The prodigiosins are reddish-colored tripyrrole antibiotics biosynthesized by several microorganisms such as Serratia,1 Pseudomonas2 and Streptomyces.3 Streptomyces griseoviridis 2464-S5 produces prodigiosin R14 and roseophilin,5 a unique prodigiosin-related compound containing two pyrrole and one furan moieties (Figure 1), and carries the rph gene cluster involved in their biosynthesis.6 Prodigiosin R1 and roseophilin possess an alkyl chain with the same length and differ in the cyclization pattern (Figure 1). Twenty-one of 25 genes in the rph cluster are homologous to red genes in Streptomyces coelicolor A3(2),7 which produces undecylprodiginine3 and butyl-meta-cycloheptylprodiginine (streptorubin B).8 In the rph cluster, rphG, rphG2, rphG3 and rphG4 showed sufficient homology to redG, a gene involved in cyclization of undecylprodiginine to butyl-meta-cycloheptylprodiginine.9 Thus, we attempted to analyze the functions of these rph genes using a heterologous expression system in S. coelicolor.

Figure 1
figure 1

Structures of prodigiosin derivatives.

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S. coelicolor M511, an actinorhodin-nonproducing mutant of S. coelicolor A3(2),10 also produced undecylprodiginine (3) and butyl-meta-cycloheptylprodiginine (4). Disruption of redG in S. coelicolor M511 resulted in the lack of 4 production. The redG disruptant (ΔredG) transformed with a redG-expressing plasmid restored the productivity of 4. To analyze the function of redG homologous genes, the redG disruptant was transformed with plasmids bearing rph genes. Two prodigiosins (1 and 2) in addition to 3 were detected in the culture of S. coelicolor M511 ΔredG carrying an rphG-expressing plasmid, whereas the strain expressing rphG2, rphG3 and rphG4 produced only 3 (Figure 2).

Figure 2
figure 2

HPLC analysis of the culture extract of S. coelicolor M511 ΔredG expressing rph genes. (a) ΔredG, (b) ΔredG+redG, (c) ΔredG+rphG2−rphG4, (d) ΔredG+rphG. 1: metacycloprodigiosin, 2: propyl-meta-cyclooctylprodiginine, 3: undecylprodiginine, 4: butyl-meta-cycloheptylprodiginine.

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Compound 1 was identified as metacycloprodigiosin (Figure 1)11, 12 by its 1H and 13C NMR spectra (Table 1) and by high-resolution FAB-MS (m/z 392.2703 [M+H]+, calcd. for C25H34N3O, 392.2702). Compound 2 had the same molecular formula (C25H33N3O) as 1, as indicated by high-resolution FAB-MS (m/z 392.2702 [M+H]+, calcd. for C25H34N3O, 392.2702). In the 1H and 13C NMR spectra, aromatic signals for 2 were almost identical to those for 1 (Table 1), thereby showing that these compounds only differ in the alkyl-chain branching position. The existence of a propyl group in 2 was required by 1H–13C long-range correlations from H3-24 to C-23 and C-22 in the HMBC spectrum. A COSY experiment revealed a proton spin network from the propyl group to H2-20 through a methine proton at δ 3.00 (H-21). The cyclization position of the alkyl chain at C-21 was confirmed by HMBC data as shown in Figure 3. The structure of 2 was thus determined as propyl-meta-cyclooctylprodiginine (Figure 1). To our knowledge, this prodigiosin has not been reported previously.

Table 1 13C and 1H NMR data for metacycloprodigiosin (1) and Propyl-meta-cyclooctylprodiginine (2) in CDCl3
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Figure 3
figure 3

NMR analysis of propyl-meta-cyclooctylprodiginine (2).

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Prodigiosins are known to show various biological properties such as antimicrobial, antimalarial, antitumor and immunosuppressive activities.13 The cytotoxic activities of linear or cyclized prodigiosins were evaluated using HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells. The IC50 values against HeLa and HT1080 cells were 1.1 and 3.7 μM for 1, 0.89 and 3.5 μM for 2, 3.5 and 4.2 μM for 3 and 3.2 and 3.9 μM for 4, respectively. The results indicate that cyclization of the alkyl chain does not significantly affect the cytotoxicity of prodigiosins.

Known prodigiosin cyclization genes, redG and mcpG, are involved in cyclization of undecylprodiginine (3) to butyl-meta-cycloheptylprodiginine (4) and metacycloprodigiosin (1), respectively.9 The rph cluster contained four redG homologous genes, rphG, rphG2, rphG3 and rphG4. Among them, rphG was identified as a prodigiosin cyclization gene based on two cyclized prodigiosins produced by an rphG-expressing strain of S. coelicolor M511 ΔredG. These prodigiosins were determined to be metacycloprodigiosin (1) and propyl-meta-cyclooctylprodiginine (2) by spectroscopic analyses. Metacycloprodigiosin possesses the same 12-membered carbocyclic ring as prodigiosin R1 and propyl-meta-cyclooctylprodiginine has an 11-membered carbocyclic ring. RphG is presumed to cyclize isotridecylprodiginine to prodigiosin R1, although no prodigiosin with an 11-membered carbocyclic ring has been isolated from S. griseoviridis 2464-S5. Neither rphG2, rphG3 nor rphG4 was able to cyclize undecylprodiginine. Since a substrate for the cyclization enzyme is not undecylprodiginine in the roseophilin producer, these rph genes might be involved in cyclization of the true substrate to form a roseophilin-type carboskeleton. Further biosynthetic studies are in progress.

Experimental procedure

General experimental procedures

UV and visible spectra were measured on a UV-1700 spectrometer (Shimadzu, Kyoto, Japan). IR spectra were obtained on a Spectrum 100 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) in the ATR (attenuated total reflection) mode. Mass spectra were measured on a JMS-SX102A spectrometer (JEOL, Akishima, Tokyo, Japan) in the FAB mode using m-nitrobenzyl alcohol as matrix and polyethylene glycol as internal standard. NMR spectra were obtained on a JNM-LA400 spectrometer (JEOL) with 1H NMR at 400 MHz and with 13C NMR at 100 MHz. Chemical shifts are given in parts per million relative to CDCl3 (δH 7.24 and δC 77.0).

Bacterial strains and culture conditions

Actinobacterial strains, S. griseoviridis 2464-S5 and S. coelicolor M511, were used in this study. Growth condition and medium of S. griseoviridis were previously described.5 S. coelicolor and its derivatives were grown in yeast extract-malt extract (YEME) medium for making protoplasts or R5 agar medium for prodigiosin production.12 Escherichia coli XL1-blue MRF′ was used for genetic engineering and grown in Lennox broth (LB) medium supplemented with 100 μg ml−1 ampicillin as required. E. coli JM110 was used for preparation of non-methylated DNA for transformation of S. coelicolor protoplasts.

DNA isolation and manipulation

Actinobacterial genetic procedures and other general procedures were carried out as described by Kieser et al.14 and Sambrook et al.,15 respectively. Isolated genomic DNA was farther purified by QIAGEN Genomic-tip (QIAGEN, Venlo, Netherlands).

Disruption of redG in S. coelicolor M511

A 1.8-kbp fragment upstream and a 1.6-kbp fragment downstream of redG were amplified from S. coelicolor genomic DNA using two sets of primers with additional restriction sites, and KOD plus PCR system (Toyobo, Osaka, Japan) and digested with XbaI/BglII and BglII/HindIII, respectively. These fragments were cloned between XbaI and HindIII sites of pGEM-11Z (Promega, Madison, WI, USA). A BglII fragment of the apramycin resistance gene apr was cloned into a BglII site between the fragment upstream and downstream of redG. The plasmid was digested with XbaI/HindIII and the apr-containing fragment was ligated into an XbaI/HindIII site of pWHM3-DIS, a gene disruption plasmid obtained by BclI digestion of pWHM3. The redG disruption plasmid was introduced in S. coelicolor M511 and apramycin-resistant/thiostrepton-sensitive mutants were selected on R5 plates.

Construction of expression plasmids for prodigiosin cyclization genes

A promoter for the rphM gene in S. griseoviridis was used for expression of redG, rphG, rphG2, rphG3 and rphG4 in S. coelicolor. A cosmid containing rphM was digested with NheI and PstI and an rphM–rphG4 region was cloned between XbaI and PstI sites of pWHM3. An 875 bp fragment containing the rphM promoter was amplified by KOD plus PCR system (Toyobo) using the rphM–rphG4-containing plasmid as template with M13 primer (5′-GTTTTCCCAGTCACGAC-3′) and a primer containing an XbaI site (5′- CATTCTAGAGTCCCTCCGCGTGTCACC-3′). The fragment was cloned between EcoRI and XbaI sites of pWHM3 to construct the expression plasmid pWHMexM. Prodigiosin cyclization genes were amplified by KOD plus or Tks Gflex (Takara Bio, Kusatsu, Japan) using S. griseoviridis genomic DNA as template with primers containing XbaI or HindIII site (rphG: 5′-GCTCTAGAATGATCCCGAATCAGTGGTACGCC-3′ and 5′-ATCAAGCTTTCATGCGGAGGCCGCGATGT-3′, rphG2–rphG4: 5′-GCTTCTAGACGCAGTACGGAAAGGCAGCTGAAC-3′ and 5′-GGGAATTCAGCATGGACACAATGAAAGCTTGGT-3′) and cloned between XbaI and HindIII sites of pWHMexM.

HPLC analysis of prodigiosins

Fermented mycelium on R5 plates with 10 μg ml−1 thiostrepton was extracted with 2 volumes of acetone. The extract was concentrated and partitioned between ethyl acetate and water. The organic layer was evaporated and dissolved in methanol. The methanol solutions were analyzed by HPLC using a Senshu-Pak PEGASIL ODS SP100 column (Senshu Scientific, Tokyo, Japan) with 90% methanol containing 5 mM disodium hydrogen citrate. Absorption peaks for prodigiosins were detected at 530 nm.

Isolation of cyclized prodigiosins

The rphG-expressing strain was cultured on R5 plates (20 ml × 300 plates) at 27 °C for 7 days. The culture was extracted with acetone and the aqueous concentrate was extracted with ethyl acetate. The extract was partitioned between hexane and 90% methanol. The 90% methanol layer was evaporated and applied to a silica gel column, which was washed with hexane-chloroform (2:1) and eluted with chloroform. The eluate was subjected to preparative silica gel TLC developed with chloroform–methanol–29% ammonia water (200:20:1). Two fractions (1: Rf 0.86, 2: Rf 0.84) thus obtained were separately purified by HPLC using a Senshu-Pak PEGASIL ODS SP100 column with 90% methanol containing 5 mM disodium hydrogen citrate. The prodigiosin fractions were evaporated and the aqueous concentrates were extracted with ethyl acetate. Each extract was acidified by addition of equivalent hydrogen chloride and concentrated to dryness to give a hydrochloride salt of prodigiosin (1: 9.9 mg, 2: 3.6 mg).

Propyl-meta-cyclooctylprodiginine (2): red amorphous powder; m.p. 73 °C; high-resolution FAB-MS m/z 392.2702 ([M+H]+, calcd. for C25H34N3O, 392.2702); UV λmax (ɛ) 535 nm (62 700) in MeOH, 535 nm (82 700) in 0.01 M HCl–MeOH, 473 nm (32 700) in 0.01 M NaOH–MeOH; IR (ATR) νmax 3150, 3100, 1600, 1260 cm−1.

Cell culture and cell viability assay

HeLa and HT1080 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 0.1% glucose. The cells were plated and incubated for 48 h with various concentrations of samples. After the cells were treated with 0.5 mg ml−1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37 °C, the relative cell number was measured as absorbance at 540 nm. IC50 values were calculated by linear interpolation between the two drug concentrations above and below the 50% inhibition line.