Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.In modern microbiology, single-strain culture is the standard method for cultivating microorganisms. However, owing to the absence of interacting microorganisms that are present in the natural environment, the growth conditions in a flask culture are significantly different from those in the natural environment.The members of the order Actinomycetales, especially the genus Streptomyces, produce a number of antibiotics and other bioactive natural products. The genomic analysis of some Streptomyces strains revealed the presence of biosynthetic gene clusters for about 30 secondary metabolites, and these data imply that a single Streptomyces strain can produce more than 30 secondary metabolites (1, 9, 12). However, some of these secondary-metabolite genes are not expressed in fermentation culture. To date, various methods (7,14,15,17) have been used to activate genes synthesizing cryptic secondary metabolites. Secondary-metabolite production is affected by environmental factors (5, 17) such as temperature, the presence of hormone-like chemicals (6), and medium composition (3). Therefore, to identify new antibiotics, the isolation of new actinomycetes should be accompanied by the study of the effects of different growth conditions on each isolated strain.Coculture is an effective method for inducing the production of cryptic metabolites. Some coculture methods have been reported ...

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

Staurosporine is a representative member of indolocarbazole antibiotics. The entire staurosporine biosynthetic and regulatory gene cluster spanning 20-kb was cloned from Streptomyces sp. TP-A0274 and sequenced. The gene cluster consists of 14 ORFs and the amino acid sequence homology search revealed that it contains three genes, staO, staD, and THE JOURNAL OF ANTIBIOTICS DEC. 2002

The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides.

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In a systematic search for microbial products active in the syncytia inhibition assay (SIA)1}, two streptomycetes strains AA6532 (ATCC 55290) and AA3891 (ATCC 55286) were found to produce new antiviral antibiotics, named siamycins I and II, respectively. They are peptide group antibiotics structurally related to each other, and both show good inhibitory activity against HIV and HSV viruses. This paper describes the fermentation, isolation, physico-chemical properties and biological activities of siamycins I and II. Streptomyces sp. AA3891and AA6532were isolated from soil samples collected in Madhya Pradesh, India and Tokyo, Japan, respectively. Siamycin I was produced by the strain AA6532 in 500-ml Erlenmeyer flasks using medium composed of lactose 2%, dextrin (Nichiden Kagaku Co.) 2%, glucose 0.5%, corn steep liquor (Oji Corn Starch Co.) 1%, rice bran 1.5%, K2HPO4 0.05% and CoCl2-6H2O 0.0002%, pH 7.0. The flasks were shaken on a rotary shaker (200 rpm) at 28°C for 88 hours. Antibiotic production in the fermentation broth was estimated by the SIA. The medium for siamycin II herpes simplex viruses to antiviral agents. Antiviral Research 3: 223-234, 1986

Streptomycetes, which belong to the Gram-positive bacteria, produce secondary metabolites and sporulate. The timing of starting the secondary metabolite production and the sporulation depends on environmental conditions such as nitrogen and carbon sources. In order to obtain a tool for understanding the regulation mechanism, we carried out screening for chemical substances that induce secondary metabolism and sporulation in streptomycetes and found an active substance from the culture broth of Streptomyces sp. TP-A0584. This substance designated goadsporin promoted the formation of red pigment and sporulation at a concentration of 1 jim in Streptomyces lividans TK23 which does not produce the pigment under normal growth conditions. Goadsporin is an oligopeptide consisting of 19 amino acids with the molecular formula C72H97N19O20S2. Sporulation and/or secondary metabolite production was induced in 36 streptomycetes strains among 42 strains tested. These results suggest that goadsporin acts on a common regulation pathwayfor sporulation and secondary metabolism in streptomycetes and can be a powerful tool to analyze the regulation mechanism.

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