Oxytetracycline Formation by Streptomyces rimosus in Chemically Defined Media

Details in the fermentation of oxytetracycline in a synthetic medium with Streptomyces rimosus have been presented. In these studies, an organic nitrogen source was shown to be essential for the production of significant amounts of antibiotic activity. Of the amino acids tested, aspartic acid, proline, threonine, valine, and beta-alanine were utilized well for both growth and antibiotic production. Markedly different fermentation patterns were observed with aspartic acid and beta-alanine. Glycerol and glucose supported antibiotic yields superior to those found with other carbohydrates tested. Short chain organic acids were not effectively utilized for growth in the absence of a readily fermentable carbohydrate.     

Oxytetracycline production by Streptomyces rimosus in solid-state fermentation of corncob

Corn-cob was used as a substrate in the production of oxytetracycline by Streptomyces rimosus TM-55 in a solidstate fermentation. Oxytetracycline was detected on day 4, and reached its maximum on day 8. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64% to 67%, supplementation with 20% (w/w) rice bran or 1.5% to 2.5% (w/w) (NH₄)₂SO₄ as sole N source, 1.0% (w/w) CaCO₃, 2% (w/w) MgSO₄.7H₂O, and 0.5% (w/w) KH₂PO₄, with incubation for 8 days at 25 to 30°C. Each g substrate produced 7 to 8 mg oxytetracycline.     

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.     

工程改造龟裂链霉菌提高土霉素产量

土霉素是由龟裂链霉菌合成的一类广谱性抗生素,前期研究工作证明其生物合成受其自身途径特异性调控蛋白OtcR的直接调节,OtcR能够激活和促进土霉素合成基因簇的转录表达。在龟裂链霉菌M4018宿主内利用强启动子单独过表达OtcR蛋白,使土霉素的产量提高到原来产量的4倍;为了进一步提高土霉素产量,在M4108宿主内表达乙酰辅酶A羧化酶基因,提高其胞内土霉素合成的前体物丙二酸单酰辅酶A的含量。对出发菌株M4018进行工程改造,同时过表达途径特异性调控蛋白OtcR和乙酰辅酶A羧化酶,发酵检测改造后的重组工程菌株土霉素的产量由1.37g/L提高到9.09g/L,该研究策略对工程改造龟裂链霉菌提高土霉素的产量具有重要的指导意义。     

Oxytetracycline production in solid state and submerged fermentation by protoplast fusants of Streptomyces rimosus

Streptomyces rimosus TM-55 was treated with 3% EMS, and 29 auxotrophic mutants (AM-1 - AM-29) were isolated from 5457 colonies at a survival rate between 6.6-66.7%. Three sets of the auxotrophic mutants, AM-3 and AM-27, AM-7 and AM-28, and AM-3 and AM-21, were chosen for protoplast fusion with 50% PEG 1000 for 30 minutes at 25 degrees C, and 25 fusants were isolated (f-1 - f-25). In solid substrate, oxytetracycline production of 20% fusants was higher than that of the wild strain, while in submerged fermentation, it was 44%. Oxytetracycline productions of fusants f-1, f-6, f-11, f-12, f-20 and f-21 were lower than that of the wild strain in solid substrate, but this was reversed in submerged fermentation. On the other hand, OTC production of fusant f-8 was higher than that of the wild strain in solid substrate, and this was reversed in submerged fermentation.     

Oxytetracycline production by Streptomyces rimosus: gas and temperature patterns in a solid-state column reactor

During oxytetracycline production by Streptomyces rimosus TM-55 on sweet potato residue in a solid-state column reactor, the moisture content increased by between 2 and 5% (w/w) during incubation, from an initial content of 70 to 73%, and pH initially increased from 6.0 to 7.3, followed by a gradual decrease to 6.2. Appropriate aeration enhanced oxytetracycline production, while mixing only once daily decreased it. The temperatures in the centre and upper layers of each reactor were higher than elsewhere in static non-aerated cultures. The maximum CO₂ concentration ranged from 2.9 to 3.2% (v/v) and the minimum O₂ concentration was 11.0 to 17.2% (v/v) in static cultures. Under optimal conditions, each gram of dry substrate produced the equivalent potency of 12 mg oxytetracycline.The authors are with the Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China     

Extracellular metalloendopeptidase of Streptomyces rimosus

Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10⁻⁴ M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH₂-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.     

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.     

Production of a novel dimeric metabolite of primaquine by Streptomyces rimosus

Primaquine, an 8-amino-6-methoxyquinoline antimalarial agent, was subjected to metabolic studies with microorganisms. Streptomyces rimosus converted primaquine to the previously reported N-acetyl derivative. Continued incubation of S. rimosus resulted in the formation of a minor dimeric metabolite. The structure of the minor dimeric metabolite was proposed based primarily on its spectral data (1H and 13C nuclear magnetic resonance spectra and mass spectrum). The proposed structure of the metabolite was confirmed by synthesis of the dimer by treatment of primaquine-N-acetate with potassium ferricyanide in a biphasic chloroform-aqueous sodium bicarbonate system with a phase-transfer catalyst. Since (+/-)-primaquine was used for both the microbial transformation and synthesis, a diastereomeric mixture of symmetrical dimers was formed in each case. The metabolite sample was identical to the synthetic sample, as shown by direct comparison (thin-layer chromatography, co-thin-layer chromatography, high-pressure liquid chromatography, co-high-pressure liquid chromatography, 1H nuclear magnetic resonance spectra, and mass spectrum).     

Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.     

Oxytetracycline production byStreptomyces rimosus in solid state fermentation of sweet potato residue

Sweet potato residue, a starchy agricultural waste, was used as a substrate to produce oxytetracycline byStreptomyces rimosus TM-55 in a solid-state fermentation. Oxytetracycline was detected on the third day, reached its maximum value on the sixth day and remained constant to the twentieth day. Optimal conditions for oxytetracycline production were an initial pH of 5.5 to 6.5, supplemented with 20% (w/v) defatted roasted peanut meal, as the sole nitrogen source, 1.0% (w/v) CaCO₃ and 2.0% (w/v) MgSO₄·7H₂O, being incubated at 26 to 35°C for 6 to 7 days. Oxytetracycline reached 12.1 mg/g substrate.

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Résumé On a utilisé des résidus de patates douces, on résidu agricole amylacé, comme substral pour la production d'oxytétracycline parStreptomyces rimosus TM-55 par fermentation en milieu solide. On a détecté foxytétracycline le 3ème jour. Celui-ci a arteint sa concentration maximum le 6ème jour et celle-ci est restée constante jusqu'au 20eme jour. Les conditions optimales pour la production d'oxytétracycline sont les staivanies: un pH initial compris entre 5.5 et 6.5, l'ajout de 20% (p/v) de farine d'arachide dégraissée, comme seule source d'azote, 1.0% (p/v) de CaCO₃ et 2.0% (p/v) de MgSO₄.7H₂O, une température d'incubation de 26 à 35°C pendant 6 à 7 jours. On a aneint 12.1 mg d'oxytetracycline par g de substrat.