Relationship between threonine dehydratase and biosynthesis of tylosin in Streptomyces fradiae.

To elucidate the repression mechanism of ammonium ions on the biosynthesis of tylosin in Streptomyces fradiae NRRL 2702, enzyme activities involved in the metabolism of the aspartate family of amino acids were evaluated in relation to the ammonium ion concentration and tylosin production. It was found that aspartate aminotransferase was essential for both cell growth and tylosin production. However, both threonine dehydratase and valine dehydrogenase were repressed by supplemented ammonium ions at concentrations higher than 50 mM. Threonine dehydratase was purified from cell-free extracts by acetone precipitation, ion-exchange chromatography and gel filtration, and its molecular mass was estimated to be 67,200 Da. The optimum pH and temperature for threonine dehydratase activity were 7.5 and 25 degrees C, respectively, and the Km value for threonine under these optimum conditions was 21 mM. The inhibition pattern of ammonium ions on the activity of threonine dehydratase appeared to be a mixed type.     

Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin

The effect of ammonium ions on growth and tylosin biosynthesis in Streptomyces fradiae NRRL 2702 cultured on a chemically defined medium was studied. Mycelial growth and tylosin production were not affected when ammonium sulphate was added to idiophase cultures to a final concentration of 10 mm or 20 mm; however, when ammonium sulphate was added to tylosin cultures to a final concentration of 20 mm before the onset of antibiotic biosynthesis (trophophase), tylosin production was severely suppressed while mycelial growth was stimulated. The activities of propionyl-coenzyme A carboxylase (EC 6.4.1.3) and methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, were depressed in high ammonium cultures. The activity of macrocin 3′-o-methyltransferase, which catalyses the methylation of macrocin to form tylosin, was also affected by high concentrations of ammonium ions added in the trophophase.     

Metabolism of l-threonine and fatty acids and tylosin biosynthesis in Streptomyces fradiae

Abstract In Streptomyces fradiae l -threonine is catabolized by threonine dehydratase or threonine aldolase to 2-ketobutyrate or acetaldehyde and glycine, respectively. Threonine dehydratase synthesis is repressed and its activity is inhibited by NH₄⁺ ions. Threonine aldolase is not repressed by NH₄⁺ ions and its activity is slightly stimulated by these ions. The addition of threonine to the medium increased pronouncedly the fraction of non-branched fatty acids with an even carbon number under conditions when threonine dehydratase was repressed and inhibited. The results indicate that threonine serves as a source of propionyl-CoA and 2-methylbutyryl-CoA and also of acetyl-CoA required for tylosin and fatty acid biosynthesis.     

Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae.

Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.1 and S. ambofaciens ATCC 15154, are approximately 80% identical over all 363 amino acids and 96% identical over a span of the first N-terminal 107 amino acids, respectively, to the deduced product of the Streptomyces coelicolor vdh gene. The organization of the regions flanking the vdh genes is the same in all three species. Inactivation of the genomic copy of the vdh gene in S. fradiae and S. ambofaciens by insertion of a hygromycin resistance (hyg) gene caused loss of the valine dehydrogenase (Vdh) activity, and thus only one enzyme is responsible for the Vdh activity in these organisms. Analysis of the culture broth by bioassay revealed that the vdh::hyg mutants produce an approximately sixfold-lower level of tylosin and an approximately fourfold-lower level of spiramycin than the wild-type S. fradiae and S. ambofaciens strains, while maintaining essentially identical growth in a defined minimal medium with either 25 mM ammonium ion or 0.05% asparagine as the nitrogen source. The addition of the valine catabolite, propionate or isobutyrate, and introduction of the wild-type vdh gene back to each vdh::hyg mutant reversed the negative effect of the vdh::hyg mutation on spiramycin and tylosin production. These data show that the catabolism of valine is a major source of fatty acid precursors for macrolide biosynthesis under defined growth conditions and imply that amino acid catabolism is a vital source of certain antibiotic precursors in actinomycetes.     

Aspartate aminotransferase and tylosin biosynthesis in Streptomyces fradiae.

Aspartate aminotransferase as well as valine dehydrogenase and threonine dehydratase was required for the biosynthesis of tylosin in Streptomyces fradiae NRRL 2702. The biosynthesis of these enzymes and tylosin production were repressed by high concentrations of ammonium ions. The change in specific tylosin production rates in batch cultures with different initial concentrations of ammonium ions showed patterns similar to those of the specific production rates of aspartate aminotransferase, valine dehydrogenase, and threonine dehydratase. Aspartate aminotransferase has been purified by acetone precipitation, DEAE-cellulose, hydroxyapatite, and preparative electrophoresis chromatographies. The purified enzyme (120 kDa) consisted of two subunits identical in molecular mass (54 kDa) and showed homogeneity, giving one band with a pI of 4.2 upon preparative isoelectric focusing. The enzyme was specific for L-aspartate in the forward reaction; the Km values were determined to be 2.7 mM for L-aspartate, 0.7 mM for 2-oxyglutarate, 12.8 mM for L-glutamate, and 0.15 mM for oxaloacetate. The enzyme was somewhat thermostable, having a maximum activity at 55 degrees C, and had a broad pH optimum that ranged from 5.5 to 8.0. The mode of action was a ping-pong-bi-bi mechanism.     

Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids

Labelled sodium isobutyrate [(CD3)2-CHCOONa] was added to the culture medium of Streptomyces fradiae and up to 14 atoms of deuterium were found to be incorporated into a molecule of tylosin aglycone (tylactone). This observation is in accordance with the data in the literature. When fatty acids were analyzed, as much as 34% of the isobutyrate incorporated into the cell was formed to be transformed into butyrate that was used for the synthesis of even, straight-chain fatty acids; 57% of the labelled isobutyrate was incorporated into the even isoacids, whereas 9% was degraded to propionate and further used for the synthesis of the odd acids.     

Improvement of tylosin fermentation by mutation and medium optimization

A tylosin-hyperproducing mutant of Streptomyces fradiae MNU20 was isolated from 3500 strains obtained from either MNNG- or u.v.-treated S. fradiae NRRL2702. With the optimal medium, S. fradiae MNU20 was able to produce 159 mg tylosin g biomass(-1), indicating the tylosin productivity in S. fradiae NRLL2702 was increased 14-fold by mutation and medium optimization. When the effect of valine, succinate and natural zeolite on tylosin production was investigated sing the optimal medium, these substances essentially enhanced tylosin production up to 349 mg g biomass(-1); their time addition during the culture period appeared to be critical for the increase.     

Ammonium ion affecting tylosin production by Streptomyces fradiae NRRL 2702 in continuous culture

Nitrogen regulation in tylosin production by Streptomyces fradiae NRRL 2702 was studied in chemostat culture using a soluble synthetic medium. The maximum value of specific tylosin formation rate ( q _TYL) was 1·13 mg g⁻¹ h⁻¹ at the specific growth rate (μ) of 0·05 h⁻¹, and q _TYL decreased with increasing levels of the specific growth rate after reaching a rate of 0·1 h⁻¹. The optimum conditions for tylosin formation were that the specific ammonium ion uptake rate ( q _N) and μ were 0·13 mmol g⁻¹ h⁻¹ and 0·05 h⁻¹, respectively. The specific formation rates of threonine dehydratase (TDT) and tylosin were repressed by high levels of specific ammonium ion uptake rate. This study showed the adaptation to chemostat cultures of the nitrogen regulation of tylosin fermentations.     

Application of atmospheric pressure chemical ionization liquid chromatography–mass spectrometry in identification of lymph triacylglycerols

Atmospheric pressure chemical ionization liquid chromatography–mass spectrometry was used in the identification of triacylglycerol molecular species in lymph samples from rats given either a structured lipid or safflower oil. The structured lipid was MLM-type (M, medium-chain fatty acid; L, long-chain fatty acid) and manufactured from caprylic acid (8:0) and the oil (safflower oil or high-oleic sunflower oil). The triacylglycerol composition of lymph varied significantly between structured triacylglycerols and safflower oil. Diacylglycerol fragment ions were found for all triacylglycerols and we could also observe the ammonium adduct molecular ion [M+NH₄]⁺ for all the triacylglycerols at the selected conditions. Protonated molecular ions were formed from triacylglycerols containing unsaturated fatty acids, and fatty acid fragment ions were also observed in the case of strong fragmentation. The lymph triacylglycerols were identified from their ammonium adduct molecular ions and diacylglycerol fragment ions. In addition to the intact MLM-type structured triacylglycerols, the MLL- and LLL-type triacylglycerols were also identified. The absorption pathway of MLM-type structured triacylglycerols is most likely the same as that of conventional long-chain triacylglycerols, i.e. they were hydrolyzed into 2-monoacylglycerol and medium-chain fatty acids, which were then used for resynthesis of triacylglycerols. The present study thereby also demonstrates the possibility to study the absorption pathway of triacylglycerol via identification of triacylglycerol species in biological samples.     

添加氧载体提高泰乐菌素发酵的得率

通过添加氧载体（如正十二烷、全氟化碳等）,提高了发酵系统中的氧传递速度,从而促进了泰乐菌素的生物合成。当加入５％的正十二烷或全氟化碳,泰乐菌素的生成量分别提高１４％和８％;在加入正十二烷和全氟化碳的同时,再加入载体Ａｉｄ—ＰｌｕｓＭＬ—５０Ｄ,可使泰乐菌素的生成量分别提高１９％和２０％。     

Obtaining of mutants of Streptomyces fradiae producing tylosin by action of nitrosomethylurea on synchronously germinating spores

The method of localized mutagenesis was applied to obtain mutants of Streptomyces fradiae producing higher amounts of tylosin. The populations of the germinating spores were subjected to a short-term treatment with nitrosomethylurea during different periods of the first DNA replication cycle. The method reveals defines periods sensitive to mutation induction and isolates a mutant producing a 60% increase in the yield of tylosin as compared to that provided by the stock strain.     

A family of r-determinants in Streptomyces spp. that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics.

Inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics in Streptomyces spp. comprises a family of diverse phenotypes in which characteristic subsets of the macrolide-lincosamide-streptogramin antibiotics induce resistance mediated by mono- or dimethylation of adenine, or both, in 23S ribosomal ribonucleic acid. In these studies, diverse patterns of induction specificity in Streptomyces and associated ribosomal ribonucleic acid changes are described. In Streptomyces fradiae NRRL 2702 erythromycin induced resistance to vernamycin B, whereas in Streptomyces hygroscopicus IFO 12995, the reverse was found: vernamycin B induced resistance to erythromycin. In a Streptomyces viridochromogenes (NRRL 2860) model system studied in detail, tylosin induced resistance to erythromycin associated with N6-monomethylation of 23S ribosomal ribonucleic acid, whereas in Staphylococcus aureus, erythromycin induced resistance to tylosin mediated by N6-dimethylation of adenine. Inducible macrolide-lincosamide-streptogramin resistance was found in S. fradiae NRRL 2702 and S. hygroscopicus IFO 12995, which synthesize the macrolides tylosin and maridomycin, respectively, as well as in the lincosamide producer Streptomyces lincolnensis NRRL 2936 and the streptogramin type B producer Streptomyces diastaticus NRRL 2560. A wide range of different macrolides including chalcomycin, tylosin, and cirramycin induced resistance when tested in an appropriate system. Lincomycin was active as inducer in S. lincolnensis, the organism by which it is produced, and streptogramin type B antibiotics induced resistance in S. fradiae, S. hygroscopicus, and the streptogramin type B producer S. diastaticus. Patterns of adenine methylation found included (i) lincomycin-induced monomethylation in S. lincolnensis (and constitutive monomethylation in a mutant selected with maridomycin), (ii) concurrent equimolar levels of adenine mono- plus dimethylation in S. hygroscopicus, (iii) monomethylation in S. fradiae (and dimethylation in a mutant selected with erythromycin), and (iv) adenine dimethylation in S. diastaticus induced by ostreogrycin B.     

Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae

The antibacterial properties of macrolide antibiotics (such as erythromycin, tylosin, and narbomycin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as narbomycin) have only single sugar substituents. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the narbomycin producer Streptomyces narbonensis. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.     

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.     

Production of tylosin byStreptomyces fradiae in palm oil medium

Streptomyces fradiae (NRRL 2702) produced tylosin when cultured on a synthetic defined medium M3. Palm oil, palm kernel oil and their fractions, as well as fatty acids and glycerol were investigated to serve as the major carbon source in shake flask culture. The lipids, glycerol and fatty acids, particularly palmitic acid but not oleic or lauric acid, were suitable for growth and tylosin production. For palmitic acid, at 168 h, the dry cell yield and tylosin production were 8.9 mg/ml and 0.84 mg/g cell mass respectively.     

Continuous-culture studies on the regulation of tylosin biosynthesis

The metabolic regulation of tylosin synthesis by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture a medium which diminished the trophophase-idiophase kinetic pattern was used to assess the activities of the enzymes involved in tylosin synthesis. The enzymes methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) showed early enzymatic derepression, both enzymes reaching their highest specific activities after 72-96 fermentation. The activity of macrocin 3' -O-methyltransferase, the enzyme catalyzing the conversion of macrocin (tylosin C) to tylosin (tylosin A). also peaked at 72 h. The specific activities of the three enzymes showed close correlation with the q(tylosin) value. In chemostat cultures the activities of the enzymes and the intracellular level of the adenylate pool and energy charge were studied as a function of dilution rate. Under steady-state conditions, increases in the specific growth rate repressed the enzymes activities with a concomitant increase in the intracellular level of the adenylate pool, while the adenylate energy charge remained almost constant and in the range 0.5-0.52. The highest specific activities of the enzymes were observed when D = 0.008 h (-1). The specific rate of tylosin synthesis was inversely proportional to the specific growth rate and the intracellular level of adenylate pool. The pool of adenylate could be a nutritional parameter which had a considerable influence on the biosynthesis of tylosin.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.