medium A (60 ml) in a 225-ml Erlenmeyer flask was sterilized at 121 °C for 30 min, cooled and then inoculated with a loopful of a slant culture of Lentzea sp. 7887. After incubation at 30 °C with shaking at 250 r.p.m. for 3 days, 3.2 ml of the seed culture was transferred to a 500-ml Erlenmeyer flask containing 160 ml medium B. The flasks were then further incubated at 30 °C with shaking at 250 r.p.m. for 3 days, and 3.2 and 4 l of the second seed culture was then inoculated into 200- and 300-l jar fermentors, respectively, containing 160 and 200 l of production medium A containing 0.1% Adekanol (Adeka Co., Ltd, Tokyo, Japan) and silicone 0.1% (Shin-Etsu Chemical Co., Ltd, Tokyo, Japan), respectively. Fermentation was performed at 30 °C with stirring at 200 r.p.m. for 3 days. After the culture period, a solution of FR901459 (180 g) in methanol (7.2 l) was added to 360 l of the cultured broth. The reaction mixture was then incubated at 30 °C with constant stirring for 13 h and the reaction products were analyzed by HPLC, as described below.

Assay for anti-HCV activity

Inhibitory activity against replication of the HCV replicon was evaluated by determining the amount of replicon RNA using real-time quantitative reverse transcription (RT)-PCR.21, 22 HCV replicon cells (#50-1, a kind gift from Dr K Shimotohno, Kyoto University) were cultured in Dulbecco's Modified Eagle medium (DMEM) containing 5% fetal bovine serum and 300 μg ml−1 G418 at 37 °C in 5% CO2.23 After incubation for 2 days with the test compounds, total RNA was isolated from cells using the RNeasy kit (Qiagen Inc., Tokyo, Japan) according to the manufacturer’s protocol. The quantitative RT-PCR was performed using the TaqMan Ez RT-PCR Core Reagent (Applied Biosystems Inc., Foster City, CA, USA). The sequences of the primer set for amplifying part of the HCV genome were as follows: (5′-CGGGAGAGCCATAGTGG-3′ and 5′-AGTACCACAAGGCCTTTCG-3′) and the complementary probe (5′-FAM-CTGCGGAACCGTGAGTACACTAMRA-3′; Takara Bio Inc., Shiga, Japan). The quantitative RT-PCR was performed using the ABI PRISM 7900HT sequence detection system (Applied Biosystems Inc.). The standard curve for this assay was calculated using a series of 10-fold dilutions of synthetic HCV RNA (a kind gift from Dr M Kohara, Tokyo Metropolitan Institute of Medical Science). The copy number was calibrated to the levels of the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase. IC50 values of the compounds were determined from the 50% replicon RNA levels corrected by the intrinsic control glyceraldehyde-3-phosphate dehydrogenase values.

Taxonomic characterization of Lentzea sp. 7887

Strain 7887 was taxonomically characterized using the methods and media described by Hamada.24 All observations were made after 14-day cultivation at 30 °C.

Morphological observations were made using an optical microscope and a scanning electron microscope (Hitachi S-2600N, Hitachi High-Technologies Corp., Tokyo, Japan) on cultures grown on 1/5 yeast extract–starch agar, which was composed of 0.4 g yeast extract (Daigo Eiyo, Osaka, Japan), 2 g soluble starch and 16 g agar in 1 l tap water (pH was adjusted to 7.2 with 1 N NaOH before sterilization). The temperature growth range of strain 7887 was also determined using 1/5 yeast extract–starch agar. Carbon utilization was evaluated on Pridham and Gottlieb’s medium.25 All color names used in this study were taken from the Methuen Handbook of Colour.26 The chemotaxonomic characterization of strain 7887 was performed by the procedure of Suzuki et al.27 Cell preparation and diaminopimelic acid isomer detection were conducted following the procedures developed by Becker et al.28

16S rDNA sequencing and phylogenetic analysis

The 16S rDNA sequence of strain 7887 was determined using the method developed by Nakagawa and Kawasaki,29 and was compared with 16S rDNA sequences of type strains obtained from the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp) by BLAST search. A phylogenetic tree was then constructed using the alignment program ClustalW.30 The DDBJ/GenBank/EMBL accession number for 16S rDNA sequence of strain 7887 is AB986227.

Evaluation of bioconversion activity of Lentzea strains

Each strain was grown in tubes (17 mm × 100 mm) containing 1.5 ml medium A. The culture tubes were inoculated with a loopful of a slant culture and then incubated at 30 °C with shaking at 250 r.p.m. for 3 days. After incubation, 0.5 ml of culture broth was transferred to a tube containing 0.5 mg ml−1 FR901459, and the reaction mixtures were further incubated at 30 °C for 9 h with shaking at 250 r.p.m. Extracts were prepared by mixing the reaction mixture with equal volumes of acetone followed by centrifugation of the extracts (3000 g, 10 min). The resulting supernatants were analyzed by HPLC (Agilent HP1100; Agilent Technologies, Inc., Santa Clara, CA, USA) using an ODS column (Mightysil RP-18 GP, 100 mm × 2.0 mm I.D.; Kanto Chemical Co., Inc.). The elution solvents were water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B). A 15-min linear gradient from 45 to 100% solvent B was used with a 2-min hold and a flow rate of 0.4 ml min−1. The detection wavelength was set at 210 nm and the column was heated at 50 °C. Under these conditions, FR901459 was eluted at a retention time of 16.9 min and compounds 1– 7 were eluted at retention times of 10.9, 8.5, 10.2, 9.4, 5.0, 4.0 and 4.2 min, respectively.