Norbert Madry

Biosynthesis of cyclosporin A

Abstract Short term feeding of the mould Tolypocladium inflatum with 14C-labelled amino acids revealed a selective incorporation of l -leucine, l -valine, glycine and d , l -alanine into cyclosporins A and C. Feeding of l -[Me-14C]methionine exclusively labelled the N-methyl moieties of the cyclosporins. The distribution of radioactivity from this substrate was directly proportional to the number of the relevant N-methyl amino acids in cyclosporin A, indicating a simultaneous methylation of these residues.

Enniatin production by Fusarium oxysporum in chemically defined media

SummaryProduction of the depsipeptide antibiotic enniatin was studied in submerged cultures of Fusarium oxysporum. The influence of various nutritional factors on growth and enniatin production was investigated. Sucrose and glucose were suitable carbon sources, NaNO3 was the best nitrogen source with respect to growth and production. Enniatin yields up to 2.5 g/l were achieved within 4 days of cultivation of a variant obtained after two subsequent mutagenic treatments. Addition of several amino acids to producing cultures enhanced product formation up to 5 g/l. External supply of the branched-chain amino acids, valine, leucine, and isoleucine triggered an increased formation of the corresponding enniatin homologues which contain the amino acid fed as a constituent.

Regulation of tylosin synthesis in Streptomyces: Effects of glucose analogs and inorganic phosphate

SummaryGlucose, 2-deoxy glucose and inorganic phosphate inhibited tylosin production and fatty acid oxidation in Streptomyces T 59–235. Glucose-6-phosphate was accumulated in high-phosphate cultures. The possible function of glucose phosphate as a common mediator of both glucose and phosphate effects is discussed.

Selective synthesis of depsipeptides by the immobilized multienzyme enniatin synthetase

SummaryThe multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or α-aminobutyric acid as sole amino acid moieties, was observed.

Incorporation of radioactive precursors into beauvericin produced by Paecilomyces fumoso-roseus

Abstract The biosynthesis of the depsipeptide antibiotic beauvericin was studied by feeding submerged cultures of Paecilomyces fumoso-roseus with [ 14 C]-labelled precursors. l -Phenylalanine, hydroxyisovaleric acid, l -valine and methyl-labelled methionine were efficiently incorporated into beauvericin, whereas no radioactive depsipeptide was formed with l -leucine and l -tyrosine. [ 14 C]Phenylalanine was found to label exclusively the N -methylphenylalanine residues of beauvericin, the N -methylgroups, thereof, were derived from methionine. Beauvericin synthesized in the presence of dl -[2- 14 C]hydroxyisovaleric acid or l -[ 14 C]valine was specifically labelled in its hydroxy valeryl moieties.

Formation of secondary metabolism enzymes in the tylosin producerStreptomyces T59-235

Production of the macrolide antibiotic tylosin byStreptomyces T59-235 was inhibited in cultures containing high phosphate concentrations (30 mM Pi). Vegetative growth (dry weight increase, DNA and RNA synthesis) was hardly affected. Tylosin production began when macromolecule synthesis had slowed down to minimum level; in cultures with 30 mM Pi the onset of antibiotic production was retarded compared to cultures with low phosphate concentration (5 mM). The activities of three enzyme systems involved in tylosin biosynthesis (dTDP-D-glucose-4,6-dehydratase; dTDP-mycarose-forming enzyme system; SAM: macrocin-O-methyl transferase) were measured and found to be significantly lower in cultures with 30 mM Pi than in low phosphate cultures.Chloramphenicol, but not rifampicin, caused a rapid decrease of both tylosin formation rate and dTDP-D-glucose-4,6-dehydratase activity when added to tylosin producing cultures.

Covalent immobilization of the multienzyme enniatin synthetase

SummaryThe multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.