Microbiological studies of some peptide antibiotics

Introduction
Among the penicillin-producing organisms which are members of the Class Fungi Imperfecti are Penicillium chrysogenum and the Cephalosporium sp. C-91. The former can produce a series of extracellular penicillins (I) with non-polar side-chains (R.CO) which vary with the side-chain precursor added to the fermentation medium.
The Cephalosporium sp. produces only two extracellular antibiotics of this type and both have a δ-(D-α-aminoadipyl) side-chain. One is penicillin N (II) and the other cephalosporin C (III).
Early studies established that the biogenetic precursors of the ring systems of both benzylpenicillin and cephalosporin C were L-cysteine and L-valine, and that the side-chains were derived from phenylacetic acid and α-aminoadipic acid respectively. The tripeptide δ-(α-aminoadipyl)cysteinylvaline (ACV,IV) was isolated from the mycelium of P. chrysogenum (Arnstein & Morris, 1960) and subsequently detected in the Cephalosporium sp. also (Smith et al., 1967). This suggested that δ-(α-aminoadipyl)cysteinylvaline might be an intermediate in the biosynthesis of the penicillins and of cephalosporin C.
The difficulty of assessing the role of possible intermediates such as ACV in penicillin or cephalosporin biosynthesis has depended partly on the existence of permeability barriers. The development of broken-cell or cell-free systems in which these barriers have been removed and in which all or part of the biosynthetic pathways can be followed is thus clearly important.
Cell-free or broken-cell systems have been used to study the biosynthesis of several peptide antibiotics. These systems have been prepared by mechanical rupture of the cells, or by enzymic digestion of the cell wall to form protoplasts, which were then easily disrupted by osmotic shock. Dissolution of the cell wall of B. licheniformis and of B. brevis by muramidase has led to cell-free systems capable of synthesising bacitracin and tyrocidine respectively (Ishihara, Sasaki & Shimura, 1968; Fujikawa, Suzuki & Kurahashi, 1966). However, protoplasts from fungi have proved more difficult to prepare (Villaneuva, 1965). Snail enzyme, an impure preparation containing approximately thirty enzymes, has commonly been used to prepare protoplasts from yeast cells. Cells from different strains and of different ages vary markedly in their susceptibility to snail enzyme, but young cells are in general more susceptible than older ones.
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Aims and Scope of the Present Investigation
The main aim was to prepare protoplasts from the Cephalosporium sp. and to study their properties with the intention of using them as the starting material for the preparation of an active cell-free extract. Towards this end it was decided to find out which phase of a culture of Cephalosporium sp. C-91 produced penicillin N and cephalosporin C at maximal rates, and the morphological appearance of the mycelium in this phase so that protoplasts could be prepared from this type of culture. Finally, it was hoped that a cell-free extract obtained from protoplasts would be able to carry out all or some of the steps associated with antibiotic biosynthesis.
Material and Methods
Cephalosporium sp. C-91 was grown in batch cultures in shake flasks in a chemically-defined medium. The cultures were in general seeded with a heterogeneous inoculum but in some experiments with a spore inoculum. The growth of the organism was plotted from dry weight measurements, antibiotic production was measured by bioassay and morphological changes were followed by phase-contrast microscopy and photo-micrography.
Protoplasts were prepared from mycelium harvested at 48 hours by resuspension in a solution of snail enzyme (40 mg./ml) and sodium chloride stabiliser (0.9M) after pretreatment with 2-mercaptoethanol or dithiothreitol. After incubation (3–4 hours) protoplasts were separated from intact mycelium and resuspended in stabiliser. The percentage yield of protoplasts was measured by comparing their content of ethanol-extractable nucleotide with that obtained from a known weight of intact mycelium. Electron micrographs were made of mycelium and protoplasts after fixation in Kellenbergers buffer (Kellenberger, Ryter & Séchaud, 1958) and staining with osmium tetroxide.
For the measurement of [14C]-valine uptake by mycelial preparations, samples from the resuspension fluids were withdrawn at intervals and centrifuged. A known volume of supernatant fluid was removed for radioactivity measurement.
Antibiotic production by the protoplasts was measured by bio-assay and by the incorporation of 14C from DL-[1-14C]-valine. Culture fluids of protoplasts and mycelium containing isotopically labelled antibiotics were desalted on Nuchar from which the antibiotics were eluted with ethanol.
Radioactive compounds in extracts of culture fluids were located after paper chromatography or electrophoresis by radioautography and counted on the paper; absolute radioactivities were calculated from the counts on paper. Small amounts of authentic cephalosporin C and penicillin N were added to samples containing radioactive antibiotics and their positions after analysis on paper were determined by scanning in ultraviolet light (Abraham & Newton, 1961).
Results and Discussion
Section I. The growth rate was divided into three phases. The first began immediately after inoculation when the growth rate increased rapidly and was accompanied by the production of young, undifferentiated hyphae, and the germination of spores. Towards the end of this phase penicillin N production began, and, as in phase two, its production appeared to be associated with growing mycelium. During phase two the growth rate reached its maximum when cultures consisted of young hyphae in an extending and branching growth phase. Cephalosporin C production began later in phase two, while in phase three both antibiotics attained maximum production rates as the growth rate was falling. Thus, maximum antibiotic production was associated with the presence of non-growing, differentiated mycelium. Penicillin N appears to be synthesised by a pathway which was not restricted to non-growing cells, and which is different from, and initiated earlier than that which leads to Cephalosporin C formation.
Section II. The cell walls of 48 hour cultures of the Cephalosporium sp. were not readily digested by snail enzyme. However, pretreatment for 1 hour with 0.02M-2-mercaptoethanol or 0.01M-dithiothreitol rendered it more susceptible to attack. In yeast cell walls 2-mercaptoethanol is thought to reduce disulphide bonds linking protein to mannan. Although there is no evidence to suggest that these linkages are present in the Cephalosporium sp. cell walls, it is possible that a similar reaction occurs, after which the walls are more rapidly degraded by the snail enzyme complex.
The electron micrographs showed that the cell wall of Cephalosporium sp. C-91 was a four layered structure. After treatment with dithiothreitol the cytoplasm withdrew from the inner layer of the wall, while the walls themselves appeared unchanged. The protoplasts were completely spherical with no detectable vestiges of wall material at their surface. They possessed vacuoles, mitochondria and ribosomes in their cytoplasm, but the latter were more diffuse than in the mycelium. This and the loss of invagination in the protoplasmic membrane suggested that the stabilising solution had entered the protoplasts causing them to swell in a manner analogous to the swelling of damaged mammalian cells (Leaf, 1956).
Section III. Pretreatment of mycelium with dithiothreitol followed by incubation in sodium chloride stabiliser reduced penicillin N production by approximately 40% and that of cephalosporin C by 60–70%. Thus, it seemed that the cephalosporin C system was more sensitive to external changes in environment, possibly because certain enzymes or intermediates involved in its formation are more labile than any of those involved in penicillin N production.
On the other hand, after treatment of mycelium with snail enzyme solution the production of cephalosporin C was inhibited less than that of penicillin N. Hence, it appears unlikely that cephalosporin C was formed from penicillin N. It is possible that co-factors or enzymes required for penicillin N synthesis were located outside the protoplast membrane and removed when the wall was dissolved. However, even when antibiotic synthesis could not be measured by bioassay, the formation of [14C]-penicillin N as well as that of [14C]-cephalosporin C could be detected after the addition of [14C]-valine to the protoplast suspensions.
The protoplasts released cephalosporin C readily after agitation, as if it were only loosely bound to the cells. The total intracellular amino acid content of the protoplasts was estimated to be approximately one tenth of that of whole mycelium. In addition, it was found that potassium ions leaked out of the protoplasts during incubation, although enough appeared to be retained to maintain an energy-generating system.
The dipeptide δ-(DL-α-aminoadipyl)-L-cysteine was able to enter the protoplasts, although it did not enter the intact mycelial cell. But once inside it did not appear to enhance antibiotic production, suggesting that the synthesis of δ-(α-aminoadipyl)cysteine was not a rate-limiting step in the biogenetic pathway in the protoplasts.
The uptake of DL-[1-14C]-valine by suspensions of protoplasts was compared with that by starved mycelium in water and DTT-pretreated mycelium in 0.9M-NaCl. A similar overall pattern was obtained, which could be divided into three phases. In the initial uptake, lasting up to 10 minutes, as much as half of the total isotope was removed. In the next phase, from 10 to 30 minutes, there was a rise in the extracellular radioactivity which was assumed to be due to the release of [14C]-valine from protoplasts and from mycelium. In the third phase, valine was taken up at a similar rate by protoplasts and mycelium, but more slowly than in the initial phase. Within this pattern, variations between suspensions were observed in the amounts of valine removed or released in each phase.
Section IV. The biosynthesis of glutathione, of δ-(α-amino-adipyl)cysteinylvaline and of penicillin N and cephalosporin C was studied in cell-free extracts prepared by grinding mycelium with sand or alumina, or from lysed protoplasts. In the presence of an ATP-generating system, [14C]-glutathione was formed from glutamic acid, cysteine and [14C]-glycine. It was synthesised more readily in extracts from 72 hour mycelium than in those from 48 hour mycelium, and more readily in the supernatant than the particulate fractions of the extracts. In a protoplast extract, synthesis of δ-(α-aminoadipyl)cysteinylvaline (ACV) was detected only when δ-(L-α-aminoadipyl)-L-cysteine was added with 14C-valine, which indicated that the formation of the dipeptide was rate-limiting in this cell-free system.
When α-aminoadipic acid, cysteine and [14C]valine were added to protoplast lysates, small amounts of labelled antibiotics were formed. Hence, if ACV is an intermediate in antibiotic formation, the protoplast extracts must have synthesised it from its constituent amino acids. It is suggested that after being formed in minute amounts, ACV is rapidly transformed to penicillin N and cephalosporin C and thus escapes detection. Unlike the intact protoplasts, cell-free extracts derived from them synthesised more penicillin N than cephalosporin C. The formation of the latter may be more dependent on the structural integrity of the cell.
Conclusions
The work described in this thesis has shown that protoplasts can be made from Cephalosporium sp. which retain some of the ability of the intact mycelium to synthesise penicillin N and cephalosporin C. Further, a cell-free system has been obtained by lysis of these protoplasts in which it has been possible to demonstrate the biosynthesis of δ-(α-aminoadipyl)cysteinylvaline from δ-(α-aminoadipyl)-cysteine and [14C]-valine. Fractionation of this lysate has produced a 20,000 g supernatant, free of whole or broken protoplasts, which is able to synthesise small amounts of the antibiotics from α-amino-adipic acid, cysteine and [14C]-valine.
It is concluded that more definitive information about biosynthesis precursors could be obtained by the use of isotopically labelled intermediates, such as δ-(α-amino-adipyl)cysteinylvaline, in the cell-free systems described here. From one of these systems it might be possible to prepare a purified enzyme complex capable of synthesising penicillin N and cephalosporin C.

Advisor: Newton, G