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, which is isolated from Streptomyces platensis MA7327, and platencin, which is isolated from S. platensis MA7339, are two recently discovered natural products that serve as important antibiotic leads. Here we report on the identification of S. platensis MA7327 as a dual producer of both platensimycin and platencin. A PCR-based approach was used to locate and clone the locus involved in platensimycin and platencin production, including ptmR1, which encodes a putative GntR-like transcriptional regulator. Deletion of this gene from the producing organism allowed us to isolate strains that overproduce platensimycin and platencin with yields of 323 ؎ 29 mg/liter and 255 ؎ 30 mg/liter, respectively. These results illustrate the effectiveness of genetic manipulation for the rational engineering of improvements in titers.The discovery of platensimycin (16,20) and platencin (8,19) as members of an entirely new class of antibacterial antibiotics with a mode of action not exploited by current drugs represents an important step in the fight against antibiotic resistance (Fig. 1a). Both compounds are potent and selective inhibitors of bacterial fatty acid synthesis. Platensimycin specifically targets the elongation ␤-ketoacyl-acyl carrier protein (ACP) synthase I/II, FabF/B (20), while platencin has a dual mode of action and targets both FabF/B and the initiation ␤-ketoacyl-ACP synthase III, FabH (19). Both natural products are effective against a broad spectrum of gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and show no cross-resistance with other classes of commercially available antibiotics (19). Although platensimycin has proven effective in clearing methicillin-resistant S. aureus infection from a mouse model, the high doses and the suboptimal delivery system required highlight the need for further refinement of its structure prior to the conduct of clinical trials. Multiple total syntheses of both compounds, as well as numerous analogs, underscore the excitement generated by these compounds as leads for novel antiinfectives (10-14, 18).Platensimycin was isolated from Streptomyces platensis MA7327 at a yield of 2 to 4 mg/liter (16), and platencin was isolated from Streptomyces platensis MA7339 at a yield of 1 mg/liter (8). Subsequent fermentation optimization has led to a recent report that S. platensis MA7327 produced platensimycin at yields up to 56 mg/liter (6). No such improvement of the platencin titer has been reported to date. Strains capable of producing higher yields of platensimycin or platencin, or both, will facilitate the development of these promising leads into clinical agents. Toward this end, we have determined an appropriate set of protocols for the genetic manipulation of platensimycinproducing strain S. platensis MA7327 and exploited the regulatory mechanism of platensimycin and platencin biosynthesis to engineer S. platensis strains that are capable of overproducing both platensimycin and platencin at titers of 32...

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-

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.

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.

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.

Summary Platensimycin (PTM) and platencin (PTN) are potent inhibitors of bacterial fatty acid synthases and have emerged as promising antibacterial drug leads. We previously characterized the PTM and PTN biosynthetic machineries in the Streptomyces platensis producers. We now identify two mechanisms for PTM and PTN resistance in the S. platensis producers - the ptmP3 or ptnP3 gene within the PTM-PTN or PTN biosynthetic cluster and the fabF gene within the fatty acid synthase locus. PtmP3/PtnP3 and FabF confer PTM and PTN resistance by target replacement and target modification, respectively. PtmP3/PtnP3 also represents an unprecedented mechanism for fatty acid biosynthesis in which FabH and FabF are functionally replaced by a single condensing enzyme. These findings challenge the current paradigm for fatty acid biosynthesis and should be considered in future development of effective therapeutics targeting fatty acid synthase.

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