Cloning and characterization of the biosynthetic gene cluster for tomaymycin, an SJG-136 monomeric analog

Abstract

Tomaymycin produced by Streptomyces achromogenes is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.

FIG. 1.

(A) Chemical structures of naturally produced PBDs. The compounds of interest are indicated in boldface. The dashed lines separate the PBDs substituted and not substituted at C-9. (B) Chemical structures of lincomycin A and SJG-136.

FIG. 2.

HPLC-ESI analysis of the secondary metabolites produced by wild-type and ΔtomA strains and by a ΔtomA strain complemented with pUWL201b/tomA.

FIG. 3.

(A) Proposed pathway for the biosynthesis of the 4-ethylidene-tetrahydropyrrole-2-carboxylic acid moiety in tomaymycin, which is the suggested substrate for the NRPS enzymes catalyzing diazepine ring formation. The branching points for the anthramycin, sibiromycin, and lincomycin A biosyntheses are indicated. (B) Proposed pathway for the biosynthesis of the 4-hydroxy-5-methoxyanthranilic acid moiety in tomaymycin, which is the suggested substrate for the NRPS enzymes catalyzing diazepine ring formation.

FIG. 4.

(A) HPLC and bioassay analyses of the secondary metabolites produced by wild-type and ΔtomO strains and by chemical complementation of the ΔtomO strain with 5-hydroxyanthranilic acid. (B) ESI spectra of the peaks labeled 1 and 2 in the HPLC chromatogram from panel A. The dideoxy analog of tomaymycin has much weaker antibiotic properties than tomaymycin.