Studies on the production of daunomycinonederived glycosides and related metabolites inStreptomyces coeruleorubidus andStreptomyces peucetius

Strains ofStreptomyces coeruleorubiduě ISP 5145,JA 10092 and 39–146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, ε-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher R_f (0.4–0.7), isolated from strain 39–146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis.Streptomyces peucetius IMI 101 335 differed fromStreptomyces coeruleorubidus in an increased production of ε-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots.Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo₃; glucose was a more useful carbon source for the remaining strains The activity ofStreptomyces coeruleorubidus was inhibited by 1-propanol, Na-propionate,5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.     

Isolation and characterization of stable mutants ofStreptomyces peucetius defective in daunorubicin biosynthesis

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced byStreptomyces peucetius. In this study we report isolation of stable mutants ofS. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, ε-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed bydnrE (mutants SPAK and SPMAG),dnrS (SPFS and SPRHO) anddoxA (SPDHC) gene products.     

Production of 4′-epidaunorubicin by metabolic engineering of Streptomyces coeruleorubidus strain SIPI-1482

Streptomyces coeruleorubidus strain SIPI-1482 is an important industrial microbial strain which produces daunorubicin, the precursor for semi-synthesis of first-line anti-tumor antibiotics doxorubicin and epirubicin. dnmV, the C4 ketoreductase gene in the biosynthetic pathway of TDP-l-daunosamine was successfully disrupted by homologue recombination. The SIPI-1482 dnmV-blocked mutant lost the ability to produce daunorubicin and aggregate the intermediate ε-rhodomycinone. By introducing dnmV, the daunorubicin biosynthetic pathway in S. coeruleorubidus was reconstituted. Further more, aveBIV from S. avermitilis, as well as oleU from S. antibiotics, and novS from S. niveus were introduced into the dnmV-blocked mutant. The SIPI-1482 dnmV::aveBIV mutant could produce 4′-epidaunorubicin instead of daunorubicin, but dnmV::oleU and dnmV::novS mutant could not. Our study showed that the genetically engineered strain had a different fermentation condition and extraction protocol compared with the wild type daunorubicin producer. These results suggest that metabolic engineering is a powerful tool to produce novel hybrid antibiotics and a good alternative to chemical synthesis.     

Production of rhodomycins inStreptomyces griseoruber 4620

The strainStreptomyces griseoruber 4620 produces, besides the anthracycline antibiotics beromycins, some other anthracyclines of the rhodomycin type. Twelve isolates exhibiting a higher antibiotic activity (up to 2.5×), as compared to the parent strain, were obtained after a spontaneous selection. The following species were isolated from the hydrolysate of mycelial extract: β-rhodomycinone, β-isorhodomycinone, α₂-rhodomycinone and 10-deoxy-β-rhodomycinone, which has not yet been described.     

Improved yields of daunomycinone glycosides in developmental mutants ofStreptomyces coeruleorubidus

When improvingStreptomyces coeruleorubidus JA 10092, a producer of antibiotics of the daunomycinone complex, the most active variants were found among isolates of morphological typesbld-1 (with a suppressed production of the aerial mycelium on organic media containing glucose) andwhi (with an asporogenic aerial mycelium on glucose media and with the bald phenotype on media containing starch). Submerged cultures of thewhi mutants produced increased quantities of daunomycinone glycosides in the antibiotic complex, the amount of free anthracyclinones being simultaneously decreased. Thewhi strains differed from the wild type also in higher demands for aeration, concentration of glucose and in an increased production capacity in starch media. The overall antibiotic activity increased more than 40 times after a six-step selection (application of UV light, γ-radiation, nitrous acid and natural spreads) combined with an altered fermentation technology.     

The study of variability and strain selection inStreptomyces atroolivaceus

Mutants ofStreptomyces atroolivaceus blocked in the biosynthesis of mithramycin were isolated both by natural selection and after treatment with mutagenic factors (UV and gamma rays, nitrous acid). Both physical factors were more effective than nitrous acid. The selection was complicated by a high instability of isolates, out of which 20–80% (depending on their origin) reversed spontaneously to the parent type. The primary screening (selection of morphological variants and determination of their activity using the method of agar blocks) made it possible to detect only potentially non-productive strains; however, the final selection had to be performed always under submerged conditions. Fifty-four stable non-productive mutants were divided, according to results of the Chromatographic analysis, in five groups differing in production of six biologically inactive metabolites (compounds A-H). The mutants did not accumulate chromomycinone, chromocyclomycin and chromocyclin. On mixed cultivation none of the pairs of mutants was capable of cosynthesis of mithramycin or new compounds differing from standard metabolites. Possible causes of the above results are discussed.     

Microbial transformation of semisynthetic derivatives of daunomycinone modified in ring a

Semisynthetic derivatives of daunomycinone with 7,9-isopropylacetal, 7-O-methyl, 7-O-(4-penten-2-yl), and 7-O-(2-hydroxyethyl) substituents were converted byStreptomyces peucetius var.caesius (an adriamycin-blocked mutant) into 7-deoxy-13-dihydrodaunomycinone, while daunomycinone was transformed into 13-dihydrodaunomycinone (predominantly) and 7-deoxy-13-dihydrodaunomycinone.S. coeruleorubidus mutants 24–74 (accumulating aclavinone derivatives instead of daunomycin and related compounds) and 96-85 (producing no anthracycline substances), andS. aureofaciens B-96 (a tetracycline-blocked mutant) transformed the above substrates into the corresponding 13-dihydro derivatives, with the exception of 7,9-isopropylacetal daunomycinone which remained intact. 7-O-Propyn-1-yl daunomycinone was not transformed by any of the strains used under the conditions.     

Developmental mutants ofStreptomyces coeruleorubidus,a producer of anthracyclines: Isolation and preliminary characterization

Abstbact The wild strainStreptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 andbld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variantbld-1 after treatment with mutagens (UV light, γ radiation, nitrous acid) and after natural selection Formation of the aerial mycelium was fully suppressed in thebld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; typesspo-2, spo-3, spo-4 andspo-5 differed in size, shape and surface structure of spores, the typewhi formed asporogenous aerial hyphae.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

Effect of DL-ethionine on the biosynthesis of anthracyclines inStreptomyces coeruleorubidus

Summary Inhibitory action of DL-ethionine on 4-0-methylation and 10-COOH-esterification of the anthracyclinone ring inStreptomyces coeruleorubidus causes the formation of substances of the carminomycinone series, a concomitant drop in the production of daunomycinone derivatives and suppression of the biosynthesis of -rhodomycinone. The extent of inhibition depends on inhibitor concentration and the time of its addition to cultures. Ho formation of anthracyclinone ethyl-derivatives was found.     

Escherichia coli mutants completely deficient in adenosylmethionine decarboxylase and in spermidine biosynthesis.

Mutants of Escherichia coli deficient in adenosylmethionine decarboxylase, an enzyme in the biosynthetic pathway for spermidine, were isolated after mutagenesis of E. coli K 12 with N-methyl-N-nitro-N-nitrosoguanidine or with the bacteriophage Mu. The mutated gene, designated speD, is at 2.7 min on the E. coli chromosome map. In several of the mutants resulting from Mu insertion both adenosylmethionine decarboxylase activity and spermidine were undetectable. The absence of spermidine from speD strains proves the essential role of adenosylmethionine decarboxylase in the biosynthetic pathway for spermidine. Despite the complete absence of spermidine, these mutants grew at 75% of the wild type rate.     

Development of a Streptomyces venezuelae-based combinatorial biosynthetic system for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.     

Hemin-dependent mutants isolated from methicillin-resistantStaphylococcus aureus strains

Mutants requiring the addition of hemin for growth were isolated from fourteen naturally occurring methicillin-resistant (penicillin-tolerant)Staphylococcus aureus strains. In two of these mutant strains, hemin could be replaced by δ-aminolevulinic acid. All the mutants were capable of growth in the absence of hemin in aerobiosis and in anaerobiosis when the media were supplemented with acetate, pyruvate or mevalonate. The resistance to cloxacillin of the mutant cultures grown without hemin was higher and much more uniform than that of the parent strains. In the presence of hemin, the resistance of the mutants was lowered and phenotypically more heterogenous. The selective mutant isolation technique used in this study was based on the heteroresistance of staphylococci to penicillins and cephalosporins.     

Streptomyces deserti sp. nov., isolated from hyper-arid Atacama Desert soil

The taxonomic position of a Streptomyces strain isolated from a hyper-arid desert soil was established using a polyphasic approach. The organism had chemical and morphological properties typical of the genus Streptomyces and formed a phyletic line at the periphery of the Streptomyces coeruleorubidus subcluster in the 16S rRNA gene tree. DNA:DNA relatedness values between the isolate and its nearest phylogenetic neighbours, Streptomyces lomondensis NRRL 3252^T and Streptomyces lusitanus NRRL B-12501^T were 42.5 (±0.48)% and 25.0 (±1.78)%, respectively. The isolate was readily distinguished from these organisms using a combination of morphological and phenotypic properties. On the basis of these results, it is proposed that isolate C63^T (CGMCC 4.6997^T, = KACC 15425^T) be classified as the type strain of Streptomyces deserti sp. nov.     

Solubilisation and mineralisation of [14C]lignocellulose from wheat straw by Streptomyces cyaneus CECT 3335 during growth in solid-state fermentation

Nine Streptomyces strains were screened for their ability to solubilise and mineralise ¹⁴C-labelled lignin during growth in solid-state fermentation. Streptomyces viridosporus was confirmed as an active lignin-degrading organism along with a new isolate, Streptomyces sp. UAH 15, further classified as Streptomyces cyaneus CECT 3335. This organism was able to solubilise and mineralise the [¹⁴C]lignin fraction of lignocellulose (44.96 ± 1.77% and 3.41 ± 0.48% respectively) after 21 days of incubation. Cell-free filtrates from Streptomyces sp. grown in solid-state fermentation were capable of solubilising up to 20% of the [¹⁴C]lignin after 2 days incubation, with most of the product detected in the acid-soluble rather than in the water-soluble fraction. Identification of the extracellular enzymes produced during growth of S. cyaneus CECT 3335 revealed that extracellular peroxidase and phenol oxidase activities were present, with the activity of phenol oxidase being 100 times greater than peroxidase activity. The activity of these two enzymes was found to correlate with both solubilisation and mineralisation rates. This is the first report of phenol oxidase activity produced by a Streptomyces strain during growth in solid-state fermentation. A role for the enzyme in the solubilisation and mineralisation of lignocellulose by S. cyaneus is suggested. Received: 12 May 1997 / Accepted: 19 May 1997     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.     

Regulation of biosynthesis of secondary metabolites

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.     

Interaction between supernodulating or non-nodulating mutants of soybean and two arbuscular mycorrhizal fungi

 Twelve nodulation mutants (seven non-nodulating and five supernodulating) of soybean [Glycine max (L.) Mirr.] were screened for arbuscular mycorrhizal colonization in the presence of either Glomus etunicatum Becker and Gerdemann or Gigaspora margarita Becker and Hall. The cultivars showed variation in colonization parameters. The two supernodulating mutants En6500 and NOD1–3 had higher frequencies of colonization with 2.5–4.5 times higher arbuscular abundance than the respective wild types. The enhanced mycorrhization resulted in significant enhancement of P uptake by En6500. The non-nodulating mutants showed decreases in mycorrhizal parameters. Mutants En1282 and Harosoy^–exhibited aborted infection after formation of typical appressorium-like structures at some sites. However, none of these had the non-mycorrhizal phenotype. Growth and nutrient-uptake parameters should be considered while studying plant mutants for mycorrhization. Accepted: 7 July 2000