Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C

Abstract

Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus.

Keywords: 3-hydroxy-4-methylanthranilic acid (4-MHA); Streptomyces anulatus Streptomyces antibioticus; Streptomyces chrysomallus; actinomycin; actinomycin C; actinomycin X; actinomycin halves; biosynthesis; evolution of biosynthetic gene cluster; genetic transmission of biosynthetic gene cluster; genomes.

Figure 1

(A) Structures of actinomycins of the X complex and the C complex. There are different designations in the literature for the various actinomycins such as for actinomycin D, which is synonymous with actinomycin C₁ or actinomycin X₁. (B) Phenoxazinone formation reaction. This reaction can be spontaneous or catalyzed by phenoxazinone synthase (PHS) for both in an oxygen dependency. Abbreviations: Thr, threonine; Val, valine; Pro, proline; HyPro, 4-trans-hydroxyproline, OxoPro, 4-oxo-proline; MeGly, N-methylglycine, Sarcosine; MeVal, N-methyl-l-valine; d-Ile, d-allo-isoleucine.

Figure 2

Biosynthesis of Acm C₁ (X₁, D) in Streptomyces chrysomallus. Upper row: reaction sequence leading from tryptophan to 3-hydroxy-4-methylanthranilic acid (4-MHA), enzyme designations (Keller et al; still non-identified 3-kynurenine-monooxygenase is indicated by a question mark). Lower row: nonribosomal ACMS assembly line of Acm half molecule (shown Acm X₁ half) and oxidative condensation of Acm X₁ halves to Acm X₁. Abbreviation: ACMS, actinomycin synthetase.

Figure 3

Alignment of the actinomycin biosynthetic gene clusters of Streptomyces chrysomallus (A) and Streptomyces antibioticus (B). Arrows designate putative start points of transcription. Stem-loop symbols indicate large inverted repeats. Gene designations Keller et al.

Figure 4

Reaction schemes of 3-HK-4-methyltransferase (AcmI or AcmL) and of 3-HA-4-methyltransferase (HAMT) in the formation of 4-MHA. The upper scheme includes the pathway specific conversion of 4-MHK to 4-MHA, whereas the lower scheme includes nonpathway specific methylation step prior conversion of 3-HK to 3-HA. Abbreviations: 4-MHA, 3-hydroxy-4-methylanthranilic acid; 3-HK, 3-hydroxykynurenine; 4-MHK, 3-hydroxy-4-methylkynurenine.

Figure 5

Model of fusion of two different single-armed biosynthetic gene clusters from an Acm X producer (lower gene cluster) and an Acm C producer, leading to the present double-armed acm C gene cluster of Streptomyces chrysomallus.

Figure 6

Physical map of flanking regions of the acm biosynthetic gene cluster of Streptomyces chrysomallus. The borders of the gene cluster are indicated by their genomic coordinates. The various IS elements or IS element fragments are symbolized by red and white checks. IS elements with inverted repeats are with arrows. Hypothetical proteins are in gray. IS elements are hatched. Terminal genes of the acm gene cluster are in blue, disrupted kdpABC is in beige, and DR are in black. Physical map is not at scale. Abbreviation: DR, directed repeat.