1. The stereoisomers of delta-(alpha-aminoadipyl)-L-cysteinylvaline (LLD, LLL and DLD) were synthesized from valine labelled with 3H in its methyl groups or in the alpha position. L-Cysteinyl-D-[4,4'-3H]valine was also synthesized. 2. 3H was incorporated into a compound that behaved like penicillin N when the LLD tripeptide containing either a methyl- or an alpha-labelled valine residue was incubated with a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium. 3. Incorporation was not observed under these conditions from the labelled all-L- or DLD-tripeptide, from L-cysteinyl-D-[4,4'-3H]valine, or of Penicillium chrysogenum appeared to be the LLD isomer, like that from C. acremonium. 5. These findings are discussed in relation to penicillin biosynthesis.

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1. 3H was incorporated into solvent-soluble penicillin from isopenicillin N and 6-aminopenicillanic acid 3H-labelled in the 2beta-methyl group when the labelled compounds were incubated with a crude extract of Penicillum chrysogenum. 2. With a soluble protein fraction of the extract incorporation from isopenicillin N occurred on addition of phenyl-acetyl-CoA. 3. Labelled benzylpenicillin was isolated after incubation of the crude extract with phenylacetyl-CoA and isopenicillin and the addition of unlabelled benzylpenicillin as a carrier. 4. No incorporation of 3H into solvent-soluble penicillin was detected on incubation of these extracts with penicillin N.

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...

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