Enhancement of oxytetracycline production after gamma irradiation-induced mutagenesis of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> CN08 strain

Streptomyces rimosus CN08 isolated from Tunisian soil produced 8.6 mg l⁻¹ of oxytetracycline (OTC) under submerged fermentation (SmF). Attempts were made for enhancing OTC production after irradiation-induced mutagenesis of Streptomyces rimosus CN08 with Co⁶⁰-γ rays. 125 OTC-producing colonies were obtained after screening on kanamycin containing medium. One mutant called Streptomyces rimosus γ-45 whose OTC production increased 19-fold (165 mg l⁻¹) versus wild-type strain was selected. γ-45 mutant was used for OTC production under solid-state fermentation (SSF). Wheat bran (WB) was used as solid substrate and process parameters influencing OTC production were optimized. Solid-state fermentation increased the yield of antibiotic production (257 mg g⁻¹) when compared with submerged fermentation. Ammonium sulphate as additional nitrogen source enhanced OTC level to 298 mg g⁻¹. Interestingly, OTC production by γ-45 mutant was insensitive to phosphate which opens the way to high OTC production even in medium containing phosphate necessary for optimal mycelia growth.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus

Summary The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic productions in strains othersise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.     

Toxicity of cadmium to twoStreptomyces species as affected by clay minerals

Summary The effect of cadmium on the growth ofStreptomyces rimosus andS. bottropensis (both isolated from soil) was investigated. The modifying effect of the presence of the clay minerals kaolinite, bentonite and vermiculte on Cd toxicity was also included. After four days no growth was observed at 100 ppm CdCl₂ ofS. bottropensis and at 150 ppm in case ofS. rimosus. After six days some growth ofS. rimosus occurred at 150 ppm CdCl₂ and ofS. bottropensis at 100 ppm. Addition of the three clay minerals decreased the Cd toxicity considerably.     

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases

Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C₁₈ columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC₅₀ in the range of 0.3–2.4 μM. IC₅₀ values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10^–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase. Received: 7 December 1998 / Accepted: 29 March 1999     

Microbial conversion of tamoxifen

Summary Screening studies of tamoxifen (TAM) have shown that Streptomyces rimosus ATCC 2234 was able to metabolize TAM to 4-hydroxytamoxifen, which was obtained in a 10% yield and identified by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Several other microorganisms were capable of biotransforming TAM to tamoxifen-N-oxide and desmethyltamoxifen. The isolated metabolites were identified by gas chromatography/mass spectrometry and by two-dimensional NMR spectroscopic techniques. This is the first report of microbial hydroxylation of TAM. Offprint requests to: S. H. El-Sharkawy     

Production of oxytetracycline and rifamycins B and SV with cells immobilized on glass wool

The antibiotic producing microorganisms Streptomyces rimosus PFIZER 18234-2 and Amycolatopsis mediterranei CBS 42575 were immobilized in glass wool for the production of oxytetracycline and rifamycins B and SV. The growth of the immobilized cells was dependent on the type of the microorganism and on the amount of glass wool as well. After 5 exchanges of the medium, 530 mg O.T.C., 2138 mg rifamycin B and 8672 mg rifamycin SV were maintained. The periodic batch culture was continued for 20 days.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

A New Hybrid Strain of an Oxytetracycline-producing Organism, Streptomyces rimosus

A new strain, Streptomyces rimosus LS-T hybrid, characterized by a lower level of foaming and higher antibiotic activity as compared to the other active strains of S. rimosus, is obtained as a result of selection among prototrophic recombinant forms of strains LS-T293 and BS-21. The studies on strain LS-T hybrid show the possibilities of combining properties of different strains of S. rimosus in the hybrid forms. The occurrence of new properties in the hybrid forms is detected.     

Physiologische Selektion auf kältebehandelten Oberflächenkulturen von Streptomyces rimosus

Zusammenfassung Overflächen- und Submerskulturen von Streptomyces rimosus zeigen nach Kältebehandlung einen raschen Vitalitätsverlust. Physiologische Untersuchungen an flüssigen und festen Nährubstraten haben ergeben, daß die Ausreifung des Mycels von einer erheblichen Alkalisierung der Medien begleitet wird; in den Flüssigkeitskulturen werden pH-Werte, von über 8,5 erreicht. Submerse Folgepassagen solcher alkalischer Aufbewahrungskulturen zeigen in Abhängigkeit von der Zeitdauer der Kältewirkung physiologische Veränderungen, die auf synthetischen Medien als gestörter Kohlenhydratstoffwechsel zu fassen sind. In Übereinstimmung mit einschlägigen Literaturangaben unterliegen die reifen Streptomycetenkulturen der Wirkung ihres selbstgebildeten Antibioticums, die entweder eine Selektion stoffwechselabweichender Organismen auslöst oder die den Stoffwechsel der Resistenten erst während der Aufbewahrungszeit prägt. In diesem Falle wäre der abweichende Stoffwechselzustand über mehrere Folgekulturen im Mycel manifestiert worden.

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Physiological selection in cold-treated surface cultures of Streptomyces rimosus

Summary Surface and submerged cultures of Streptomyces rimosus show a rapid loss of viability after storage at + 4°C. Physiological tests with fluid and solid nutrient substrates indicate that as the mycelium ripens, the medium becomes strongly alkaline (fluid cultures show a pH of over 8,5). Subsequent submerged passages of such alkaline stored cultures show physiological changes dependent on the duration of the cold treatment which identified on synthetic media as disturbances of the carbohydrate metabolism.From the literature it is known that cultures of ripe Streptomycetes are affected by their own antibiotic. This either causes a selection of organisms with unusual metabolism, or affects the metabolism of the resistant strains during cold storage. In this case, the deviant metabolism should persist in the mycelium through several culture passages.

     

Performance of industrial scale bioreactors with modified RUSHTON turbine agitators

The data presented here with respect to the behaviour of industrial scale stirred tank bioreactors equipped with modified RUSHTON turbine agitators in the biosynthesis processes of antibiotics are valid for that case that the power consumption is the same as it is in standard RUSHTON turbine agitators. Each modified RUSHTON turbine agitator was obtained through the variation of the blade surface by adding perforations so that the ratio between the perforation surface area and the full surface area (or the surface fraction of the perforations) is 0.36. In the fermentations of Streptomyces aureofaciens, Streptomyces rimosus and Penicillium chrysogenum producing tetracycline, oxytetracyline and penicillin, respectively, in bioreactors equipped with modified RUSHTON turbine agitators, the relative antibiotic production is increased by more than 30% compared to standard bioreactors.     

The Use of Phase Inversion Temperature (PIT) Microemulsion Technology to Enhance Oil Utilisation during <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> Fed-batch Fermentations to Produce Oxytetracycline

The use of a rapeseed oil emulsion feed, produced by a phase inversion temperature (PIT) process, produced more biomass, gave a 3-fold increase in oil utilisation and a higher oxytetracycline titre but a higher residual oil concentration when compared to a conventional fed-batch Streptomyces rimosus process fed with crude rapeseed oil. Importantly, microbial utilisation of the surfactant was confirmed for the first time.     

Effect of the culture filtrates ofStreptomyces on growth and productivity of wheat plants

Streptomyces olivaceoviridis, S. rimosus andS. rochei proved to possess a high capacity for the production of auxins, gibberellins and cytokinin-like substances, together with substantial levels of α-amylase and proteinase. Grain priming with culture filtrates ofS. olivaceoviridis, S. rimosus orS. rochei appeared to enhance growth vigor and crop yield of wheat plants. In the majority of cases, the culture filtrate ofS. olivaceoviridis appeared to be the most effective in this respect. The present results are discussed in relation to the indirect role played by these bacteria in producing plant growth-regulating substances and their effects on growth and yield of wheat.     

Purification and chemical characterization of antimicrobial compounds from a new soil isolate Streptomyces rimosus MTCC 10792

A new isolate of Streptomyces sp. from soil of state Chhattisgarh (India) having broad spectrum antibacterial and antifungal activity was obtained. The active strain was identified as Streptomyces rimosus subsp. rimosus with accession number MTCC 10792 based on physiological, biochemical characteristics and 16S rRNA sequence homology studies. Antimicrobial compound produced by S. rimosus was tested against the drug resistance pathogens by the Bauer and Kirby method. The crude active metabolite was extracted using solvent n-butanol and purified by silica column chromatography and HPLC method. The physicochemical characteristics of the one purified compound viz. color, melting point, solubility, elemental analysis, ESIMS, IR, UV, 1HNMR, ¹³CNMR and chemical reactions have been investigated. Purified antimicrobial compound produced by S. rimosus MTCC 10792 at concentration 25 μg/mL showed antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37R as well as broad activity against all tested bacterial and fungal pathogens.     

Rheological behaviour of some antibiotic biosynthesis liquids

This paper prsents the results of teh study of rheological behaviour of antibiotic biosynthesis liquids obtained by submerged aerobic cultivation of microorganisms belonging to the actinomycete and fungi classes, in stirred tank bioreactors with turbine impellers. These liquids have a non-Newtonian behaviour which follows the power-law rhcological model with a correlation index of over 0.95. The studied liquids are pseudoplastic, and alter their rheological properties, such as consistency index, (K), flow index, (n), apparent viscosity, (η_a), maximum Newtonian viscosity (η₀), with the culture age, microrganism strain and batch conditions. Also, these liquids are time dependent, exhibiting thixotropy. The most viscous liquids are produced by Streptomyces aureofaciens and Streptomyces rimosus cultivation, while that produced by Streptomyces griseus is the least viscous. A higher pseudoplasticity appears after 30 hours culture age. Since all these biosynthesis are aerobic, a careful observation of the rhelogical behaviour dynamics is necessary to avoid the oxygen culture supply limitation and the decrease of the bioreactor performance during biosynthesis.     

Streptomyces daliensis sp. nov. from soil

A novel actinomycete strain YIM 31724^T was isolated from a soil sample collected from Dali, Yunnan Province, People’s Republic of China. The strain is characterized by white to yellow white aerial mycelia, spiral spore chains and smooth spore surface. The cell wall of strain YIM 31724^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell hydrolysates contained mainly glucose and small amounts of galactose and xylose. The menaquinones were MK-9(H₆) (31%) and MK-9(H₈) (69%). Phosphatidylethanolamine was the diagnostic phospholipid. The DNA G+C content of strain YIM 31724^T was 67.2 mol%. Phylogenetic analysis indicated that the strain belongs to the genus Streptomyces, with highest similarity to Streptomyces rimosus subsp. rimosus JCM 4667^T (rRNA gene sequence similarity value of 98.9%) and Streptomyces erumpens DSM 40941^T (rRNA gene sequence similarity value of 98.7%). Based on its phenotypic and genotypic characteristics, including low DNA–DNA hybridization results, strain is proposed as the type strain of a novel species, Streptomyces daliensis sp. nov.     

Continuous production of oxytetracycline by immobilized growing Streptomyces rimosus cells

Summary Streptomyces rimosus cells were immobilized with urethane prepolymers and used in the production of oxytetracycline. Based on the criteria for oxytetracycline productivity, cell growth in gels, cell leakage from gels and mechanical strength of gel, a hydrophilic prepolymer, PU-1, the main chain of which was polyethylene glycol (molecular weight, approximately 1500) was employed as gel material among 11 kinds of urethane prepolymers. Use of glucose-free medium for cultivation of PU-1-entrapped cells increased the production rate of oxytetracycline and minimized cell leakage from the gels. When the gel-entrapped cells lost activity, treatment of the cell-entrapping gels with saline or 70% ethanol resulted in recovery of the oxytetracycline productivity. Continuous oxytetracycline fermentation using PU-1-entrapped growing cells was successfully achieved in air-bubbled reactor for at least 35 days with reactivation of the cells.     

Extracellular α-amylase from Streptomyces rimosus

Summary A purification procedure for an extracellular -amylase from Streptomyces rimosus, oxytetracycline-producing strain, is described. The enzyme obtained was shown to be an acidic (pI 4.75) monomer with a relative molecular mass (M_r) of 43 000, containing three cysteines involved in the catalytic activity of the enzyme. Its amino-terminal part has 57–67% homology with amylases from other Streptomyces species. S. rimosus -amylase is sensitive to higher temperatures, and partially stabilized by Ca²⁺ ions. It hydrolyses starch (optimum at pH 5.0–6.0) in an endohydrolase manner giving rise to maltotriose, maltotetraose and higher oligosaccharides. Starch granules, except those from rice, were not significantly affected by the isolated -amylase.     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Ein zellfreier Extrakt aus Streptomyces rimosus katalysiert die Synthese von TDP-Mycarose aus TDP-d-Glucose und S-Adenosyl-l-methionin. Die Reaktion benötigt NADPH. Das Reaktionsprodukt enthält einen weiteren methylierten TDP-Zucker unbekannter Struktur. Die Reaktion verläuft über TDP-4-Keto-6-desoxy-d-glucose als Zwischenprodukt.

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Metabolic products of microorganisms

Summary A cell-free extract from mycelium of Streptomyces rimosus producing the antibiotic tylosin, catalyses the formation of TDP-mycarose from TDP-d-glucose and S-adenosyl-l-methionine. The reaction requires NADPH. The product contains a second methylated TDP-sugar with a presently unknown structure. TDP-4-Keto-6-deoxy-d-glucose is an intermediate in the reaction.

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112. Mitteilung: T. Anke und H. Diekmann: Biosynthesis of Sideramines in Fungi. Rhodotorulic Acid Synthetase from Extracts of Rhodotorula glutinis. FEBS Letters (im Druck).