The relationship was studied between the level of the intracellular adenylates and the biosynthesis of tylosin by Streptomyces fradiae NRRL 2702. The adenylate level was observed to be inversely related to the rate of tylosin biosynthesis and hence the final concentration of the antibiotic. The concentrations of the adenylates were maximal during the trophophase, dropped quickly before the onset of tylosin biosynthesis, and remained at low levels throughout the idiophase. The adenylate energy charge was almost constant throughout the fermentation and was in the range of 0.4 to 0.55. Glucose addition in the idiophase suppressed tylosin biosynthesis, accompanied by a rapid increase in the adenylate levels. The biosynthesis of tylosin resumed after a rapid drop in the adenosine triphosphate concentration. Two enzymes catalyzing the interconversion of propionyl-coenzyme A and methylmalonyl-coenzyme A were found in this organism: methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3). The activity of the former was two orders of magnitude higher than that of the latter. The activities of both enzymes were affected by the increased glucose addition in the idiophase.Tylosin, a macrolide antibiotic synthesized by Streptomyces fradiae, was first described by McGuire et al. in 1961 (9). Later investigations showed that the organism coproduced four antibiotics with structures closely related to tylosin (6). Seno et al. (14) presented data showing that the major factors formed by S. fradiae were tylosin, macrocin, and relomycin (Fig. 1). These compounds occur in the terminal stages of tylosin biosynthesis, and a pathway relating the compounds has been proposed (14).In a previous study (P. P. Gray and S. Bhuwapathanapun, Biotechnol. Bioeng., in press), chemostat cultures were used to determine the relationship between the specific uptake rates of glucose, glycerol, phosphate, and sodium glutamate on the biosynthesis of tylosin and related compounds.In this study, shake flask cultures have been used to investigate the relationship between tylosin biosynthesis and the intracellular concentrations of adenosine triphosphate, diphosphate, and monophosphate (ATP, ADP, and AMP) and the adenylate energy charge in normal batch culture and after the addition of glucose to idiophase cultures. The 16-membered lactone ring of tylosin has been shown (11) to be derived from two acetates, five propionates, and one t Present address:

SummaryThe production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase-idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (qtylosi,,) was found to be a function of the growth rate. The maximum value of (qtylosi,,) was observed when D = 0.017 hr-l. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr-l, the maximum value of 9tyiosin was 0.71 mg tylosidg dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW/hr. This value of qtylosin was 40% greater than the maximum qtylosln observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of qtYlosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of qtylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased uptake of phosphate decreased the values of qtyiosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of qtylosln of 1.1 rng/g/hr being obtained at a growth rate of 0.03 hr-l. Batch fermentations with this medium did not follow a trophophase-idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.

1. Six new metabolites of quinine have been identified in urine of man by methane chemical ionization g.l.c.--mass spectrometry. 2. Metabolites identified were: unchanged quinine and dihydroquinine, 3-hydroxyquinine, 3-hydroxydihydroquinine, 6'-hydroxycinchonidine, 6'-hydroxydihydrocinchonidine, quinine-10,11-epoxide and quinine-10,11-dihydrodiol. 3. 10-Chloro-11-hydroxydihydroquinine was identified as an artifact of the isolation procedure. 4. Chloroquine and desethylchloroquine (artifacts) were identified in the urine, 17 days after completion of a 48 h treatment with chloroquine.

The effects of increased concentration of inorganic phosphate on the biosynthesis of tylosin, the level of the intracellular adenylates, the energy charge, and the activities of enzymes involved in the synthesis of tylonolide precursors were studied in Streptomycesfradiae NRRL 2702. No metabolic response was observed when elevated levels of inorganic phosphate were added in idiophase. Increased initial levels of inorganic phosphate suppressed tylosin production and markedly increased the levels of the adenylates, although the adenylate energy charge was unchanged. Higher growth and glucose uptake rates were also observed. The activities of methylnalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) were suppressed by the increased concentration of inorganic phosphate. The results indicated that the rate of tylosin synthesis was inversely related to the absolute level of the adenylates rather than to the energy charge.Inorganic phosphate is well known for its depressive effects on the synthesis of many antibiotics (7). It has been reported that phosphate concentrations which are optimal to growth often suppress antibiotic production (13). Antibiotics of different groups are subject to phosphate regulation, including macrolide antibiotics, peptide antibiotics, tetracyclines, and other complex antibiotics.In a chemostat study, increasing the specific uptake rate of inorganic phosphate suppressed tylosin synthesis (1). A previous paper (12) described the adenylate level, energy charge, and the enzymes involved in the synthesis of tylonolide precursors during the batch cultivation of Streptomycesfradiae; the effect on these parameters of the metabolic stress caused by glucose addition in idiophase was also described.In this report the effect of increased levels of inorganic phosphate on batch fermentations of tylosin is described. The effect ofincreased phosphate on the levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge, and the activities of propionyl-coenzyme A (CoA) carboxylase (EC 6.4.1.3) and methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, is also reported. MATERIALS AND METHODS S. fradiae NRRL 2702 was used in this study.Growth conditions and assay procedures were as previously described (12). RESULTSEffect of inorganic phosphate on metabolism of S. fradiae and on tylosin biosynthesis. Elevated levels of inorganic phosphate added to 5-day idiophase cultures of S. fradiae did not suppress the synthesis of tylosin or cause changes in cell dry weight (Fig. 1). However, a concentration of 4.6 g/liter (2.3 g/liter in the control) of inorganic phosphate added at the beginning of the fermentation caused a severe suppression of tylosin synthesis (Fig. 2) and a more rapid growth of the culture. Rapid utilization of glucose and retarded consumption of methyloleate were also observed.Effect ofinorganic phosphate addition on adenylate level and...

Tylosin is a member of the macrolide group of antibiotics and contains a sixteen-membered lactone ring. In common with many macrolide antibiotics it is a multi-component antibiotic, the structure of the major components being shown in Fig. 1. In order to study the regulation controlling the factor distribution in a fermentation it would be convenient to have an assay procedure that could separate and quantitate the various factors. Current assay techniques require the estimation of total tylosin factors by optical density measurements on a solvent extract of the broth followed by thin-layer chromatography to determine the various factors. High pressure liquid chromatography (HPLC) seemed to be a new technique that warranted investigation as an alternative assay technique.Reports on the application of HPLC to the assay of antibiotics are rather limited and most are listed in a review by Tswt1). For the macrolide antibiotics, the separation of erythromycin and anhydroerythromycin is mentioned in the above review and there has been a report" on the use of HPLC for the determination and factor separation of leucomycin. The report by OMURA et al.21 also mentioned the use of HPLC for the determination of spiramycin, magnamycin, erythromycin and tylosin, however no details were given on the separation of the various tylosin factors. We have investigated the separation of tylosin factors using a Du Pont Model 841 HPLC. Initial work indicated that using filtered fermentation broth adversely affected both the resolution and column life, so subsequent work was carried out using a chloroform extract of the broth adjusted to pH 5.0. The Du Pont columns examined for the resolution of pure factors of tylosin A, B, C, D (and of tylosin present in fermentation broths) were ODS Permaphase and Sorbax SIL.With the ODS-Permaphase column tylosin B could be separated from the other factors (Fig. 2) but tylosins A, C, D all had equivalent retention times and could not be separated even with a wide range of mobile phase compositions.