Pneumocandins from Zalerion arboricola. II. Modification of product spectrum by mutation and medium manipulation.

Zalerion arboricola ATCC 20868 produces pneumocandin A0 (L-671,329), a cyclic hexapeptide with a dimethylmyristic acid side chain. This compound has anti-candida and anti-pneumocystis activities. We were interested in looking for other related compounds produced by this organism. To facilitate this search, a simple medium (S2) composed of D-mannitol, peptonized milk, lactic acid, glycine, KH2PO4 and trace elements, which supported the production of a number of such compounds, was designed. For the isolation of mutants, either spores or growing mycelia were treated with N-nitroso-N-methylurethane or N-methyl-N'-nitro-N-nitrosoguanidine and survivors were screened for changes in the product spectrum. From approximately 1,500 survivors tested, 5 mutants were isolated. Mutants ATCC 20957, 74030, 20958 and 20988 exclusively produce various pneumocandins other than A0. These compounds were active against Candida and Pneumocystis carinii. The yield of A0 was found to be increased 2.5-fold over that of the parent in the fifth mutant, MF5415. Further medium studies indicated that the addition of soybean oil to S2 medium improved the yields. Subsequent development of another series of media containing Pharmamedia as a nitrogen source resulted in increase in production by 10- approximately 20-fold. Overall, these studies resulted in substantial improvement in the production of A0 as well as discovery and/or facile production of 7 other related compounds.

Impaired penicillin production in lysine regulatory mutants of Penicillium chrysogenum.

Lysine-regulatory mutants of Penicillium chrysogenum which excrete lysine were found to be deficient in production of penicillin. A revertant recovered the ability to produce penicillin.

Insensitivity of homocitrate synthase in extracts of Penicillium chyrosogenum to feedback inhibition by lysine.

We previously reported that lysine inhibits in vivo homocitrate synthesis in the lysine bradytroph, Penicillium chrysogenum L(1), and that such feedback inhibition could explain the known lysine inhibition of penicillin formation. In the present study, it was found that dialyzed cell-free extracts of mutant L(1) converted [1-(14)C]acetate to homocitrate. This homocitrate synthase activity was extremely labile but could be stabilized by high salt concentrations. The pH optimum of the reaction was 6.9, and the K(m) was 5.5 mM with respect to alpha-ketoglutarate. The reaction was also dependent upon the presence of Mg(2+), adenosine 5'-triphosphate, and coenzyme A. Surprisingly, the activity in these crude extracts was not inhibited by lysine. Benzylpenicillin at a high concentration (20 mM) partially inhibited the enzyme, an effect that was enhanced by lysine. Casein hydrolysate also partially inhibited the enzyme.

Mutagenesis and enrichment of auxotrophs in Penicillium chrysogenum.

An efficient method for isolation of auxotrophs of Penicillium chyrysogenum involving mutagenesis with ethyl methanesulfonate followed by enrichment with sodium pentachlorophenate was developed. The auxotroph frequencies obtained were 30 to 40%.