Zalerion Arboricola Research Articles

Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina.

A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0.03-0.07 microgram ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.

Reclassification of a pneumocandin-producing anamorph, Glarea lozoyensis gen. et sp. nov., previously identified as Zalerion arboricola

The importance of pneumocandin BO as the fermentation-derived starting material for the antifungal drug candidate, MK-991, along with the identification of our production strain as Z. arboricola (ATCC 20868) as CBS prompted a search for other strains of Z. arboricola or Zalerion species with improved titres or that might produce natural pneumocandin analogues. Analysis of morphology, secondary metabolites profiles, and DNA fingerprinting demonstrated that ATCC 20868 was not congeneric with Z. arboricola. Ribosomal DNA sequences were compared among Zalerion species and pneumocandin-producing fungi and with rDNA sequences in GenBank. No good matches with sequences in GenBank were obtained for Z. arboricola or Z. maritimum, but for Z. varium, P. carpinea and ATCC 20868, relevant similarities were observed with ITS1 sequences from fungi of Leotiales. ATCC 20868 was phylogenetically more akin to P. carpinea, another pneumocandin producer, than initially suspected. The closest relative of ATCC 20868 seemed to be Hymenoscyphus monotropae. We conclude that the genus Zalerion is artificial; its species bear no phylogenetic relation among themselves. ATCC 20868 and Z. varium were related to fungi of the Leotiales. We propose a new anamorph genus and species, Glarea lozoyensis, to accommodate ATCC 20868.

Isolation and sequence analysis of the cDNA encoding Δ1-pyrroline-5-carboxylate reductase from Zalerion arboricola

A cDNA encoding Δ 1-pyrroline-5-carboxylate reductase (P5CR) was isolated from the pneumocandin(Pmo)-producing fungus, Zalerion arboricola (Za), by complementation of a P5CR-deficient mutant ( pro3) of Saccharomyces cerevisiae (Sc). The cloned cDNA was placed under control of the Sc galactokinase ( GAL1) promoter and restored P5CR activity to the pro3 mutant. Sequence analysis revealed that the Za P5CR-encoding cDNA encodes an approx. 35 kDa protein with substantial amino acid (aa) identity to P5CR from another filamentous fungus, Neurospora crassa (Nc). Za P5CR exhibits a moderate degree of aa identity to P5CR from plants, bacteria, human and Sc. This is the first gene to be isolated from Za.

5426038 Process for production of an antibiotic compound with Zalerion arboricola : Fountoulakis Jimmy M; Masurekar Prakash S; Kaplan Louis Westfield, NJ, United States Assigned to Merck & Co Inc

5426038 Process for production of an antibiotic compound with Zalerion arboricola : Fountoulakis Jimmy M; Masurekar Prakash S; Kaplan Louis Westfield, NJ, United States Assigned to Merck & Co Inc

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

We 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 micrograms/ml are attained. For the synthesis of a related compound, pneumocandin B0, by a mutant strain of Z. arboricola, the medium gives similar production kinetics and a titer of 800 micrograms/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.

Pneumocandins from Zalerion arboricola. V. Glutamic acid- and leucine-derived amino acids in pneumocandin A0 (L-671,329) and distinct origins of the substituted proline residues in pneumocandins A0 and B0.

Pneumocandins from Zalerion arboricola. V. Glutamic acid- and leucine-derived amino acids in pneumocandin A0 (L-671,329) and distinct origins of the substituted proline residues in pneumocandins A0 and B0.

Pneumocandins from Zalerion arboricola. IV. Biological evaluation of natural and semisynthetic pneumocandins for activity against Pneumocystis carinii and Candida species.

A series of lipopeptide compounds co-produced during the fermentation of pneumocandin A0 (L-671,329) and related semisynthetic compounds were evaluated in vivo against Pneumocystis carinii pneumonia and systemic candidiasis. In addition, they were tested in vitro against a panel of pathogenic Candida species and in a Candida membrane 1,3-beta-D-glucan synthesis assay. The results of these studies demonstrate that pneumocandin A0 and pneumocandin B0 (L-688,786) are the most potent compounds when considering both antipneumocystis and anticandida activity. Other compounds in the series are selectively more potent against P. carinii or Candida albicans suggesting a diverging structure-activity relationship. Evaluation of these compounds for their ability to inhibit C. albicans 1,3-beta-D-glucan synthesis in vitro demonstrates that they inhibit this process. A positive correlation between 1,3-beta-D-glucan synthesis inhibition and in vitro antifungal activity was also demonstrated for some of the pneumocandins.

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.

Pneumocandins from Zalerion arboricola. I. Discovery and isolation.

HPLC bioautography of the directed biosynthesis of Zalerion arboricola led to the discovery of pneumocandin B0 (L-688,786), a new antifungal and anti-Pneumocystis carinii lipopeptide. Isolation techniques were developed to separate this component from pneumocandin A0 (L-671,329) in fermentations of a mutant of Zalerion arboricola. A number of related compounds were also isolated, which differ from pneumocandins A0 and B0 in the hydroxylation patterns on the ornithine, homotyrosine, and proline.

Pneumocandins from Zalerion arboricola. III. Structure elucidation.

Pneumocandin B0 (6) and six related lipopeptides are antifungal and anti-Pneumocystis carinii agents from mutants of Zalerion arboricola, whose structures were determined mainly on the basis of spectroscopic analysis. They belong, along with pneumocandin A0 (L-671,329) previously isolated from these laboratories, to the echinocandin class of antifungal agents. The product from base-catalyzed ring opening involving the hemiaminal position of the dihydroxyornithine residue of B0, has been clearly defined as 6b. Modifications were limited to the 3-hydroxy-4-methylproline, 3,4-dihydroxyhomotyrosine and 4,5-dihydroxyornithine residues of pneumocandin A0.

An echinocandin from an endophytic Cryptosporiopsis sp. and Pezicula sp. in Pinus sylvestris and Fagus sylvatica

Fermentation broths of Cryptosporiopsis sp. and Pezicula sp. yielded the same echinocandin as is produced by the unrelated fungus Zalerion arboricola. The compound showed antimicrobial properties against certain yeasts.

ChemInform Abstract: Biosynthesis of L‐671,329, an Echinocandin‐Type Antibiotic Produced by Zalerion arboricola: Origins of Some of the Unusual Amino Acids and the Dimethylmyristic Acid Side Chain.

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Antifungal/Echinocandin B Derivatives: Novel Fungal Cell Wall Glucan Synthetase Inhibitors

SummarySummaryNovelty: New antibiotic substances produced by the cultivation of Zalerion arboricola are described. The compounds are cyclic lipopeptides and are claimed as potential agents for Pneumocystis carinii, Candida albicans and Candida parapsilosis infections.Biology: The tested compound has an IC50 = 0.5μg/ml against Candida albicans (MY 1055), 16-128 μg/ml against Candida parapsilosis (MY 1009). One compound gave 66% reduction of Pneumocystis carinii cysts in C3H/He mice at 2 mg/kg (given for seven days).Chemistry: 1-[N2-(10,12-dimethyl-1-oxotetradecyl)ornithine]-4-[4-hydroxyphenyl)-2-aminobutanoic acid]-5-(3-hydroxyglutamine) echinocandin B is one exemplified compound.

Biosynthesis of L-671,329, an echinocandin-type antibiotic produced by Zalerion arboricola: origins of some of the unusual amino acids and the dimethylmyristic acid side chain

The biosynthesis of L-671,329, an antibiotic of the echinocandin class, was studied in Zalerion arboricola by stable isotope tracer techniques and high-field NMR spectroscopy. The organism incorporates DL-(2-13C)tyrosine into the antibiotic with the label appearing as C-3 of the homotyrosine residue; the C-2 position of this residue can be labeled by (2- 13C)acetate. Thus, homotyrosine arises from tyrosine by a chain elongation mechanism involving condensation with acetate. (2-13C) Acetate also labels all the even-numbered carbon atoms (C-2-C-14) of the myristic acid side chain. L-(13CH3)Methionine does not donate its methyl group to 3-hydroxy-4-methylproline but is the origin of both methyl moieties of the 10,12-dimethylmyristoyl side chain. L-(l-13C)Proline is incorporated into only one of the two substituted proline residues, viz, 4-hydroxyproline. Label from L-(2-13C)leucine enriches the 3-hydroxy-4-methylproline residue, suggesting that this proline moiety is formed by cyclization of leucine.

L-671,329, a new antifungal agent. I. Fermentation and isolation.

In screening for new antifungal agents from fungi, a new lipopeptide antifungal agent, L-671,329, similar to echinocandin B, has been isolated from Zalerion arboricola. Studies indicate that L-671,329 is produced under both solid and liquid fermentation conditions.

A microecological approach to larch canker biology

In a comprehensive study of the microflora of larch cankers, it was shown that Trichoscyphella willkommii (Hart.) Nannf. was the only fungus consistently associated with canker extension tissue. A total of eighty-three other fungal species was isolated from 208 cankers and a sequential colonization of cankers by these fungi was demonstrated. The spread within the canker of other species coincided with disappearance of T. willkommii. The four most frequently isolated canker invading fungi, Cryptosporiopsis abietina, Zalerion arboricola, Tympanis laricina and a Phialophora sp. were shown to be able to replace T. willkommii in bark blocks, while Z. arboricola and Phialophora sp. brought about the death of its hyphae on agar plates. Inoculation of the four species either into branch lesions in which T. willkommii was established, or jointly with T. willkommii into new incisions, showed that lesion extension was markedly decreased in their presence. It was suggested that the natural healing of larch cankers is largely initiated because T. wilkommii is placed at an ecological disadvantage through having to compete with other fungi.