Penicillium chrysogenum Takes up the Penicillin G Precursor Phenylacetic Acid by Passive Diffusion

Abstract

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.

Uptake of phenylacetic acid by two strains ofPenicillium chrysogenum

“…2B). These data indicate that uptake of PA occurs through simple diffusion, in accordance with earlier conclusions (Hillenga et al, 1995). However, at low external PA concentrations in the range from 2.3-100 M, the kinetics of accumulation in the two strains differed, as in the Wisconsin strain accumulation of total PA and PA-derived carbon showed saturation kinetics with apparent K m of 15 M and V max of 109 pmol/mg dry weight per min ( Fig.…”

“…In contrast, early results suggested that PA and other weak acids penetrate the plasma membrane of P. chrysogenum by simple diffusion of the undissociated acids (Hunter and Segel, 1973), and recently Hillenga et al (1995) concluded that uptake of PA in P. chrysogenum Wisconsin 54-1255 and Panlabs P2 occurs through simple diffusion of the undissociated PA across the plasma membrane. One reason for the conflicting conclusions on kinetics of PA uptake in P. chrysogenum may be the different concentration ranges employed; low external concentrations of PA (1.4-100 M) were used by Fernández-Cañón et al (1989a,b), whereas higher PA concentra-tions (60-30,000 M) were applied by Hillenga et al (1995).…”

“…To investigate if the conversions of PA have an impact on the PA-uptake experiments, the nature of the radiolabeling in cell extracts was analyzed after fractionation by HPLC. For extraction of intracellular radiolabeled compounds a procedure involving extraction from whole cells in chloroform-methanol (Hillenga et al, 1995), was tried. By this method, however, less than 50% extraction of the labeled material was obtained, and furthermore the presence of chloroform interfered with subsequent fractionation by HPLC because the chloroform-containing samples were not miscible with the mobile phase.…”

“…Phenoxyacetic acid is transported into the cells of P. chrysogenum by simple diffusion across the plasma membrane (Eriksen et al, 1995), whereas for uptake of PA contradicting conclusions have been drawn (Fernández-Cañón et al, 1989a;Hillenga et al, 1995). Fernández-Cañón et al (1989a,b) suggested that an inducible and energy-dependent specific PA-transport system is present in P. chrysogenum Wisconsin 54-1255.…”

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

“…With respect to nitrate it is assumed that uptake is similar to nitrate uptake in Neurospora crassa; that is, active transport through proton symport with a stoichiometry of 2H + per NO 3 − anion transported across the membrane (Blatt et al, 1997). Transport of phenylacetic acid in P. chrysogenum has been reported to occur through passive diffusion (Hillenga et al, 1995).…”