Richard L. Monaghan

Avermectins, New Family of Potent Anthelmintic Agents: Producing Organism and Fermentation

The avermectins are a complex of chemically related agents which exhibit extraordinarily potent anthelmintic activity. They are produced by a novel species of actinomycete, NRRL 8165, which we have named Streptomyces avermitilis. The morphological and cultural characteristics which differentiate the producing organism from other species are described. The avermectins have been identified as a series of macrocyclic lactone derivatives which, in contrast to the macrolide or polyene antibiotics, lack significant antibacterial or antifungal activity. The avermectin complex is fully active against the gastrointestinal nematode Nematospiroides dubius when fed to infected mice for 6 days at 0.0002% of the diet. Fermentation development, including medium modification and strain selection, resulted in increasing the broth yields from 9 to 500 μg/ml. Images

Avermectins, New Family of Potent Anthelmintic Agents: Isolation and Chromatographic Properties

The avermectins, a family of new anthelmintic agents, were isolated from the mycelia of Streptomyces avermitilis. Four closely related major components and four homologous minor components were separated from the complex. Solvent extraction, solvent partition, and adsorption methods were used to isolate and purify the complex; novel partition chromatography systems using Sephadex LH-20 were used to separate the components. A reverse-phase high-pressure liquid chromatography assay for the quantitative determination of all components was used extensively to monitor the purification methods.

Improvement in the titer of echinocandin-type antibiotics: a magnesium-limited medium supporting the biphasic production of pneumocandins A0 and B0.

SummaryWe have developed a liquid fermentation medium for the submerged culture of the fungus,Zalerion arboricola, which supports the rapid production of an echinocandin-type antibiotic, pneumocandin A0 (formerly L-671, 329), in yields increased at least 4-fold over those reported previously. The improvements were achieved through medium simplification, substitution of high levels of mannitol for glycerol as the major source of carbon, and restriction of available magnesium. Antibiotic formation in batch cultures with this mannitol-based medium is not confined to the idiophase; rather production appears to be biphasic, with synthesis beginning during growth (i.e., at day 3) and increasing in rate at day 11, well after rapid growth has ended. Accumulation of antibiotic continues beyond 14 days, and by 21 days titers greater than 500 μg/ml are attained. For the synthesis of a related compound, pneumocandin B0, by a mutant strain ofZ. arboricola, the medium gives similar production kinetics and a titer of 800 μg/ml. Although supplementation of the medium with magnesium ions stimulates growth, it decreases titer by preferentially affecting the second phase of antibiotic synthesis. This decline in synthesis in the magnesium-supplemented medium is explained by the depletion of mannitol before the second phase of synthesis can begin. In contrast, mannitol in the magnesium-limited medium is used more slowly with approximately half still available at day 11 to support continued antibiotic formation.

History of yield improvements in the production of asperlicin byAspergillus alliaceus

SummaryThe natural product asperlicin is the first nonpeptide antagonist of cholecystokinin isolated from a microbial source. At discovery, production of asperlicin by the original soil isolate ofAspergillus alliaceus was between 15 and 30 mg/l. Selection of natural variants ofA. alliaceus, use of Plackett & Burman and Simplex experimental designs; formulation of synthetic media; amino acid supplementation of production media; analysis of complex nitrogen sources for their amino acid content; evaluation of promising media in fermentors; substitution of glycerol for glucose as a carbon source and rational mutant selection all contributed to titer increases to >900 mg/l.

Microbial transformation of immunosuppressive compounds III. Glucosylation of immunomycin (FR 900520) and FK 506 byBacillus subtilis ATCC 55060

SummaryThe regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells ofBacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived fromBacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.

Microbial transformation of N-heptyl physostigmine, a semisynthetic alkaloid inhibitor of cholinesterase.

The microbiological transformation ofN-heptyl physostigmine (L-693, 487) (1), a semisynthetic physostigmine cholinesterase inhibitor, was investigated usingVerticillium lecanii MF 5713 (ATCC 74148),Acremonium sp MF 5723 (ATCC 74164) andActinoplanes sp MA 6559 (ATCC 53771). Nine microbial metabolites (2–10) of 1 were isolated and purified using reversed-phase HPLC. The structures of the metabolites were established using spectroscopic techniques including MS and NMR. Some of the microbial metabolites were identical to metabolites present in urine of a dog treated with 1.

STUDIES ON THE AVERMECTIN FERMENTATION

ABSTRACT The avermectins are a group of potent anthelmintic and insecticide active compounds produced by Streptomyces avermitilis. Synthesis of the avermectins in complex medium is regulated by added carbon, nitrogen, phosphorous and sulfur. The antifoam polyglycol P-2000 also influences the fermentation. The yield of nonproducing bald isolates is increased from 2% to as much as 60% by treatment with intercalating dyes. The possible involvement of extrachromosomal elements in controlling aerial mycelia, pigment and avermectin production is suggested.