The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus: a genetic hall of mirrors for synthesis of a molecule with mirror symmetry

Abstract

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.

FIG. 1.

Structure of actinomycin C₁. Actinomycin C₁ (I) is the main component of the actinomycin C mixture (C₁, C₂, and C₃) elaborated by S. chrysomallus. Actinomycins C₂ and C₃ differ from C₁ by the replacement of one or both d-valine residues with d-allo-isoleucine. (II) Structure of 4-MHA pentapeptide lactone (half molecule of actinomycin C₁). (III) Structure of 4-MHA. Sar, sarcosine (N-methylglycine); MeVal, N-methyl-l-valine.

FIG. 2.

Actinomycin biosynthetic gene cluster. The functions of the 28 genes that make up the gene cluster, including the 4 already known NRPS genes acmA to -D (66), are represented by arrows as follows: black, regulation; dark gray, 4-MHA biosynthesis; white, unknown role or function; gray, NRPS genes. In the lower part is shown the assembly line of the four NRPS subunits which catalyze the formation of 4-MHA pentapeptide lactone.

FIG. 3.

Pathways of tryptophan metabolism. The catabolic pathway leading from tryptophan to anthranilic acid, found in a number of bacteria, is inducible by tryptophan (56). The pathway leading from kynurenine to 3-HA is constitutive and found in all eukaryotes, including S. cerevisiae (64) and mammals (69). In the eukaryotic cell, 3-HA serves as a precursor of NAD⁺ or can be metabolized further. In actinomycin synthesis, 3-HA is converted to 4-MHA. NAD⁺, nicotine adenine dinucleotide.

FIG. 4.

Alignments of substrate-binding pocket sequences of eukaryotic and prokaryotic kynureninase-like enzymes. The alignments show the locations of the kynureninase and hydroxykynureninase active-site signatures based on the structural and functional analysis of the human (hydroxy)kynureninase sequence (48). Accession numbers: NP_003928.1, kynureninase isoform A (Homo sapiens); YP_257899.1, Pseudomonas fluorescens Pf-5 kynureninase; NP_627839.1, S. coelicolor A3(2) hydrolase.

FIG. 5.

Disruption of 4-MHA biosynthetic genes in the actinomycin gene cluster. Disruption was performed with a hygromycin cassette using an E. coli pUC18 derivative as a suicide vector as described in Materials and Methods.

FIG. 6.

Kynureninase and hydroxykynureninase activities in S. chrysomallus strains Sc1 and Sc-white. Gray bars, kynureninase activity; white bars, hydroxykynureninase activity. Specific activities were measured in crude extracts from 60-h-old mycelia grown in glutamate-mineral salts medium supplemented with maltose as described in Materials and Methods. The ratio of the specific activities of hydroxykynureninase and kynureninase in a culture of strain Sc1 reproducibly increased concomitantly with actinomycin production in culture up to an approximate value of 2 to 2.5:1 after 55 to 65 h of growth (not shown). Crude extracts from strain Sc-white and S. coelicolor strain M145, which does not produce actinomycins, were obtained from mycelium grown under the same conditions as strain Sc1 of S. chrysomallus. Ind, extract prepared from each strain after induction with 2.5 mM tryptophan. For details, see Materials and Methods.

FIG. 7.

Phylogenetic tree of kynureninase-like enzyme sequences from various bacteria and fungi, including AcmH and AcmK. Accession numbers: S. coelicolor A3(2), NP_627839.1; S. avermitilis, NP_825704.1; S. griseus subsp. griseus, YP_001824925.1; S. refuineus, ABW71847.1; S. viridochromogenes_1, ZP_05533056.1; S. viridochromogenes_2, ZP_05534763.1; Streptosporangium sibiricum, ACN39740.1; Arthrobacter sp., YP_830926.1; Saccharopolyspora erythraea, YP_001106253.1; Rhodococcus opacus, YP_002778666.1; Penicillium chrysogenum, XP_002565957.1)], Neurospora crassa, XP_956782.2; Aspergillus fumigatus, XP_755369.1; Pseudomonas fluorescens, YP_257899.1; Micromonospora sp. ATCC 39149, EEP69912.1; Geodermatophilus obscurus DSM 43160, YP_003408639.1; Burkholderia thailandensis, YP_441266.1; Ralstonia eutropha, CAJ93893.1; X. campestris pv. campestris strain B100, YP_001904112.1; S. chrysomallus, HM038107. The various clades are denoted with their kynureninase/hydroxykynureninase signatures.

FIG. 8.

Kynurenine formamidase activities in S. chrysomallus strains Sc1 and Sc-white. Specific activities were measured in crude extracts as described in Materials and Methods. Crude extracts from S. coelicolor strain M145, which does not produce actinomycins, were also tested for comparison. Ind, extract prepared from each strain after induction with 2.5 mM tryptophan. For details, see Materials and Methods.

FIG. 9.

Phylogenetic trees of kynurenine formamidases and tryptophan dioxygenases from different streptomycetes and bacteria. (Left) Kynurenine formamidases from S. coelicolor A3(2) (CAB42031.1), S. avermitilis (NP_825708.1), S. griseus subsp. griseus (YP_001824924.1), S. refuineus (ABW71851.1), S. viridochromogenes_1 (ZP_05533057.1), S. viridochromogenes_2 (ZP_05534764.1), Streptosporangium sibiricum (ACN39734.1), Saccharopolyspora erythraea (YP_001104168.1), Rhodococcus opacus (YP_002779035.1), Pseudomonas fluorescens (YP_257889.1), and Ralstonia eutropha (CAJ95273.1). There are two sister clades (shaded) containing streptomycete kynurenine formamidase, one containing catabolic sequences and the other containing sequences involved in secondary metabolism, such as AcmF in 4-MHA biosynthesis. (Right) Tryptophan dioxygenases from S. coelicolor A3(2) (NP_627840.1), S. avermitilis (NP_825703.1), S. griseus subsp. griseus (YP_001824926.1), S. refuineus (ABW71848.1), S. viridochromogenes_1 (ZP_05533055.1), S. viridochromogenes_2 (ZP_05534761.1), Streptosporangium sibiricum (ACN39739.1), Arthrobacter sp. (A0JUV5.1|T23O_ARTS2), Saccharopolyspora erythraea (A4FH01.1|T23O_SACEN), Rhodococcus opacus (YP_002778667.1), Pseudomonas fluorescens (AAY96155.1), Burkholderia thailandensis (YP_441265.1), and Ralstonia eutropha (YP_727262.1). There are two sister clades (shaded) containing streptomycete tryptophan dioxygenases, one containing catabolic sequences and the other containing sequences apparently involved in secondary metabolism, such as AcmG in 4-MHA biosynthesis.

FIG. 10.

Chromosomal clustering of genes related to the anthranilate pathway and the 4-MHA biosynthetic pathway in S. chrysomallus and other streptomycetes. The upper five gene clusters are catabolic sequences. They are pinned around the orthologues of SCO3645 (kynureninase like). The lower clusters represent the three 4-MHA-related clusters of tryptophan degradation. Orthologues and homologues of the various pathways are labeled with matching patterns as follows: dark gray, kynurenine formamidase-like enzyme; gray, tryptophan dioxygenase-like enzyme; white, kynureninase-like enzyme. Interspersed genes are indicated by small arrows.