The discovery and industrial production of penicillin G by high-yielding
Penicillin G is synthesized by cellular condensation of activated L-α-aminoadipic acid (A), L-cysteine (C) and L-valine (V) to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV), formed by the ACV synthetase (ACVS) – encoded by the
Peroxisomes also known as microbodies are single bilayer membrane bound highly dynamic organelles ubiquitous to most eukaryotic cells. The peroxisomal membrane is a dynamic structure modified according to the environmental needs. In contrast to the nucleus, mitochondria, or chloroplasts, peroxisomes do not contain DNA. Proliferation by division of preexisting organelles and the role of endoplasmic reticulum in the biogenesis of these organelles is now well established [11, 15, 16]. Mitochondria are also involved in the biogenesis of the peroxisomal membrane . It has long been regarded that the primary function of fungal peroxisomes is limited to the β-oxidation of fatty acids. However, studies in filamentous fungi have revealed that peroxisomes have diverse functional activities. A variety of fungal metabolites are at least partially synthesized inside peroxisomes, including different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters. Peroxisomes are versatile organelles that play an important role in the growth and survival processes of filamentous fungi, e.g. Woronin body that plugs the septal pore upon hyphal damage to prevent excessive cytoplasmic loss. A number of reports have demonstrated that Woronin bodies are derived from peroxisomes . Peroxisomes contain a variety of proteins depending on the organism. These organelles accumulate toxic metabolites and act as a barrier protecting the cytosol from the hazardous compounds by degrading of toxic metabolites. Peroxisome acts as a waste furnace of toxic organic compounds, where they are oxidated, and secreted out of the cells coupled to carrier molecules  or in industrial amounts by pexophage and exocytosis [11, 15, 16]. A good example is the process of penicillin G biosynthesis and its secretion from the mycelial cells of
The immunoelectron microscopic arrangement of IPNS in apical, young sub-apical, mature (adult) subapical, and senescing/degrading hyphal cells of the tested industrial mycelium is summarized in Fig. 1. In the hyphal apex a total lack of the immunlabel of IPNS could be observed (Fig. 1A) which was more visible in the young sub-apical cytosol (Fig. 1B). In mature productive non-growing highly vacuolated cells the immune-gold localization of IPNS was mainly concentrated in channel-like structures of the cell wall and at the periphery of the cytoplasm as well as around the vacuoles. The immune-gold marker of IPNS was also abundantly arranged at polyribosomes surrounding the peroxisomes (Fig. 1C). Such a cellular co-localization of cytosolic IPNS with the peroxisomal IAT and PCL might be a precisely adopted structural arrangement enabling the withdrawal of ACV substrate of IPNS immediately from the fermentation broth and from the cytoplasm as well as from the vacuolar pool to increase the efficacy and yield in penicillin G biosynthesis. In line with this argumentation, it was recently found that ACV is present in the cytosol and vacuoles, and also accumulates in the fermentation broth [17, 25]. In senescing cells, the immunelabel of IPNS was rarely located at the periphery of the vacuoles and in the cytosol (Fig. 1D). Control experiments have been performed in order to check the specificity of immunegold-labeling. The control sample with IgG-gold conjugate alone was essentially devoid of label. The control sample incubated with pre-immunoserum, followed by goat anti-rabbit IgG – 15 nm gold conjugate exhibited only very light labeling of the hyphal cell . In the mature hyphal cells of the low-penicillin-producing strain
The IAT is entrapped in peroxisomes of penicillin G yielding strains . For this reason its ultrastructural location refers to the peroxisome deployment in the hyphal cells (Fig. 2). In sections through the hyphal apex typical tip bodies (Spitzenkörper) composed of small vesicles could be observed. The apical 1.0–3.0 μm is occupied by ribosomes and mitochondria. This hyphal region is characterized by the electron dense cytoplasm, lack of peroxisomes and vacuoles as well as cross-walls (Fig. 2A). The young sub-apical hyphal cells are densely packed with ribosomes and mitochondria. Abundant
Pexophagy is a pathway in which peroxisomes are degraded inside of vacuoles in response to specific environmental conditions. The last step in penicillin G production is located in peroxisome (Fig. 3). Secretion of this antibiotic in industrial scale from the peroxisomes across the plasma membrane is poorly understood  and requires further explanation [20–22, 26, 27]. The lack of clear evidence that any of the ABC transporters are involved in penicillin G secretion is at present intriguing. It may indicate that the ABC transport is not enough sufficient or the antibiotic secretion does not proceed through the classical ABC pumps. The results of our experiments exhibit that the abundant pexophagy and exocytosis characterized by large vacuolar budding as well as the presence of numerous vacuolar vesicles which fuse with the plasma membrane are important structural features characterizing the non-growing productive cells of the high-yielding strain [9, 11, 15, 16]. This structural arrangement is closely combined with the period of high penicillin of G secretion in an industrial scale. The vacuolar pH of about 5 is suitable for the stability of penicillin G. The abundant pexophagy and exocytoses could not be observed in the mature cells of the low-penicillin-producing strain
The overproduction of penicillin G is associated with a strictly adjusted cellular organization. The young sub-apical and mature non-growing peroxisomal cells of the industrial hyphae are privileged in overproduction of penicillin G. The co-location of IPNS and peroxisomes at the periphery of the cells and around the vacuoles may increase the enzyme supplying efficacy in penicillin G biosynthesis from the fermentation broth and from the cytosol as well as from the vacuolar pool. In penicillin G biosynthesis the structurally grouped organelles build a well organized assembly line composed of cytosol concentrated and membrane encompassed enzymes, substrates, intermediates, precursors (PA, A, C, V) side- and end-products. Penicillin G biosynthesis in an industrial scale is a cellular detoxification process protecting the mycelial cell from the toxicity of the PA. Pexophagy and exocytoses should be currently considered in large-scale secretion of penicillin G as a putative alternative for active secretion by the ABC transporters. The novelty of described data is the differentiation of the hyphal cells in penicillin G biosynthesis and the discovered collocation of IPNS and peroxisomes. It is important, because the knowledge concerned with the cellular arrangements in overproduction of penicillin G is of great economical importance.