Pexophagy in Penicillin G Secretion by Penicillium chrysogenum PQ-96

Abstract

A b s t r a c tPenicillin G oversecretion by Penicillium chrysogenum PQ96 is associated with a strictly adjusted cellular organization of the mature and senescent mycelial cells. Abundant vacuolar phagy and extended cellular vacuolization combined with vacuolar budding resulting in the formation of vacuolar vesicles that fuse with the cell membrane are the most important characteristic features of those cells. We suggest as follows: if the peroxisomes are integrated into vacuoles, the penicillin G formed in peroxisomes might be transferred to vacuoles and later secreted out of the cells by an exocytosis process. The peroxisomal cells of the mycelium are privileged in penicillin G secretion. led us to the conclusion that the immunogold marker of isopenicillin N synthase is abundantly arranged at polyribosomes surrounding the peroxisomes. Such a cellular accumulation of isopenicillin N synthase may enhance the selective, continuous and sufficient sub strate supply in penicillin G biosynthesis. It was recently found that δ(Lαaminoadipyl)LcysteinylDvaline tripeptide is present in the cytoplasm and accumulates in the fermentation medium to concentrations of up to 2 mM (for review see Kurzątkowski et al., 2014a). The affinity of isopenicillin N synthase for this tripeptide is in the submM range. The high concentration of the tripeptide in the fermentation broth might explain our unexpected results concerning the localization of isopenicillin N synthase at the periphery of the cyto plasm and in channellike structures of the cell wall. This location might be a precisely adopted structural arrangement enabling the withdrawal of the tripeptide from the fermentation broth and from the cytoplasm for the peripherally located isopenicillin N synthase to increase the efficacy and yield in penicillin G bio synthesis. On the contrary, in the mature nongrowing hyphal cells of the low penicillinproducing strain P. chrysogenum Q176 the total lack of peroxisomes about 0.1 μm in diameter were visible.Peroxisomes play a crucial role in the production of penicillin G and cephalosporin C by industrial strains (Kurzątkowski and GębskaKuczerowska, 2015). High penicillin G producing strains show increasing num bers of large peroxisomes mainly at the period of the Fig. 1. P. chrysogenum PQ96, 72 h culture, highpenicillin producing strain, activity of penicillin G biosynthesis (U/ml): total yield at 72 h of cultivation -8300 (4.98 g/1000 ml), increase of yield between 48 h and 72 h of fermentation -4850. The results of our experiments exhibit that the abundant vacuolar pexophagy of large peroxisomes combined with vacuolar budding and the presence of numerous vacuolar vesicles which fuse with the plasma membrane are the most important structural features characterizing the nongrowing productive cells as well as the lateapical degenerating highly vacuolated cells of the tested industrial strain. This structural arrange ment is closely combined with the period of largescale secretion of penicillin G. Such a cellular organization was not visible...

Detection of Penicillin G Produced byPenicillium chrysogenumKF 425 in Vivo with Raman Microspectroscopy and Multivariate Curve Resolution-Alternating Least Squares Methods

“…It is also known that increases in vacuoles and pexophagy (autophagy of peroxisomes) result in excessive secretion of penicillin. 28 For the secretion of penicillin G, a mechanism involving exocytosis has been proposed as follows: penicillin G is taken up into vacuoles by pexophagy, and vacuolar vesicles are formed, by vacuolar budding, which fuse to the cell membrane and are finally secreted via exocytosis. According to this hypothesis, penicillin G is contained and localized in compartments (peroxisomes, vacuoles, and vacuolar vesicles) within the mycelial body.…”

Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi

“…Similarly, production of the aflatoxins by Aspergillus parasiticus has been reported to be associated with specific cells in the filamentous hyphae [60,92]. Electron microscopy studies of the hyphae of P. chrysogenum in a high penicillin producing mutant as compared to a low producer strain showed that the active penicillin producing cells in the subapical region contain a high number of large peroxisomes and are partially vacuolated but in the old parts of the hyphae the cells are more highly vacuolated and do not produce penicillin [58]. The subapical penicillin producing cells contain isopenicillin N acyltransferase (IAT) in peroxisomes as shown by immunoelectron microscopy [32,81,82].…”

“…The subapical penicillin producing cells contain isopenicillin N acyltransferase (IAT) in peroxisomes as shown by immunoelectron microscopy [32,81,82]. All the available evidence suggests that the entire process of penicillin compartmentalization and transport between intracellular organelles and the cell membrane/cell wall is highly structured and has a precise spatial organization [58]. This article describes the present status of knowledge of the localization and organization of different penicillin and cephalosporin biosynthetic steps and the traffic systems between the different organelles including the controversial final penicillin secretion step.…”

“…However, protein-protein interaction studies are required to support this hypothesis. Recently, Kurzatkowski and Kuczerowska [58] using immunodetection of IPN synthase provided evidence indicating that this enzyme is indeed located in the cytoplasm but it is not uniformly distributed; rather, it localizes in a few specific subcellular sites in the cytosol; namely the IPN synthase is associated with the endoplasmic reticulum and strongly concentrated around the peroxisomes and near the vacuole tonoplasts. In addition, a surprising observation of these authors revealed that some IPN synthase is present in tube-like structures in the cell wall.…”

“…The autophagy process affects different organelles, in particular the autophagy of peroxisomes is named pexophagy. If peroxisomes are engulfed by a pexophagy mechanism the penicillin and the biosynthetic enzymes will be discharged into the vacuoles [58]. Using electron transmision macroscopy, Kurzatkowski and Kuczerowska [58] have also observed the accumulation of debris material inside the vacuole tonoplasts and budding structures in the vacuoles that might be involve in exocytosis.…”