INTRODUCTION

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Secondary metabolic pathways of Streptomyces species sometimes allow cells to produce not only one end product but also structurally related compounds (3, 6, 23). The diversity of metabolites is attributed to the low substrate specificity of some of the secondary metabolic enzymes and to chemical instability of some of the intermediates (5). Such a situation is frequently encountered as an accumulation of shunt products instead of intermediates when a step in the secondary metabolic pathway is blocked (15, 20). Some of these shunt products form pigments that are structurally similar to the desired end product of the pathway.

Chlortetracycline (CTC) biosynthesis in Streptomyces aureofaciens has been well investigated (2, 20, 26). A type II polyketide synthase constructs the CTC skeleton, and 11 subsequent reactions modify the CTC skeleton. The final products in the pathway are four tetracycline antibiotics, CTC, tetracycline (TC), 6-demethylchlortetracycline (6-DCT), and 6-demethyltetracycline (6-DMT). TC and 6-DCT are formed when the 7-chlorination step and the 6-methylation step, respectively, in the pathway are blocked, and 6-DMT is formed when both of these steps are blocked. Deficiencies in the other steps of the CTC-biosynthetic pathway result in cells that cannot synthesize these four tetracycline antibiotics.

Mutants of S. aureofaciens that lack the 6-methylation step produce the yellow compound 6-DCT, which is an industrial material used for production of semisynthetic tetracyclines, and simultaneously produce dark brown pigments that accumulate in the media (4, 21). Recently, the dark brown melanin-like pigment (MP) produced in a culture of S. aureofaciens NRRL 3203, an organism that produces 6-DCT, was purified and was shown to have absorption and electron spin resonance spectra similar to those of melanin (12, 25a). However, the parent strain does not contain tyrosinase, suggesting that the pigment is derived from TC-related compounds (18). In this study, we examined the relationship between 6-DCT and MP, and below we discuss the mechanism of MP production in 6-DCT-producing S. aureofaciens strains.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. S. aureofaciens NRRL 2209 (wild type) was used as the DNA donor for cloning DNA that complemented the 6-methylase deficiency. Strain H-7591, which was derived through several rounds of mutagenesis from S. aureofaciens NRRL 3203, was used for mutation analysis. Other strains which we used are described in Table 1. Escherichia coli JM110 (29), purchased from Funakoshi (Tokyo, Japan), was used as a host strain for preparation of a cosmid library and for isolation of dam and dcm plasmid DNAs used for integrative transformation of S. aureofaciens. Cosmid pHC79 (11), purchased from Boehringer Mannheim (Indianapolis, Ind.), was used for preparation of a cosmid library. Integrative vector pSE119 was constructed by inserting a 1.1-kb BclI fragment containing the tsr gene prepared from plasmid pIJ702 (13) into the BglII site of E. coli plasmid pUC19-Bgl (7).

Media and culture condition. SK2 medium and PK2 medium were used during cultivation of Streptomyces strains as the seed and production media, respectively. SK2 medium contained (per liter) 20 g of soluble starch (stabilose K; Matsutani Kagaku Kogyo, Hyogo, Japan), 5 g of glucose, 5 g of yeast extract, 5 g of peptone, 3 g of Ehrlich meat extract, 0.2 g of KH₂PO₄, and 0.6 g of MgSO₄ · 7H₂O in deionized water (pH 7.6). PK2 medium contained (per liter) 50 g of corn starch, 60 g of soybean meal, 40 g of soybean oil, 5 g of KCl, 0.05 g of MgSO₄ · 7H₂O, 0.05 g of CuSO₄, 0.05 g of ZnSO₄, and 20 g of CaCO₃ in deionized water (pH 6.5). To produce tetracyclines and MP, frozen stock mycelium or spores in 20% glycerol were inoculated into a small test tube containing 4 ml of SK2 medium and cultivated aerobically at 28°C to the stationary phase (40 h). A portion (0.5 ml) of the seed culture was transferred to a large test tube containing 10 ml of SK2 medium. After 24 h of cultivation at 28°C, 1 ml of the seed culture was transferred into a 300-ml flask containing 30 ml of PK2 medium. Cultivation was carried out aerobically at 28°C for 6 days.

General DNA techniques. Conventional DNA manipulations were carried out by using standard procedures (19). Labelling of DNA probes, hybridization, and detection were performed by using the DIG system (Boehringer Mannheim). Genomic DNAs were isolated from Streptomyces strains by the method of Hopwood et al. (13). The plasmids used to transform S. aureofaciens were prepared from E. coli JM110 with a Qiagen (Hilden, Germany) plasmid kit.

Isolation of blocked mutants. Vegetative mycelia of S. aureofaciens H-7591 grown in 10 ml of SK2 medium were harvested, resuspended in 30 ml of 50 mM Tris-maleate buffer (pH 6.0) containing N-methyl-N'-nitro-N-nitrosoguanidine (500 µg/ml), and incubated at 30°C for 30 min. The mutagenized mycelia were washed twice with sterile water and spread onto ISP medium 2 (Difco). After incubation at 28°C for 2 weeks, spores were harvested, and appropriate dilutions were spread onto antibiotic medium 4 (Difco). Mutants that did not produce 6-DCT were selected by performing a bioassay with E. coli JM110, followed by an HPLC analysis of the fermentation products.

Analytical methods. Cell growth was monitored by determining the packed-cell volume ratio of the culture broth after centrifugation (1,600 × g, 10 min). The total sugar content was determined by the method of Dubois et al. (10).

1

Chemicals. All of the tetracyclines except 6-DMT were purchased from Sigma-Aldrich (St. Louis, Mo.). 6-DMT and MP are reference compounds of the Research Laboratories of Kyowa Hakko Kogyo. Cl-DMATC (28) was purified from the NP6 culture as described by Dairi et al. (8).

	RESULTS AND DISCUSSION

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Properties of MP production by S. aureofaciens NRRL 3203. It is generally accepted that in S. aureofaciens production of tetracyclines lags behind cell growth and is reduced by excess ammonia or inorganic phosphate in the culture medium (9, 14). We cultivated S. aureofaciens NRRL 3203 under various conditions and compared MP production with 6-DCT production (Fig. 1). Under normal conditions in PK2 medium, MP production started at the same time as 6-DCT production, and the level of production increased during the exponential and stationary phases (Fig. 1A). Production of both compounds decreased similarly in response to NH₄Cl addition (Fig. 1B). Similar decreases were also observed in the presence of excess K₂HPO₄ (Fig. 1C). These results showed that the fundamental characteristics of MP production were very similar to the fundamental characteristics of 6-DCT production, suggesting that MP production is related to 6-DCT production in S. aureofaciens.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Fermentation kinetics of S. aureofaciens NRRL 3203 in PK2 medium. (A) Time course for production of 6-DCT and MP. Symbols: , MP; , 6-DCT; , residual starch; , packed cell volume (PCV). (B) Influence of excess ammonia on production of 6-DCT (open symbols) and MP (solid symbols). Symbols:  and , no addition;  and , 1 g of NH4Cl per liter;  and , 3 g of NH4Cl per liter. (C) Influence of excess phosphate on production of 6-DCT (open symbols) and MP (solid symbols). Symbols:  and , no addition;  and , 1 g of K2HPO4 per liter;  and , 3 g of K2HPO4 per liter. K2HPO4 and NH4Cl were added at the time indicated by arrows after the pH was adjusted to 6.3. The values are means of values from duplicate experiments.

Requirement for MP production in tetracycline antibiotic producers. Six tetracycline antibiotic producers were inoculated into PK2 medium and examined to determine whether pigment and tetracyclines were produced. As shown in Table 1, MP was produced not only in cultures of the 6-DCT producer NRRL 3203 but also in cultures of the 6-DMT producer S. aureofaciens H-8979 and Streptomyces psammoticus ATCC 14125. In contrast, no dark pigment was produced by the CTC producer S. aureofaciens NRRL 2209 or by the TC producer Streptomyces avellaneus ATCC 23730. These results suggested that MP production is dependent on a defect in the 6-methylation step but is independent of the 7-chlorination step. To investigate the correlation between MP production and a defect in the 6-methylation step, we derived CTC-producing recombinants from 6-DCT-producing mutants by replacing the defective genes for the 6-methylation reaction with functional genes from NRRL 2209.

Construction of CTC-producing gene replacement recombinants and verification of the influence of the defect in the 6-methylation reaction on MP production. Genes for the 6-methylation reaction are located in a 32-kb region of the S. aureofaciens chromosome (25). To obtain the CTC-biosynthetic gene cluster, including functional genes for the 6-methylation reaction, we constructed a cosmid library of the chromosome of NRRL 2209 and isolated cosmid pGLA144 (37-kb insert) by colony hybridization as described previously (8). A physical map of cosmid pGLA144 is shown in Fig. 2.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Physical map of the CTC-biosynthetic gene cluster of S. aureofaciens NRRL 3203. The solid bars under the physical map show the DNA fragments carried on cosmids pGLA2 and pGLA144 and integrative plasmids pKN14419, pKN2, pKN4, and pKN108. DNA fragments which were inserted into pGLA144 and pKN14419 were cloned from strain NRRL 2209, and the other fragments were cloned from strain NRRL 3203. tcrA and tcrC, tetracycline resistance genes; chl, 7-chlorination gene; tcsC, ATC oxygenase gene (previously chl-ORF2); pks, CTC minimal polyketide synthase genes.

Mutation analysis of the correlation between the 6-DCT-biosynthetic pathway and MP production. To further examine the possibility that MP may be synthesized via the 6-DCT-biosynthetic pathway, we isolated mutants that did not produce 6-DCT from cultures of the 6-DCT producer H-7591 by selecting for cosynthesis (22) and by performing genetic complementation experiments with integrative plasmids containing either BamHI-digested or KpnI-digested fragments of cosmid pGLA2 (Fig. 2). The mutants selected could be divided into three classes based on their metabolites. Characteristics of representative mutants are summarized in Table 2. Class Ia mutant NP41, which produced neither MP nor 6-DCT but accumulated the penultimate intermediate Cl-DMATC of the 6-DCT-biosynthetic pathway was a mutant that was defective in anhydrotetracycline (ATC) oxygenase (1), the enzyme that catalyzes the penultimate step in the 6-DCT-biosynthetic pathway. The altered production properties of NP41 were simultaneously restored by single-crossover integration of pKN108 DNA containing the ATC oxygenase gene (tcsC; previously designated chl-ORF2), which is located in the 1.8-kb SacI segment (Fig. 2) (8). Two other ATC oxygenase-deficient mutants had the same phenotype as NP41 (data not shown). Class Ib mutant NP104 was a colorless mutant which did not produce MP or any 6-DCT-related compound but still could convert Cl-DMATC into 6-DCT, which suggested that an early step in the 6-DCT-biosynthetic pathway was blocked in this mutant. 6-DCT production and MP production were simultaneously restored by single-crossover integration of pKN4 DNA containing the minimal CTC polyketide synthase genes into the chromosome. Similar results were obtained with two other class Ib mutants (data not shown). In contrast, class II mutant NP733 was able to produce MP. The ability of this strain to produce 6-DCT, accompanied by a decrease in MP production, was restored by single-crossover integration of pKN2 DNA containing the 7-kb KpnI-BamHI fragment of the 6-DCT biosynthesis gene cluster into the chromosome. Another class II mutant, NP71, produced similar results (data not shown). These results indicate that some of the 6-DCT-biosynthetic enzymes, including ATC oxygenase, are essential for MP production, and they strongly support the hypothesis that MP is formed from a certain intermediate in the 6-DCT-biosynthetic pathway.

Branch point in the 6-DCT-biosynthetic pathway. The simplest interpretation of the finding that ATC oxygenase is indispensable for MP production is that class II mutants lack the final step of the 6-DCT-biosynthetic pathway and the last intermediate of the pathway is the branch point that leads to MP and 6-DCT. However, the results of the following experiments contradict this interpretation. As shown in Table 2, class II mutant NP733, as well as class Ib mutant NP104, could convert Cl-DMATC into 6-DCT, while class Ia mutant NP41 could not convert Cl-DMATC into 6-DCT. Similar results were obtained when we performed cosynthesis tests with NP41 as the secretor strain. Another class II mutant, NP71, could also convert Cl-DMATC into 6-DCT (data not shown). These results indicated that class II mutants were defective in some step before Cl-DMATC in the 6-DCT-biosynthetic pathway, not in the final step. This implies that an intermediate before Cl-DMATC is the branch point and that after this point the ATC oxygenase reaction is involved in MP formation.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Schematic diagram of the proposed pathway for MP production in S. aureofaciens. Abbreviations: PKS, polyketide synthase reaction; 6-CH3, 6-methylation reaction; 6-OH, ATC oxygenase reaction; 7-Cl, 7-chlorination reaction; CoA, coenzyme A.