Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.antibiotic | metabolic pathway engineering | biosynthetic gene cluster | ent-copalyl diphosphate | terpene synthase I nfectious disease is the second leading cause of death worldwide, and the growing number of antibiotic-resistant microbes threatens to worsen this problem; only two previously undescribed classes of antibiotics have been introduced into the clinic since the 1960s (1, 2). Diabetes affects nearly 24 million people in the United States, and current therapies suffer from serious limitations (3). Platensimycin (PTM) and platencin (PTN) are recently discovered natural products (4) that are potent and selective inhibitors of bacterial (5, 6) and mammalian (7) fatty acid synthases. Remarkably, they have emerged as promising drug leads for both antibacterial (5,6,8,9) and antidiabetic (7) therapies. The efficacy of PTM and PTN in treating bacterial infections (5, 6), including those that are resistant to commercially available drugs, and the efficacy of PTM in treating diabetes and related metabolic disorders (7) have been demonstrated in mouse models.Structurally, PTM and PTN are composed of two distinct moieties-a substituted benzoic acid and an aliphatic cage moiety joined together by a flexible propionamide chain (Fig. 1A) (Fig. 1A) and diterpenoid natural products of both ent-kaurene and ent-atiserene origin are well known (SI Appendix, Fig. S1 C and D). Although numerous terpene synthase genes have been cloned from eukaryotes, only a few have been cloned from prokaryotes (17-19). The only ent-kaurene synthase of bacterial origin was reported in 2009 (20), and no gene or enzyme of eukaryotic or prokaryotic origin for ent-atiserene biosynthesis has ever been reported. Interestingly, ent-kaurene synthase-catalyzed biosynthesis of ent-kaurene from ent-copalyl diphosphate (ent-CPP) can produce ent-atiserene as a minor metabolite (21). Minor mutations to terpene synthases in general (22) and CPPutilizing terpene synthases in particular (21,23,24) are also known to alter product specificity. These observations, together with the fact that no ent-atiserene synthase is known, has become the basis of the current proposal th...

Platensimycin (1) and platencin (2) are novel antibiotic leads against multi-drug resistant pathogens. The production of 2 in Streptomyces platensis MA7339 is under the control of ptnR1, a GntR-like transcriptional regulator. Inactivating ptnR1 afforded S. platensis MA7339 mutant strain SB12600 that overproduces 2 at titer ~100-fold greater than that from the wild-type strain and accumulates platencin A 1 (3) and eight new congeners platencin A 2 -A 9 (4-11). The isolation, structural elucidation, and antibacterial activity of 4-11, in comparison to 1-3, are described.The recently discovered platensimycin (1) and platencin (2) represent one of only a few new classes of antibiotics that have been discovered since the early 1960's. 1-4 They potently inhibit the growth of a range of Gram-

Lymphatic filariasis is caused by the parasitic nematodes Brugia malayi and Wuchereria bancrofti and asparaginyl-tRNA synthetase (AsnRS) is considered an excellent antifilarial target. The discovery of three new tirandamycins (TAMs), TAM E (1), F (2), and G (3), along with TAM A (4) and B (5), from Streptomyces sp. 17944 was reported. Remarkably, 5 selectively inhibits the B. malayi AsnRS and efficiently kills the adult B. malayi parasite, representing a new lead scaffold to discover and develop antifilarial drugs.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. We have previously cloned and sequenced the PTM-PTN dual biosynthetic gene cluster from S. platensis MA7327 and the PTN biosynthetic gene cluster from S. platensis MA7339, the latter of which is composed of 31 genes encoding PTN biosynthesis, regulation, and resistance. We have also demonstrated that PTM or PTN production can be significantly improved upon inactivation of the pathway specific regulator ptmR1 or ptnR1, in S. platensis MA7327 or MA7339, respectively. We now report engineered production of PTN and congeners in a heterologous Streptomyces host. Expression constructs containing the ptn biosynthetic gene cluster were engineered from SuperCos 1 library clones and introduced into five model Streptomyces hosts, and PTN production was achieved in Streptomyces lividans K4-114. Inactivation of ptnR1 was crucial for expression of the ptn biosynthetic gene cluster, thereby PTN production, in S. lividans K4-114. Six PTN congeners, five of which were new, were also isolated from the recombinant strain S. lividans SB12606, revealing new insights into PTN biosynthesis. Production of PTN in a model Streptomyces host provides new opportunities to apply combinatorial biosynthetic strategies to the PTN biosynthetic machinery for structural diversity.

Lymphatic filariasis is caused by the Brugia malayi parasite. Three new congeners of the depsipeptide WS9326A (1), WS9326C (2), WS9326D (3) and WS9326E (4), were isolated from Streptomyces sp. 9078 by using a B. malayi asparaginyl-tRNA synthetase (BmAsnRS) inhibition assay. WS9326D specifically inhibits the BmAsnRS, kills the adult B. malayi parasite, and does not exhibit significant general cytotoxicity to human hepatic cells, representing a new lead scaffold for antifilarial drug discovery.

Natural products remain the best sources of drugs and drug leads and serve as outstanding small molecule probes to dissect fundamental biological processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here we report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to our approach is the innovative preparation, by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from our actinomycete collection, for their biosynthetic potential of four classes of natural products, aromatic polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products.

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Botrytis cinerea is a serious phytopathogen affecting a wide range of crops around the world. Many fungicides targeting Botrytis cinerea have failed due to the pathogen's genetic plasticity. In an effort to search for new fungicides from natural products, two new diterpenoids, named chloroxaloterpin A, 1, and B, 2, were isolated from culture broth of Streptomyces sp. SN194 along with four known diterpenoids, viguiepinol, 3, and oxaloterpins C-E, 4-6. Their structures were elucidated based on extensive MS, NMR, and X-ray crystallography analyses. Both the [(2-chlorophenyl)amino]carbonyl carbanic acyl group in 1 and the 2-[(2-chlorophenyl)amino]-2-oxo-acetyl group in 2 are discovered in natural products for the first time. All six compounds were tested against Botrytis cinerea, and chloroxaloterpin A, 1, and B, 2, demonstrated strong inhibitory activity against spore germination with EC of 4.40 and 4.96 μg/mL, respectively.