Selective and Accurate Quantification of N-Acetylglucosamine in Biotechnological Cell Samples via GC-MS/MS and GC-TOFMS.

N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived 13C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.

The filamentous fungus Penicillium chrysogenum analysed via flow cytometry-a fast and statistically sound insight into morphology and viability.

Filamentous fungi serve as production host for a number of highly relevant biotechnological products, like penicillin. In submerged culture, morphology can be exceptionally diverse and is influenced by several process parameters, like aeration, agitation, medium composition or growth rate. Fungal growth leads to several morphological classes encompassing homogeneously dispersed hyphae and various forms of hyphal agglomerates and/or clump structures. Eventually, the so-called pellet structure can be formed, which represents a hyphal agglomerate with a dense core. Pellet structures can hinder oxygen and substrate transport, resulting in different states of viability, which in turn affects productivity and process control. Over the years, several publications have dealt with methods to either gain morphological insight into pellet structure or determine biomass viability. Within this contribution, we present a way to combine both in a flow cytometry-based method employing fluorescent staining. Thereby, we can assess filamentous biomass in a statistically sound way according to (i) morphology and (ii) viability of each detected morphological form. We are confident that this method can shed light on the complex relationship between fungal morphology, viability and productivity-in both process development and routine manufacturing processes.

A 9-pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum.

A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.

Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry-the fast alternative to microscopic image analysis.

An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments.

Pexophagy in Penicillin G Secretion by Penicillium chrysogenum PQ-96.

Penicillin G oversecretion by Penicillium chrysogenum PQ-96 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.

Intact cell mass spectrometry as a progress tracking tool for batch and fed-batch fermentation processes.

Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth. After the optimization of the ICMS method to penicillin V fermentation broth samples, the obtained ICMS data were evaluated by hierarchical cluster analysis or an in-house software solution written especially for ICMS data comparison. Growth stages of a batch and fed-batch fermentation of Penicillium chrysogenum are differentiated by one of those statistical approaches. The application of two matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instruments in the linear positive ion mode from different vendors demonstrated the universal applicability of the developed ICMS method. The base for a fast and easy-to-use method for monitoring the fermentation progress of P. chrysogenum is created with this ICMS method developed especially for fermentation broth samples.

Improved sample preparation for intact cell mass spectrometry (biotyping) of mycelium samples taken from a batch fermentation process of Penicillium chrysogenum.

RATIONALE: Penicillium chrysogenum is an important species in biotechnology and an improved production rate for penicillin drug variants is of utmost interest. Intact cell mass spectrometry (ICMS) or biotyping can be a novel and time-saving tool to monitor a fermentation process of Penicillium strains for fast intervention during penicillin production. METHODS: Fermentation broth was collected directly from a fermenter at specific time points known to show significantly different penicillin production rates. The mycelium was purified by washing multiple times with water and recovered by centrifugation. The mycelium was further mixed with matrix-assisted laser desorption/ionization (MALDI) MS matrix and immediately spotted on different types of targets. ICMS spectra were obtained by MALDI time-of-flight (TOF) MS in the positive ion linear mode in the m/z range 3000 to 16 000. RESULTS: An ICMS method for culture broth samples of P. chrysogenum was developed. It was shown that ferulic acid mixed with sinapinic acid (2.5 mg and 22.5 mg/mL) is the most appropriate matrix combination. The matrices were dissolved in acetonitrile/0.1% trifluoroacetic acid (70/30, v/v) and spotted together with the sample on various target types. Sample preparation was thoroughly studied for homogeneity and reproducibility. CONCLUSIONS: Culture broth directly collected from a bioreactor could be analyzed applying the optimized approach. The ideal choice of matrix, the adequate preparation technique and the type of target were the focus of this work showing that samples collected at different times during fermentation exhibit a characteristic pattern using the developed method.

Combining light microscopy, dielectric spectroscopy, MALDI intact cell mass spectrometry, FTIR spectromicroscopy and multivariate data mining for morphological and physiological bioprocess characterization of filamentous organisms.

Along with productivity and physiology, morphological growth behavior is the key parameter in bioprocess design for filamentous fungi. Lacking tools for fast, reliable and efficient analysis however, fungal morphology is still commonly tackled by empirical trial-and-error techniques during strain selection and process development procedures. Bridging the gap, this work presents a comprehensive analytical approach for morphological analysis combining automated high-throughput microscopy, multi-frequency dielectric spectroscopy, MALDI intact cell mass spectrometry and FTIR spectromicroscopy. Industrial fed-batch production processes were investigated in fully instrumented, automated bioreactors using the model system Penicillium chrysogenum. Physiological process characterization was based on the determination of specific conversion rates as scale-independent parameters. Conventional light microscopic morphological analysis was based on holistic determination of time series for more than 30 morphological parameters and their frequency distributions over the respective parameter range by automated high-throughput light microscopy. Characteristic protein patterns enriched in specific morphological and physiological states were further obtained by MALDI intact cell mass spectrometry. Spatial resolution of molecular biomass composition was facilitated by FTIR spectromicroscopy. Real-time in situ monitoring of morphological process behavior was achieved by linking multi-frequency dielectric spectroscopy with above outlined off-line methods. Data integration of complementing orthogonal techniques for morphological and physiological analysis together with multivariate modeling of interdependencies between morphology, physiology and process parameters facilitated complete bioprocess characterization. The suggested approach will thus help understanding morphological and physiological behavior and, in turn, allow to control and optimize those complex processes.

Quantitative quenching evaluation and direct intracellular metabolite analysis in Penicillium chrysogenum.

Sustained progress in metabolic engineering methodologies has stimulated new efforts toward optimizing fungal production strains such as through metabolite analysis of Penicillium chrysogenum industrial-scale processes. Accurate intracellular metabolite quantification requires sampling procedures that rapidly stop metabolism (quenching) and avoid metabolite loss via the cell membrane (leakage). When sampling protocols are validated, the quenching efficiency is generally not quantitatively assessed. For fungal metabolomics, quantitative biomass separation using centrifugation is a further challenge. In this study, P. chrysogenum intracellular metabolites were quantified directly from biomass extracts using automated sampling and fast filtration. A master/slave bioreactor concept was applied to provide industrial production conditions. Metabolic activity during sampling was monitored by 13C tracing. Enzyme activities were efficiently stopped and metabolite leakage was absent. This work provides a reliable method for P. chrysogenum metabolomics and will be an essential base for metabolic engineering of industrial processes.

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum.

Chitin synthases catalyze the formation of ß-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.

Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene.

Chitin synthases, that catalyze the formation of chitin the major component of cell walls in most filamentous fungi, play crucial roles in the growth and morphogenesis. To investigate the roles of chitin synthase in Penicillium chrysogenum, we developed an RNAi system to silence the class III chitin synthase gene chs4. After transformation, mutants had a slow growth rate and shorter but highly branched hyphae. All transformants either were unable to form conidia or could form only a few. Changes in chs4 expression could lead to a completely different morphology and eventually cause distinct penicillin yields. In particular, the yield of one transformant was 41 % higher than that of the original strain.

Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in deltalig4 or deltaku70 mutants.

Inactivating the non-homologous end-joining (NHEJ) pathway is a well established method to increase gene targeting (GT) efficiencies in filamentous fungi. In this study we have compared the effect of inactivating the NHEJ genes ku70 or lig4 on GT in the industrial penicillin producer Penicillium chrysogenum. Deletion of both genes resulted in strongly increased GT efficiencies at three different loci but not higher than 70%, implying that other, yet uncharacterized, recombination pathways are still active causing a part of the DNA to be integrated via non-homologous recombination. To further increase the GT efficiency we applied the bi-partite approach, in which the DNA fragment for integration was split in two non-functional overlapping parts that via homologous recombination invivo can form a functional selection marker. The combined NHEJ mutant and bi-partite approach further increased GT frequencies up to approximately 90%, which will enable the efficient high throughput engineering of the P. chrysogenum genome. We expect that this combined approach will function with similar high efficiencies in other filamentous fungi.

Applicability of Penicillium chrysogenum rheological correlations to broths of other fungal strains.

Existing correlations of Power law consistency index with Penicillium chrysogenum biomass concentration and morphology were revised using a microscope magnification of 50 times to characterize the latter, rather than the 80 times used previously. This allowed tests of the correlations on broths of Aspergillus oryzae and Aspergillus niger, which have such large mycelial sizes that a lower magnification is required for accurate morphological analysis. The new correlations were successful at predicting the rheology of A. oryzae broths but not A. niger broths, which may be because of a change in the exponent on the biomass concentration in the correlations for the latter. Because the mean maximum dimension of clumps is magnification independent, the preferred correlation was K = C²m x [4 x 10⁻5D - 9 x 10⁻4] where K is the consistency index (Pa s(n)), C(m) is the biomass concentration as dry cell weight (g l(-1)) and D is the mean maximum dimension (µm).

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Antifungal and anti-mycotoxin efficacy of biogenic silver nanoparticles produced by Fusarium chlamydosporum and Penicillium chrysogenum at non-cytotoxic doses.

The cell-free culture filtrate (CFF) of the fungi Fusarium chlamydosporum NG30 and Penicillium chrysogenum NG85 was tested to synthesize silver nanoparticles (AgNPs). The synthesized AgNPs were further characterized by means of transmission electron microscopy (TEM), dynamic light scattering (DLS) and Fourier transform infra-red (FTIR) spectroscopy. TEM revealed their spherical shape, homogeneity and a size range between 6 and 26 nm for F. chlamydosporum AgNPs (FAgNPs) and from 9 to 17.5 nm for P. chrysogenum AgNPs (PAgNPs). DLS showed that the diameter of FAgNPs was narrower than that of PAgNPs. FTIR spectroscopy indicated that the functional groups present in the CFF might be responsible for the reduction of silver ions to form stabilized protein-capped AgNPs. In addition, the AgNPs showed notable antifungal activity and potency in thwarting mycotoxin production. Thus, using Aspergillus flavus as a test microorganism the minimum inhibitory concentration (MIC) was 48, 45 and 50 µg/mL for FAgNPs, PAgNPs and the antifungal compound itraconazole, respectively. Also, when testing Aspergillus ochraceus FAgNPs, PAgNPs and itraconazole led to MIC values of 51, 47 and 49 µg/mL, respectively. The statistical MIC values to inhibit completely the total aflatoxin production by A. flavus were 5.9 and 5.6 µg/mL for FAgNPs and PAgNPs, respectively, and to inhibit the ochratoxin A production by A. ochraceus 6.3 and 6.1 µg/mL for FAgNPs and PAgNPs, respectively. The cytotoxicity assay of the AgNPs on human normal melanocytes (HFB 4) revealed a cell survival of 80% and 75% at a concentration of 6 µg/mL for FAgNPs and PAgNPs, respectively.

Heterotrimeric Galpha protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism.

Fungal heterotrimeric G proteins regulate different processes related to development, such as colony growth and asexual sporulation, the main mechanism of propagation in filamentous fungi. To gain insight into the mechanisms controlling growth and differentiation in the industrial penicillin producer Penicillioum chrysogenum, we investigated the role of the heterotrimeric Galpha subunit Pga1 in conidiogenesis. A pga1 deleted strain (Deltapga1) and transformants with constitutively activated (pga1G42R) and inactivated (pga1G203R) Pga1 alpha subunits were obtained. They showed phenotypes that clearly implicate Pga1 as an important negative regulator of conidiogenesis. Pga1 positively affected the level of intracellular cAMP, which acts as secondary messenger of Pga1-mediated signalling. Although cAMP has some inhibitory effect on conidiation, the regulation of asexual development by Pga1 is exerted mainly via cAMP-independent pathways. The regulation of conidiation by Pga1 is mediated by repression of the brlA and wetA genes. The Deltapga1 strain and transformants with the constitutively inactive Pga1G203R subunit developed a sporulation microcycle in submerged cultures triggered by the expression of brlA and wetA genes, which are deregulated in the absence of active Pga1. Our results indicate that although basic mechanisms for regulating conidiation are similar in most filamentous fungi, there are differences in the degree of involvement of specific pathways, such as the cAMP-mediated pathway, in the regulation of this process.

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing.

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (beta subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.

Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro.

BACKGROUND: Invasive aspergillosis, which is mainly caused by the fungus Aspergillus fumigatus, is an increasing problem in immunocompromised patients. Infection occurs by inhalation of airborne conidia, which are first encountered by airway epithelial cells. Internalization of these conidia into the epithelial cells could serve as a portal of entry for this pathogenic fungus. RESULTS: We used an in vitro model of primary cultures of human nasal epithelial cells (HNEC) at an air-liquid interface. A. fumigatus conidia were compared to Penicillium chrysogenum conidia, a mould that is rarely responsible for invasive disease. Confocal microscopy, transmission electron microscopy, and anti-LAMP1 antibody labeling studies showed that conidia of both species were phagocytosed and trafficked into a late endosomal-lysosomal compartment as early as 4 h post-infection. In double immunolabeling experiments, the mean percentage of A. fumigatus conidia undergoing phagocytosis 4 h post-infection was 21.8 +/- 4.5%. Using combined staining with a fluorescence brightener and propidium iodide, the mean rate of phagocytosis was 18.7 +/- 9.3% and the killing rate 16.7 +/- 7.5% for A. fumigatus after 8 h. The phagocytosis rate did not differ between the two fungal species for a given primary culture. No germination of the conidia was observed until 20 h of observation. CONCLUSION: HNEC can phagocytose fungal conidia but killing of phagocytosed conidia is low, although the spores do not germinate. This phagocytosis does not seem to be specific to A. fumigatus. Other immune cells or mechanisms are required to kill A. fumigatus conidia and to avoid further invasion.

Isolation and identification of Penicillium chrysogenum strain Y5 and its copper extraction characterization from waste printed circuit boards.

Biohydrometallurgy is generally considered as a green technology for the recycling of industrial solid waste. In this study, an indigenous fungal strain named Y5 with the ability of high-yielding organic acids was isolated and applied in bioleaching of waste printed circuit boards (PCBs). The strain Y5 was identified as Penicillium chrysogenum by morphological and molecular identification. Meanwhile, we investigated that an optimal set of culturing conditions for the fungal growth and acids secretion was 15 g/L glucose with initial pH 5.0, temperature 25°C and shaking speed 120 rpm in shaken flasks culture. Moreover, three bioleaching processes such as one-step, two-step and spent medium processes were conducted to extract copper from waste PCBs. Spent medium bioleaching showed higher copper extraction percentage and it was 47% under 5%(w/v) pulp density. Transmission electron microscope (TEM) observation combining with energy dispersive analysis of X-rays (EDAX) showed that the leached metal ions did not obviously damage the hypha cells. All above results indicated that P.chrysogenum strain Y5 has the tolerance to metal ions, suggesting its potential in recycling of metals from waste PCBs in industry.

Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.