Actinomycin Production Persists in a Strain of Streptomyces antibioticus Lacking Phenoxazinone Synthase

Abstract

Truncated fragments of the phenoxazinone synthase gene, phsA, were prepared by the PCR. The resulting fragments were cloned into conjugative plasmid pKC1132 and transferred to Streptomyces antibioticus by conjugation from Escherichia coli. Two of the resulting constructs were integrated into the S. antibioticus chromosome by homologous recombination, and each of the resulting strains, designated 3720/pJSE173 and 3720/pJSE174, contained a disrupted phsA gene. Strain 3720/pJSE173 grew poorly, and Southern blotting suggested that genetic changes other than the disruption of the phsA gene might have occurred during the construction of that strain. Strain 3720/pJSE174 sporulated well and grew normally on the medium used to prepare inocula for antibiotic production. Strain 3720/pJSE174 also grew as well as the wild-type strain on antibiotic production medium containing either 1 or 5.7 mM phosphate. Strain 3720/pJSE174 was shown to be devoid of phenoxazinone synthase (PHS) activity, and PHS protein was undetectable in this strain by Western blotting. Despite the absence of detectable PHS activity, strain 3720/pJSE174 produced slightly more actinomycin than did the wild-type parent strain in medium containing 1 or 5.7 mM phosphate. The observation that strain 3720/pJSE174, lacking detectable PHS protein or enzyme activity, retained the ability to produce actinomycin supports the conclusion that PHS is not required for actinomycin biosynthesis in S. antibioticus.The actinomycins are chromopeptide antibiotics produced by a number of Streptomyces strains and by some strains of Micromonospora (14,17). In actinomycin D, the pentapeptide chains contain several methylated amino acids and one Damino acid, but it was shown some years ago that the L-forms of the relevant amino acids serve as precursors for the synthesis of the forms found in the antibiotic (19). These pioneering studies by Katz and Weissbach also demonstrated that the chromophore of actinomycin D, a phenoxazinone ring, is derived from the catabolism of tryptophan (19). Thus, 3-hydroxykynurenine is converted to 3-hydroxyanthranilic acid, and the latter intermediate is methylated to form 4-methyl-3-hydroxyanthranilic acid, the precursor of the phenoxazinone ring (28).The enzymes required for the methylation of 3-hydroxyanthranilic acid, for the activation of the chromophore precursor, and for the activation of the amino acids in the pentapeptide chains and their incorporation into those chains have been isolated and characterized (7,22,23). Schauwecker and coworkers have recently cloned the gene cluster for actinomycin production from Streptomyces chrysomallus (27). Thus, many of the biochemical and molecular genetic details regarding the biosynthesis of actinomycin have been elucidated. One unanswered question regards the synthesis of the actinomycin chromophore. Some years ago, Katz and Weissbach (18) identified an enzyme in Streptomyces antibioticus that catalyzes the oxidative condensation of 2-aminophenol derivatives to produce phenoxazinones (Fig. 1). This en...

A Thermostable Laccase fromStreptomyces lavendulaeREN-7: Purification, Characterization, Nucleotide Sequence, and Expression

“…29) Both B. subtilis cotA 30) and S. antibioticus PHS existed in their spores. 31) In the case of STSL, all of the activities were found in the cell extract, similar to the case of other bacterial laccases.…”

“…31) Since PHS is a component of S. antibioticus spores and has sequence similarity with cotA, it is thought that PHS as well as cotA may be involved in the process of spore formation.…”

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

“…Actinomycin production by S. antibioticus strains. Acm levels were measured as the A 443 of neutral ethyl acetate extracts of growth medium and converted to concentrations using the conversion factor 1 A 443 =50?2 mg ml "1 (Jones, 2000). The data represent the means of duplicate samples taken from each of two independent cultures for each strain and are expressed as mg acm produced (mg mycelial dry weight) "1 ±SEM.…”

“…No apramycin was added to GGA cultures. Growth of strains was measured as mycelial dry weight and actinomycin production was assayed as previously described (Jones, 2000). ppGpp levels were measured by HPLC as described previously (Hoyt & Jones, 1999).…”

A Novel o-Aminophenol Oxidase Responsible for Formation of the Phenoxazinone Chromophore of Grixazone

“…Hsieh and Jones (10) reported a phenoxazinone synthase in S. antibioticus that catalyzes the six-electron oxidative coupling of o-aminophenol compounds derived from tryptophan through 3-hydroxyanthranilic acid. However, disruption of the phenoxazinone synthase gene in S. antibioticus does not affect actinomycin D synthesis, showing that the phenoxazinone skeleton in actinomycin D is biosynthesized in vivo by a still unknown enzyme or non-enzymatically (11). On the other hand, michigazone with a hydroxymethyl group at the 8-position of the phenoxazinone and texazone with a carboxyl group at this position are assumed to be synthesized from a precursor(s) different from that of actinomycin D with no functional group at this position (7).…”