Abstract
The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.
Highlights
From the Abteiluwf u r Mikrobielle Biochemie, Znstitutf u r Biochemische Technologieund Mikrobiologie, Technische Universitat Wien, Getreidemaikt9, A-1060 Vienna,Austria
Ipate used in penicillin biosynthesis and the first en- Some attempts toanalyze the metabolic regulation of the zymic step in Penicillium chrysogenuminvolved, &(a- a-aminoadipate poolsize in Penicilliumchrysogenum have aminoadipy1)-L-cysteinyl-D-valinesynthetase(ACVS), been published
We have studied the localization of the initial step of the pathway of penicillin biosynthesis by cell-fractionation experiments
Summary
With regard to control of channeling of amino Organism and Cultivation-ThePenicilliumchrysogenumstrain acid precursors into the penicillin biosynthetic pathway, the used throughoutthe present study wasBC 1505, which is proprietary cellular concentration of a-aminoadipate, an intermediate of to BiochemieGmbH(Kundl,Tyrol, Austria) It wasobtainedbilysine biosynthesis in fungi and the starting amino acid for penicillin formation (Fig. l),has been shown to be critical; higher penicillin-producing strains were shown to accumulate higher intracellular concentrations of a-aminoadipate Protoplasts were removed genum, SDS-PAGE followed by Western blotting and immunostainfrom the myceliaby centrifugation at low speed For this purpose, equal volumes of samples from.
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