Formation and Properties of Protoplasts from Antibiotic-producing Strains of Penicillium chrysogenum and Cephalosporium acremonium

Abstract

SUMMARYOsmotically fragile protoplasts have been prepared by the action of lytic enzymes on the mycelium of Penicillium chrysogenum and Cephalosporium acremonium.The yield of protoplasts, based on DNA content, was up to 18 %. Pretreatment of the mycelium with a thiol compound was necessary with Cephalosporium but not with Penicillium. Electron micrographs indicated that the protoplasts contained all the intracellular organelles of the mycelium and were bounded only by a cytoplasmic membrane. The metabolic activity of the protoplasts, as measured by their respiration, ability to maintain intracellular amino-acid pools, and antibiotic production, was similar to that of control mycelium. L-Valine and L-a-aminoadipic acid, which are precursors of penicillin N and cephalosporin C, were taken up and concentrated by the protoplasts of C. acremonium, although the latter transported L-valine less rapidly than did the corresponding mycelium. Protoplasts of P. chrysogenum took up ~-valine but not L-a-aminoadipic acid or its 8-ester. There was little or no transport of the corresponding D-amino acids by protoplasts of either organism. I N T R O D U C T I O NOne of the difficulties encountered in studies of penicillin and cephalosporin biosynthesis has been associated with the permeability barriers of mycelium. Thus, the interpretation of some experiments has been limited by the failure of a substance added to mycelial suspensions to reach the site of synthesis. For example, no detectable amount of [14C]penicillin N was found to enter the mycelium of Cephalosporium acremonium from the extracellular fluid (Smith, Warren, Newton & Abraham, I 967). The dipeptide 6-(a-aminoadipyl)-cysteine was not taken up intact by the mycelium but hydrolysed to its constituent amino acids (Loder, Abraham & Newton, 1969).A fragmented hyphal preparation has been obtained by ultrasonic treatment of the mycelium of Cephalosporium acremonium which is able to synthesize a possible biosynthetic intermediate, the tripeptide 8-(a-aminoadipyl)cysteinylvaline, from 6-(L-a-aminoadipyl)-Lcysteine and ~~-[ l~C ] v a l i n e (Loder & Abraham, 1971). However, no indication of penicillin or cephalosporin formation was obtained in fragmented hyphal systems of this type and it seemed that a study should be made of gentler methods of disrupting the mycelium. The first step of a possible approach was the enzymatic removal of the hyphal walls to form osmotically fragile protoplasts. For example, a bacterium-free extract was prepared from protoplasts of Bacillus licheniformis which was able to synthesize bacitracin from its constituent amino acids in the presence of an energy-generating system (Ishihara, Sasaki & Shimura, 1968), whereas an extract prepared by ultrasonication was less effective (Shimura, P. A. F A W C E T T A N D O T H E R SSasaki & Sugawara, 1964). This paper describes the preparation of protoplasts from strains of Penicillium chrysogenum and Cephalosporium acremonium and some of the properties of these protoplasts which are relevant to the biosynt...

Naturally Occurring β-Lactams

“…Neither the' carbon label nor the tritium label of D-[2-3 H, 1-14 C]-serine was incorporated. The authors also synthesised serines labelled with a deuterium atom on C-3 in the form of the enantiomeric pairs (247) and (248), and (249) and (250). These pairs were not resolved as only the L-isomer would be f" f" f" f" (249).…”

“…HUANG et al (248) In order to demonstrate that 8-( (X-aminoadipyl)-cysteinylvaline is the penicillin precursor, cell-free biosynthetic systems had to be employed as the tripeptide is not taken up by intact cells. FAWCETT et al (244,250) were the first to prepare such a cell-free system using enzymic digestion of the cell wall of C. acremonium in an isotonic medium to give pro top lasts, which were then lysed by dilution. Again, an energy-generating system was added.…”

Penicillium and Acremonium

“…Protoplasts can be prepared from A. strictum quite readily utilizing either the Cytophaga lytic enzyme Ll (Fawcett et at., 1973) or a cellulase preparation (Cellulose CP, from Penicillium funicuiosum; Supplied by John and E. Sturge, Ltd., Selby, N. Yorks, UK), as described by Hamlyn et ai., 1981. Mycelium from 48-hr cultures, pretreated with dithiothrietol, exposed to either of these enzymes in the presence of O.7M sodium chloride will yield 2-3 x lOB protoplasts/50 mg fresh mycelium (Hamlyn, 1982).…”

Protoplast Transformation for Genome Manipulation in Fungi

“…Protoplasting is one of the methods commonly used to prepare cells in order to genetically modify fungi. More than six decades have passed since the fi rst reports appeared on protoplast isolation from yeasts (Bachmann and Bonner 1959 ;Eddy and Williamson 1959 ) and fi lamentous fungi (Bachmann and Bonner 1959 ;Fawcett et al 1973 ). Since then, the interest in the improvement of this technique has enabled scientists to achieve higher transformation rates and more effi cient targeting to the appropriate genomic locus.…”