Evidence for a compartmentation of penicillin biosynthesis in a high- and a low-producing strain of Penicillium chrysogenum

Abstract

Pulse-chase experiments using (U14C]valine were done with P2 and 4176, high-and low-penicillin-producing strains of Penicillium chrysogenum. The metabolic flux of this amino acid into protein and penicillin was measured, and compartmentation of penicillin biosynthesis was assessed. Strain P2 took up 14C-valine more slowly than strain Q176, but their rates of incorporation into protein were comparable. Incorporation of 14C-valine into penicillin occurred immediately with the high-producer P2, but exhibited a lag with Q176. After 14C-valine had been removed from the medium, the specific radioactivity of penicillin continued to increase in 4176 but started to decrease immediately in P2. The specific radioactivities of 4C-valine in protein and in penicillin were significantly different in both strains: Q176 had a higher specific radioactivity of valine in penicillin than P2, whereas P2 had a higher specific radioactivity of valine in protein than 4176. Moreover, the specific radioactivity of 14C-valine in penicillin was 20-fold higher in strain 4176 than in P2. These results indicate that penicillin and protein biosynthesis use different pools of cellular valine, and that exchange of valine between the two compartments is slow in the low-producer, but rapid in the high-producer strain. Hence these results indicate a further control point of penicillin biosynthesis in P. chrysogenum.

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

“…The vacuole may, however, play an important role in amino acid retention and, likely, the regulated supply of the amino acids to the cytosol where the ACV synthetase resides. It is believed that during penicillin biosynthesis the amino acid precursors are withdrawn from the vacuolar lumen (Affenzeller and Kubicek, 1991;Lendenfeld et al, 1993). Recently, the bidirectional amino acid transport systems in the vacuolar membrane of S. cerevisiae have been analyzed genetically (Russnak et al, 2001).…”

“…This reaction is catalyzed by a 420-kDa ACV synthetase (Mart ı ın and Liras, 1989;Zhang and Demain, 1992). During penicillin biosynthesis the amino acid precursors are thought to be withdrawn from storage pools in the vacuole (Affenzeller and Kubicek, 1991;Lendenfeld et al, 1993). Next, a cytosolic enzyme, isopenicillin N synthase, catalyzes the formation of the b-lactam backbone from the ACV tripeptide yielding isopenicillin N (M€ u uller et al, 1991;Ramos et al, 1985).…”

Biotechnical Genetics of Antibiotic Biosynthesis

“…J(L-a-aminoadipyl)-Lcysteinyl-D-valine synthetase (ACVS) is located either within or bound to the vacuolar membrane (Lendenfeld et al 1993). A large portion of cellular L-a-aminoadipic acid, which is most likely used for P-lactam synthesis, is contained in the vacuoles as well and is sequestered from the cytosol (Honlinger and Kubicek 1989b;Affenzeller and Kubicek 1991). Isopenicillin N synthase (IPNS) has been found in the cytoplasm.…”