Engineered Streptomyces platensis Strains That Overproduce Antibiotics Platensimycin and Platencin

Abstract

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...

Dedicated ent -kaurene and ent -atiserene synthases for platensimycin and platencin biosynthesis

“…Finally, we demonstrated the production of PTN by overexpressing the cloned ptn cluster in selected heterologous Streptomyces hosts. We have previously shown that ptmR1 and ptnR1 are transcriptional regulators, and upon inactivation of ptmR1 in MA7327 (25) or ptnR1 in MA7339 (28), respectively, we have yielded mutant strains that significantly overproduce PTN, PTM, or both. We isolated a plasmid pBS12615 that harbors the entire ptn cluster from MA7339, inactivated ptnR1 within the ptn cluster to yield the expression plasmid pBS12619, and introduced pBS12619 into a PTN-nonproducing heterologous host Streptomyces lividans K4-114 (29) to afford the recombinant strain SB12606 (SI Appendix).…”

“…Targeted inactivation of orf4, orf5, ptmB2, ptmO1, ptmT1, and ptmT3 in S. platensis MA7327 was carried out by following the λRED-mediated PCR-targeting mutagenesis method (30). Genetic manipulation of PTM biosynthesis in S. platensis MA7327 (25) and PTN biosynthesis in S. platensis MA7339 (28) in vivo followed previously described procedures. Heterologous expression of the ptn gene cluster from S. platensis MA7339 was carried out in S. lividans K4-114 (29).…”

“…PTM was originally isolated from Streptomyces platensis MA7327, a strain found in a soil sample collected in South Africa (5, 10), whereas PTN was first isolated from Streptomyces platensis MA7339, a strain found in a soil sample collected in Spain (6, 11); we subsequently established MA7327 as a PTM and PTN dual producer (25). Because the structural differences between PTM and PTN lie in their diterpene moieties, most likely resulting from catalytic specificity of key terpene synthases, the distinct chemical profiles of these closely related strains present a unique opportunity to investigate the evolutionary and molecular mechanisms of how terpene synthases control regiochemistry and dictate the fate of the ultimate cyclization products in terpenoid biosynthesis (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp

“…Molecular genetic manipulation, including directed mutagenesis, expression of antibiotic activators, and heterologous expression, is also an efficient approach for activating target gene clusters and improving the yields of secondary metabolites (20,21). In particular, altering the expression of transcription factors that control biosynthetic pathways and amplifying biosynthetic gene clusters have resulted in the dramatically increased production of secondary metabolites and the isolation of novel compounds and their congeners (22)(23)(24).…”

Platensimycin and platencin: promising antibiotics for future application in human medicine

“…They found that deletion or inactivation of ptmR1 in strain MA 7339 greatly altered the production of platensimycin. 23 The resulting over-producing strain was S. platensis SB12002, with a platensimycin titer of 323 mg l À1 , representing about a 100-fold greater titer than the original strain.…”