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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.

SUBMITTER: Smanski MJ

PROVIDER: S-EPMC2663125 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin.

Smanski Michael J MJ Peterson Ryan M RM Rajski Scott R SR Shen Ben B

Antimicrobial agents and chemotherapy 20090121 4

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 trans ...[more]

PMID: 19164156

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Project description:BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

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Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin.

Publications

Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin.

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