Studies on malbranchea pulchella - A penicillin forming thermophilic fungus

Abstract

Chapter I. Introduction The recent developments in the biochemistry of penicillins have been briefly reviewed. The discovery of 6 aminopenicillanic acid has led to the preparation and testing of several semi synthetic penicillins, particularly on resistant bacterial strains which seem to pose a serious problem among the staphylococci. Another notable advance in the field pertains to the characterisation of new penicillins and penicillin like compounds, e.g., cephalosporin N (penicillin N), penicillin F (isopenicillin F) and cephalosporin C. Our knowledge of the biosynthesis and mode of action of penicillin has been greatly extended, and the various mechanisms suggested for penicillin formation by Penicillium chrysogenum are referred to. The property of producing penicillins is shared by members of several genera of fungi, including a strain of Streptomyces. Its formation by the thermophilic fungus Malbranchea pulchella seems rather unique, in that it demonstrates the possibility of penicillin formation-albeit in small quantities-at elevated temperatures. The objective of the present investigation has been stated. During screening of thermophilic microorganisms of interest to fermentations in tropical countries, a strain apparently similar to M. pulchella ATCC 9949 was encountered in local composts, which forms penicillin in stationary cultures. Earlier work by the author disclosed a striking departure of M. pulchella from P. chrysogenum in producing no marked elevation in penicillin titres in the presence of added phenylacetic acid. The iodometric assays nevertheless gave higher penicillin titres compared with bioassays, and addition of phenylacetic acid did not materially reduce the discrepancy. These studies have therefore been undertaken to characterise the substance or substances that interfere with iodometric assays of penicillin in these cultures.

Chapter II. Materials and Methods The microbiological, chemical, and analytical procedures, including radiotracer techniques used presently, have been described. Fractionation and characterisation of the S 35-labelled constituents of M. pulchella broths and mycelia harvested at 120 and 168 hours-corresponding to end of growth and penicillin forming phases in synthetic media with and without added phenylacetic acid-have essentially been carried out according to the procedures of Ballio et al.

Chapter III. Characterisation of Sulphur Containing Compounds Elaborated by M. pulchella The main features of sulphur metabolism of M. pulchella in the presence and absence of added phenylacetic acid have been elucidated using S 35 sodium sulphate as the sole sulphur source in modified Soltero and Johnson’s medium containing glucose. The organism is grown in stationary cultures at 45°C. Compared with P. chrysogenum in shake cultures, the uptake of inorganic sulphate by this organism is much lower. There is also less accumulation of inorganic sulphate in the mycelium; whatever is taken up is mostly converted into organic forms. The contribution of penicillin sulphur, however, is very small and is not materially influenced by phenylacetic acid. It resembles high yielding strains of P. chrysogenum in the considerable uptake of inorganic sulphate but differs in not converting the organic sulphur largely to penicillin. Uptake of inorganic sulphate-particularly in the presence of phenylacetic acid-distinguishes M. pulchella sharply from low yielding P. chrysogenum strains. Examination of the labelled constituents of broth and mycelia at 120 and 168 hours revealed all compounds reported by Ballio et al. in P. chrysogenum cultures-except 6 aminopenicillanic acid. By fractionation on Dowex 1×8 and Dowex 50×8, these were separated and identified as:

cystine methionine sulphuryldicholine glutathione(s) 2 hydroxy 4 methylmercaptobutyric acid 2,2 dimethyl 5 oxo hexahydroimidazo(5,1 b)thiazole 3,7 dicarboxylic acid

Phenylacetyl chloride treatment (Amstein’s conditions) did not increase penicillin titres, confirming the absence of 6 APA. Additionally, a tripeptide, previously described by Arnstein in P. chrysogenum, and a new substance termed Compound B were found. Evidence suggests the tripeptide may be identical with ( aminoadipyl)cysteinylvaline. Compound B:

corresponds to sharp radiochemical peaks overlapping known penicillin by product peaks is ether extractable from acidified broths yields benzylpenicilloic acid on alkali treatment appears consistent with the assigned structure: N phenylacetyl 2,2 dimethyl 5 oxo hexahydroimidazo(5,1 b)thiazole 3,7 dicarboxylic acid

Compound B interferes with iodometric assays of penicillin, whereas Compound G (2,2 dimethyl 5 oxo hexahydroimidazo thiazole derivative) reacts with iodine even before alkali treatment and thus does not contribute to such assay values.

Chapter IV. Malbranchins A and B In addition to penicillin, M. pulchella forms small quantities of two sulphur free antibiotics-malbranchins A and B. Malbranchin A is obtained in larger amounts and has been examined in detail. Malbranchin A:

is concentrated mainly in the mycelium is partially stimulated by phenylacetic or phenoxyacetic acids may also occur in P. notatum 832, but not P. chrysogenum 0176 or Wis 20 123

Properties:

pale brown amorphous powder, m.p. ~205°C (dec.) weak acid; soluble in sodium carbonate but not sodium bicarbonate UV maximum at 262 nm IR band at 5.75 gives negative ferric chloride, Tollens’, Molisch’s, and ninhydrin tests positive periodic acid reaction yields alanine, valine, leucine, and glycine on acid hydrolysis

Malbranchin A appears to be an acyclic peptide linked molecule, though whether the amino acids are integral or impurity derived is unresolved. It is active against Gram positive bacteria including some mycobacteria. Its intraperitoneal toxicity in mice is low (no mortality at 500 mg/kg).

Chapter V. Malbranchasterol: A Sterol from M. pulchella Ether extracts obtained during malbranchin extraction contain two sterol like components. One major component-malbranchasterol-crystallises in long, colourless needles from aqueous acetone and is the first sterol reported from a thermophilic organism. Malbranchasterol resembles ergosterol in:

UV absorption colour reactions (Liebermann-Burchard, Tortelli-Jaffé) behaviour on paper and thin layer chromatography

However, it differs in:

melting point optical rotation (malbranchasterol is dextrorotatory; ergosterol is levorotatory) molecular formula (contains an additional C H O unit)

Upon UV irradiation, absorption in the 270-290 nm region increases, unlike ergosterol (which decreases); a transformation analogous to ergosterol calciferol appears unlikely. Infrared data indicate:

a homoannular , diene system a free hydroxyl group equatorial OH at C 3 (9.4 band) possible oxide linkage as the second oxygen function

Malbranchasterol shows several unusual features; ongoing work aims to elucidate its structure and metabolic role.