Identification of the Actinomycin D Biosynthetic Pathway from Marine-Derived Streptomyces costaricanus SCSIO ZS0073

Abstract

Bioactive secondary metabolites from Streptomycetes are important sources of lead compounds in current drug development. Streptomyces costaricanus SCSIO ZS0073, a mangrove-derived actinomycete, produces actinomycin D, a clinically used therapeutic for Wilm's tumor of the kidney, trophoblastic tumors and rhabdomyosarcoma. In this work, we identified the actinomycin biosynthetic gene cluster (BGC) acn by detailed analyses of the S. costaricanus SCSIO ZS0073 genome. This organism produces actinomycin D with a titer of ~69.8 μg mL^-1 along with traces of actinomycin X_oβ. The acn cluster localized to a 39.8 kb length region consisting of 25 open reading frames (ORFs), including a set of four genes that drive the construction of the 4-methyl-3-hydroxy-anthranilic acid (4-MHA) precursor and three non-ribosomal peptide synthetases (NRPSs) that generate the 4-MHA pentapeptide semi-lactone, which, upon dimerization, affords final actinomycin D. Furthermore, the acn cluster contains four positive regulatory genes acnWU4RO, which were identified by in vivo gene inactivation studies. Our data provide insights into the genetic characteristics of this new mangrove-derived actinomycin D bioproducer, enabling future metabolic engineering campaigns to improve both titers and the structural diversities possible for actinomycin D and related analogues.

Keywords: Streptomyces costaricanus; actinomycin D; bioproducer; biosynthesis; biosynthetic gene cluster.

Figure 1

Gene organization of the actinomycin D cluster in S. costaricanus SCSIO ZS0073. The direction of transcription and the proposed functions of individual ORF are indicated.

Figure 2

Proposed model for actinomycin D assembly (A) and 4-MHA precursor production (B) in S. costaricanus SCSIO ZS0073. A: adenylation domain; C: condensation; PCP: peptidyl carrier protein; TE: thioesterase.

Figure 3

HPLC analysis of S. costaricanus strains fermentation extracts. (i) WT producer; (ii) ∆acnDE mutant; (iii) ∆acnN1 mutant; (iv) ∆acnN3 mutant; (v) ∆acnGHLM mutant; (vi) ∆acnW mutant; (vii) ∆acnU4 mutant; (viii) ∆acnR mutant; (ix) ∆acnO mutant; (x) ∆acnT1 mutant; (xi) ∆acnT2 mutant; (xii) ∆acnT3 mutant. 1, actinomycin D; 2, actinomycin X_oβ.; 3, actinomycin X_2.

Figure 4

HPLC analyses of S. costaricanus strains fermentation extracts. (i) WT producer; (ii) ∆acnA mutant; (iii) ∆acnB mutant; (iv) ∆acnC mutant; (v) ∆acnU1 mutant; (vi) ∆acnU2 mutant; (vii) ∆acnU3 mutant; (viii) ∆phs mutant; (ix) ∆acnQ mutant; (x) ∆acnF mutant. 1, actinomycin D; 2, actinomycin X_oβ.

Figure 5

HPLC analysis of S. costaricanus strains fermentation extracts. (i) WT producer; (ii) ∆acnP mutant. 1, actinomycin D; 2, actinomycin X_oβ.