New Data On Isopenicillin N Synthase And Peroxisome Co-Location In The Hyphal Cells Of Penicillium Chrysogenum PQ-96 – Pexophagy And Exocytosis

The discovery and industrial production of penicillin G by high-yielding Penicillium chrysogenum strains was a great success which allowed to open the era of antibiotic therapy. Penicillin G has saved millions of human beings from annihilation. The β-lactams are some of the oldest and most widely used antibiotics in human society. In industrial strains penicillin G is secreted in amounts of 45–50 g per liter of the fermentation broth. The biosynthesis and secretion of such unnatural amounts of this antibiotic requires a specially adopted ultra-structural organization of the industrial mycelium. Schematic arrangement of penicillin G biosynthesis in hyphal cells of P. chrysogenum was presented previously [1–16].

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 pcbAB gene. The ACV tripeptide is converted to isopenicillin N (IPN) by action of IPN synthase (IPNS) – encoded by the pcbC gene. In this oxidative ring closure reaction the bi-cyclic penam nucleus is formed consisting of β-lactam and thiazolidine rings [27]. In the last step, the CoA:isopenicillin N acyltransferase (IAT) – encoded by the penDE gene synthesizes penicillin G by substitution of the L-α-aminoadipyl side chain of IPN by the phenylacetyl side chain. The phenylacetic acid (PA) which is the precursor of penicillin G biosynthesis requires previous activation by specific phenylacetyl-CoA ligase (PCL) – encoded by phl gene [27]. The ACVS and IPNS are cytosolic enzymes, and IAT as well as PCl are peroxisome entrapped enzymes [25].

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 [23]. 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 [19]. 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 [18] 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 P. chrysogenum into the fermentation broth. In high concentrations PA is toxic to the mycelia cells [18]. During industrial production of penicillin G, PA is fed in appropriate amounts to the fermentation broth to avoid its toxic effect [4]. The fungal cytoplasmic acidification results in the inhibition of enzymatic reactions and amino acid transport, leading to cell death. The last step of penicillin G biosynthesis is located in peroxisomes where the PA is coupled to 6-aminopenicillanic acid. It suggests that the penicillin G biosynthesis is a cytosol detoxification process. It was also suggested that the large peroxisomes abundantly arranged at the periphery of the cytoplasm (Fig. 2C) seem to build a PA-barrier protecting the hyphal cells from the toxicity of the precursor of penicillin G biosynthesis [15, 16]. Based on electron microscopy and biochemical data, there are now solid evidences confirming that IAT and PA are cumulated in peroxisomes [18]. The remarkable variability of activities suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive [2, 24].

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 [14]. In the mature hyphal cells of the low-penicillin-producing strain P. chrysogenum Q-176 the large peroxisomes and pexophagy as well as exocytoses could be observed much less often.

Fig. 1.

The IAT is entrapped in peroxisomes of penicillin G yielding strains [18]. 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 de novo peroxisome formation in association with osmophilic membranes is characteristic for this hyphal region (Fig. 2B). A lack of large vacuoles and mature cross-walls associated with Woronon bodies characterizes this region. In mature productive non-growing highly vacuolated cells, the polyribosomes are frequently located at the periphery of the cytoplasm and in the neighborhood of the vacuolar tonoplast as well as around the peroxisomes. The membranes of the endoplasmic reticulum abundantly transform into peroxisomes from 0.1 μm up to 1.0 μm in diameter. Moreover, peroxisome multiplication by fission of preexisting organelles is also a significant feature of the productive non-growing mycelial cells of the high-yielding strain. These cells exhibited numerous large peroxisomes frequently arranged at the periphery of the cytoplasm and around the vacuoles (Fig. 2C). Massive pexophagy and exocytosis was detected predominantly in the latemature highly vacuolated cells. The mature cross-walls are accompanied by Woronin bodies. In senescing cells very large vacuoles could be observed (Fig. 2D). The particular cells of the hyphae are separated by mature cross-walls which are accompanied by Woronin bodies. In the cells an advanced degradation process was detected, including pexophagy and mitophagy.

Fig. 2.

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 [18] 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 P. chrysogenum Q-176. It suggests that the pexophagy and exocytosis might be directly involved in penicillin secretion by industrial strains.

Fig. 3.

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.