Gérard Lefebvre

Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus

SummaryProduction of cephamycin and clavulanic acid by Streptomyces clavuligerus is controlled by the phosphate concentration. Phosphate represses the biosynthesis of “cephamycin synthetase”, expandase and “clavulanic acid synthetase”. In the presence of 2 mM phosphate, the specific activities of expandase, “cephamycin synthetase” and “clavulanic acid synthetase” were higher than in the presence of 75 mM phosphate. The specific activity of cephamycin synthetase is maximal with an initial phosphate concentration of 10 mM, whereas the specific activity of expandase is maximal with 1 mM phosphate. A correlation between cephamycin synthetase specific activity and expandase specific activity was established at phosphate concentrations higher than 10 mM. This shows that the expandase is an important enzyme in the mechanism by which the phosphate concentration affects the biosynthesis of cephamycin.

Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains.

Abstract.Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins.

Regulation of spiramycin synthesis in Streptomyces ambofaciens: effects of glucose and inorganic phosphate

The production of the 16-membered macrolide antibiotic, spiramycin, in Streptomyces ambofaciens is inhibited by glucose, 2-deoxyglucose and inorganic phosphate. The role of intracellular ATP content and phosphorylated metabolites as common regulating signals of both glucose and phosphate inhibitory effects is discussed. Two enzymatic targets of the effect of phosphate on spiramycin biosynthesis were studied. Valine dehydrogenase, the first enzyme of valine catabolism (supplier of aglycone spiramycin precursors), and alkaline phosphatase, which cleaves phosphorylated intermediates, were repressed in the presence of excess phosphate.

Effect of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens

SummaryThe effect of ammonium on growth and spiramycin biosynthesis in Streptomyces ambofaciens cultured on a chemically defined medium was studied. Spiramycin biosynthesis was better in the presence of valine and isoleucine than in the presence of ammonium. This production was reduced in the presence of excess ammonium (100 mm). The addition of catabolic intermediates of valine and isoleucine reserved the negative effect of ammonium. Valine dehydrogenase (VDH), the enzyme responsible for valine, leucine and isoleucine catabolism, was repressed when excess ammonium was present in the medium. This repression was approximately 25% when the ammonium concentration was increased from 50 to 100 mm. In addition to the repression of VDH biosynthesis, ammonium inhibited the activity of this enzyme. This inhibition was 45 and 65% in the presence of 50 and 100 mm ammonium, respectively.

Carbon catabolite regulation of cephamycin C and expandase biosynthesis in Streptomyces clavuligerus

SummaryStreptomyces clavuligerus produces cephamycin C while growing on chemically defined basal medium. Cephamycin C production takes place during the exponential growth phase and is accompanied by vigorous activity of the “cephamycin C synthetase” system and of expandase. An excessive amount of glycerol decreases cephamycin C production. Its negative effect appears to be greatest when it is added in the first phase of fermentation either alone or in the presence of starch. Starch excess also reduces cephamycin C production, but its effect is slight compared with glycerol. Glycerol hinders cephamycin C production by the repression of the cephamycin C synthetase system and particularly expandase biosynthesis. Starch and glycerol inhibit neither cephamycin C synthetase nor expandase activities. However, the phosphorylated intermediates of the glycolytic pathway, glucose 6-phosphate and fructose 1,6-phosphate, strongly inhibit expandase activity.

Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction

Evidence was demonstrated for the occurrence of a membrane-bound cholesterol oxidase (EC 1.1.3.6) in cells of Rhodococcus sp. GK1, a soil-isolated strain. This enzyme catalyzed the conversion of 3β-OH-Δ5-sterols to their 3-Keto-Δ4 derivatives with concomitant reduction of O2 to H2O2. Growth of the microbe in a mineral medium, containing either phytosterols or hexanoate as sole carbon substrate, was studied. Under these conditions, the membrane-bound cholesterol oxidase was induced in large amounts exceeding 100 U g−1 dry wt. Enzyme solubilization was achieved by cell treatment with either Triton X-100, Emulphogen BC-720, Lubrol PX, Lubrol WX, or Brij 76, all are nonionic detergents and have low critical micelle concentration (CMC). Enzyme solubilization was due to mixed micelle formation. An additional evidence for the mixed micelle formation was provided from both, enzyme filtration on Sephadex G-200 in the presence of Triton X-100, and enzyme activity stimulation by nonionic detergents. This type of cholesterol oxidase was found to be an integral membrane protein. It appeared that the enzyme active domain is located at the outer surface of the membrane. Substrate catalysis in vivo occurs externally to the cytoplasm. A topology model at the membrane level was suggested for cholesterol oxidases of Rhodococcus and related taxa. The detergent-extracted cholesterol oxidase was active on sterols with the 3β-OH-Δ5 configuration. Neither sterols with modified A-ring or B-ring nor the 3α-OH of cholic acid were catalyzed by it. With cholesterol as the substrate, the optimal pH was in a broad region, from 6.0 to 8.2 at 30°C, and the Km value was 1.9 × 10−5M at pH 7.0 and 30°C. With β-sitosterol, a Km of 2.1 × 10−5M was estimated for the enzyme under similar conditions. Preparations of the detergent-extracted enzyme were stable upon maintenance at either 2–4°C for 6 months or at −20°C for 1 year.

Extracellular cholesterol oxidase from Rhodococcus sp. cells

Abstract Evidence was demonstrated for the occurrence of an extracellular cholesterol oxidase (EC 1.1.3.6) in the culture broth of Rhodococcus sp. GK1. This enzyme catalyzed the transformation of 3 β -hydroxy- Δ 5 -sterols to their 3-keto- Δ 4 derivatives with concomitant formation of hydrogen peroxide. The influence of various carbon sources, including steroids, on enzyme biosynthesis was investigated. Among the carbon sources so far tested, hexanoic acid was the most effective inducer. Triton X-100 was used in the enzyme assay medium to dissolve the substrate, cholesterol or other sterols. The enzyme was stimulated by this nonionic detergent, and optimal activity was obtained in the presence of 0.1 to 0.2% detergent. A similar effect on enzyme activity was observed with another nonionic detergent, Lubrol PX. The enzyme was active with cholesterol (100%), β-sitosterol (70%) and stigmasterol (40%). Neither sterols with modified A-ring and B-ring, nor the 3 α-OH of cholic acid were catalyzed by it. With cholesterol as the substrate, the optimal pH was 7.0 to 7.5 at 30°C, and the K m value was about 2 × 10 −5 M at pH 7.0 and 30°C. Filtration of the enzyme from the spent medium on a Sephadex G-200 column caused it to be aggregated and eluted in the void volume. However, the enzyme was separated into three distinct peaks when gel filtration was performed in the presence of 0.5% Triton X-100. In fact, the enzyme has the ability to interact with detergent to form mixed micelles. Consequently, the resolved peaks correspond to enzyme micellar forms. The values > 200, 160 and 120 kDa were estimated for their apparent molecular masses.

Glycerol effect on spiramycin production and valine catabolism in Streptomyces ambofaciens

Spiramycin production by Streptomyces ambofaciens in a chemically defined medium, with valine as nitrogen source, was controlled by the nature and the concentration of the carbon source. The production of this antibiotic was better in dextrins than in glycerol-containing medium. The negative effect of glycerol could be attributed in part to an excess of energy and a high specific growth rate. The intracellular ATP content, at the start of spiramycin production, was twofold higher in glycerol than in dextrin-containing medium. Increasing the initial concentrations of glycerol led to an increase in the specific growth rate and a drop in spiramycin production. Comparison between glycerol and a protein synthesis inhibitor effects and the use of resting cell systems (RCS) proved that glycerol exerted both inhibitory and repressive actions on spiramycin production independently from the growth. At the enzymatic level, glycerol interfered with valine catabolism by repressing partially valine dehydrogenase (VDH) and α-ketoisoisovalerate dehydrogenase (KIVDH), generator of spiramycin aglycone precursors.

Cephamycin C biosynthesis in Streptomyces cattleya: nitrogen source regulation.

SummaryThe production of cephamycin C by Streptomyces cattleya varies with the use of asparagine, glutamine or ammonium as nitrogen sources. hydroxylase and expandase activities were demonstrated for the first time with this species. A study of the biosynthetic regulation of these enzymes by two different nitrogen sources, glutamine and asparagine, was carried out. Asparagine proved to be a better nitrogen source, both for enzymatic biosynthesis and production of cephamycin C. Moreover, an excess of asparagine in the culture environment provokes, simultaneously, a reduction in cephamycin C production and a decrease in the biosynthesis of expandase and hydroxylase.

Effect of nitrogen/carbon ratio on the specific production rate of spiramycin by Streptomyces ambofaciens

Abstract Streptomyces ambofaciens was grown on a synthetic medium with glycerol (10 g litre −1 ) and ammonium (20 m m ) as carbon and nitrogen sources, respectively. Batch fermentation produced a specific production rate of spiramycin ( q sp ) of 0·3 mg h −1 g −1 DCW. Improvement of spiramycin production was achieved by fed-batch culture. The strong influence of the initial uptake rates of glycerol ( q gly ) and ammonium ( q NH 4 + ) on the specific production rate of spiramycin ( q sp ) was demonstrated by conducting several fermentations with continuous feeding of glycerol and ammonium, in different ratios. The specific production rate of spiramycin was increased 10 fold. A low specific growth rate was also necessary for spiramycin biosynthesis.