Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants

Abstract

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.

Artificial Reconstruction of Two Cryptic Angucycline Antibiotic Biosynthetic Pathways

“…S. lividans TK24 was grown in YEME [48] for generation of protoplasts. E1 [49] was used for production of metabolites both with and without added XAD-7 (20 g L) absorbent. Streptomyces cultures were grown in shaking flasks (330 rpm) at 28 8C for 3-7 days.…”

Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp. The GenBank accession number for acmA reported in this paper is AF043550.

“…However, using the S. galilaeus blocked mutants and S. steffisburgensis as the hosts, the functionality of the various constructs was analysed. Plasmid pSY21 was introduced into the S. galilaeus mutant H039, which produces aklavinone with attached rhodinose residues (Ylihonko et al, 1994). When S. nogalater minPKS in pSY21 was expressed in H039, the hydrolysed products obtained were aklavinone and auramycinone, whereas aklavinone was obtained after 2.…”

“…The choice of Streptomyces lividans as the host for expression cloning was based on evidence that it does not itself accumulate anthracycline metabolites in the culture conditions used for fermentation. (Bhuyan & Dietz, 1965), Streptomyces steffisburgensis ATCC 27466 (Dietz, 1967), S. galilaeus ATCC 31615 (Oki et al, 1975), the blocked S. galilaeus mutants H036, H039 and H061 (Ylihonko et al, 1994), Streptomyces peucetius ATCC 27952 (Arcamone et al, 1969) and S. lividans TK24 (Hopwood et al, 1985). The plasmids used are listed in Table 1.…”

Identification of a Cyclase Gene Dictating the C-9 Stereochemistry of Anthracyclines from Streptomyces nogalater

“…Streptomyces strains were grown at 30°C in tryptone soya broth containing thiostrepton (50 g/ml) for preparation of plasmid DNA and in E1 medium containing glucose (20 g/liter), soluble starch (20 g/liter), Farmamedia (5 g/liter), yeast extract (2.5 g/liter), CaCO 3 (3 g/liter), NaCl (1 g/liter), MgSO 4 ⅐ 7H 2 O (1 g/liter), and K 2 HPO 4 ⅐ 3H 2 O (1 g/liter) in tap water (pH 7.5) for production of anthracycline metabolites (19). A DNA fragment derived from a nogalamycin biosynthetic cluster containing a cyclase gene was cloned in a pIJ486-based plasmid and introduced into S. lividans TK24 (6).…”

“…A DNA fragment derived from a nogalamycin biosynthetic cluster containing a cyclase gene was cloned in a pIJ486-based plasmid and introduced into S. lividans TK24 (6). Streptomyces galilaeus mutant H039 producing aklavinone-(rhodinose) [2][3] (19) was used as a host in attempts to produce C-9 stereoisomers of aklavinone. DNA isolation and manipulation were carried out by standard methods (6,14).…”