Characterization of the saframycin A gene cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribosomal peptide synthetase system for assembling the unusual tetrapeptidyl skeleton in an iterative manner

Abstract

Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

FIG. 1.

Structures of saframycins, safracins, and ecteinascidin 743.

FIG. 2.

Genetic organization and comparison of the saf (SFM-Mx1), sfm (SFM-A), and sac (SAC-B) biosynthetic gene clusters. The proposed functions of individual ORFs are shown and summarized in Table 1.

FIG. 3.

Proposed biosynthetic pathways for 3-hydroxy-5-methy-O-methyltyrosine (A) and saframycin A (B).

FIG. 4.

HPLC analysis of saframycin and safracins. (A) Saframycin A (a) isolated from an authentic standard (I), S. lavendulae wild-type strain (II), strain TL2003 (ΔsfmB) (III), and strain TL2004 (PermE*::sfmB) (IV). (B) Safracin B (b) isolated from a P. fluorescens wild-type strain (I), cyano-substituted safracin B (c) isolated from a P. fluorescens wild-type strain treated with 1 mM KCN (II), aminated saframycin S (d) isolated from strain TL2102 (Ptac::sfmO4) (III), and saframycin Y3 (e) isolated from strain TL2102 (Ptac::sfmO4) treated with 1 mM KCN (IV). Absorbance of UV at 270 nm (A) or 268 nm (B) in milliabsorbance units (mAU) is shown on the y axes, and time (in minutes) is shown on the x axes.

FIG. 5.

(A) Purified SfmA-C1-A1-PCP1 (lane 1), SfmB-C2-A2-PCP2 (lane 2), and SfmC-C3-A3-PCP3-RE (lane 3) as analyzed by electrophoresis on a 7.5% sodium dodecyl sulfate-polyacrylamide gel. The positions of molecular mass markers (lane 4) (in kilodaltons) are shown to the right of the gel. (B) Substrate specificities as determined by the ATP-PP_i exchange reaction with the amino acids predicted to be incorporated into SFM-A (100% relative activity corresponds to 51,540 cpm for SfmA-C1-A1-PCP1, 44,300 cpm for SfmB-C2-A2-PCP2, and 51,500 cpm for SfmC-C3-A3-PCP3-RE).

FIG. 6.

Domain organizations and proposed enzymatic mechanisms of NRPS systems for SFM-A (A), SFM-Mx1 (B) (33), and SAC-B (C) (46) biosynthesis.