Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium

Abstract

A defined medium was developed for Streptomyces peucetius that optimally contained 0.5 mM magnesium, 1 mM phosphate, 75-125 mM glucose, 10 mM nitrate, and microelements. Poorer results were obtained with nitrite, aspartate, or ammonia as sole nitrogen sources. Other carbon sources which supported best growth and highest anthracycline titers were fructose, maltose, and soluble starch. In each case, substantial residual carbon remained at the end of 6 days, suggesting a lack of catabolite repression by the carbohydrate carbon sources on anthracycline biosynthesis. Studies involving limiting and nonlimiting concentrations of glucose supplemented with arabinose, a poorly utilizable carbon source, indicated that high carbon concentrations were not necessary for osmotic stabilization. Inorganic phosphate was found to have an inhibitory effect on anthracycline production. Furthermore, when cultures at early stages of anthracycline production were spiked with inorganic phosphate, a delay in further anthracycline production resulted until the added phosphate was depleted. A 10% inoculum of stationary phase cells yielded the best growth and most consistent anthracycline production. Spectrophotometric analyses at 495 nm of chloroform--methanol-extracted material were also found to be useful for the determination of total anthracyclines in culture extracts.

Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin biosynthesis

“…1) has been described (McGuire er al., 1980a, b, c). Nitratedefined medium (NDM; see accompanying paper: Connors et al, 1990a), yeast/malt agar medium (YMA; Dekleva et al, 1985) and R2YE medium (Hopwood et al, 1985) have also been described.…”

The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-production gene cluster

“…DNR biosynthesis is unique in this regard since a nonfunctional gene is present, but another gene product from outside the biosynthetic gene cluster complements the missing activity. In erythromycin biosynthesis, the cluster of production genes, as currently defined, lacks genes encoding glucose-1 -phosphate thymidylyl transferase and TDP-D-glucose 4,6-dehydratase, but genes for these enzymes have been found elsewhere in S. eytbraea (Linton et al, 1995 The unglycosylated intermediate RHO is approximately 10-fold more prevalent than DNR when S. pegcetizls is grown in GPS medium (Dekleva et al, 1985). Therefore, it appears quite likely that one of the steps of daunosamine biosynthesis or attachment is rate-limiting.…”

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

“…These strains are maintained in the laboratory as described previously (12). R2YE, R5, and YEME media, described by Hopwood et al (25), were typically used for normal growth of the strains.…”

“…The neutral proteinase (SnpA) was purified from 10-liter fermentation cultures of S. lividans (pANT42) and from 500-ml shake flask cultures of Streptomyces galilaeus(pANT42). For shake-flask cultures, spores were scraped off of MAT solid medium and used to inoculate a 250-ml flask containing 50 ml of milk broth containing 15 jig of thiostrepton per ml (MBT [MAT lacking agar]) and a coiled spring for mycelial dispersion (12). After incubation for 24 h at 30°C with rotary shaking (250 rpm), the entire culture (primary seed culture) was used to inoculate a 2-liter flask containing either 450 ml [for S. galilaeus(pANT42)] or 950 ml of MBT and a coiled spring.…”