This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

In Streptomyces griseus, A‐factor (2‐isocapryloyl‐3R‐hydroxymethyl‐γ‐butyrolactone) at an extremely low concentration triggers streptomycin biosynthesis and cell differentiation by binding a repressor‐type receptor protein (ArpA) and dissociating it from DNA. An A‐factor‐responsive transcriptional activator (AdpA) able to bind the promoter of strR, a pathway‐specific regulatory gene responsible for transcription of other streptomycin biosynthetic genes, was purified to homogeneity and adpA was cloned by PCR on the basis of amino acid sequences of purified AdpA. adpA encoding a 405‐amino‐acid protein containing a helix‐turn‐helix DNA‐binding motif at the central region showed sequence similarity to transcriptional regulators in the AraC/XylS family. The −35 and −10 regions of the adpA promoter were found to be a target of ArpA; ArpA bound the promoter region in the absence of A‐factor and exogenous addition of A‐factor to the DNA–ArpA complex immediately released ArpA from the DNA. Consistent with this, S1 nuclease mapping showed that adpA was transcribed only in the presence of A‐factor and strR was transcribed only in the presence of intact adpA. Furthermore, adpA disruptants produced no streptomycin and overexpression of adpA caused the wild‐type S. griseus strain to produce streptomycin at an earlier growth stage in a larger amount. On the basis of these findings, we propose here a model to demonstrate how A‐factor triggers streptomycin biosynthesis at a late exponential growth stage.

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

Staurosporine isolated fromThe subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.heme ͉ staurosporine ͉ rebeccamycin ͉ secondary metabolism S taurosporine and rebeccamycin (Fig. 1A) are natural products that have attracted much attention because of their strong inhibitory activity for protein kinase or DNA topoisomerase, which makes them therapeutically important anticancer agents. These natural products are members of a family of indolocarbazole alkaloids which have a similar structure, including an indole[2,3-a]carbazole core with a C-N linkage to a sugar moiety.The staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and the rebeccamycin biosynthetic gene cluster from Lechevalieria aerocolonigenes (39243; American Type Culture Collection, Manassas, VA) have been cloned and characterized. The staurosporine biosynthetic gene cluster consists of 15 ORFs spanning 22 kb (1), and the rebeccamycin biosynthetic gene cluster consists of 11 genes spanning 17 kb (2, 3). Gene disruption and/or heterologous gene expression experiments revealed that four genes (staO, staD, staP, and staC in Streptomyces sp. TP-A0274 and the homologous genes rebO, rebD, rebP, and rebC in L. aerocolonigenes) are responsible for the biosynthesis of the indolocarbazole skeleton (2, 3). In staurosporine biosynthesis, StaO initiates the synthesis by catalyzing the reaction of tryptophan to the imine form of indole-3-pyruvic acid (IPA imine), and StaD then catalyzes the coupling of two molecules of IPA imine to yield chromopyrrolic acid (CPA). Finally, the key skeleton structure referred to as the indolocarbazole core is constructed through the following two oxidation steps by StaP and StaC (2, 4) (Fig. 1B).StaP (CYP245A1) is a member of the cytochrome P450 family (5), which includes heme enzymes involved in steroid hormone biosynthesis, drug metabolism, and many other physiologically

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.

Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH 2 -terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH 2 -terminal portion of ArpA contained an ␣-helixturn-␣-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.