Genomic insights into Penicillium chrysogenum adaptation to subseafloor sedimentary environments.

BACKGROUND: Penicillium chrysogenum is a filamentous fungal species with diverse habitats, yet little is known about its genetics in adapting to extreme subseafloor sedimental environments. RESULTS: Here, we report the discovery of P. chrysogenum strain 28R-6-F01, isolated from deep coal-bearing sediments 2306 m beneath the seafloor. This strain possesses exceptional characteristics, including the ability to thrive in extreme conditions such as high temperature (45 °C), high pressure (35 Mpa), and anaerobic environments, and exhibits broad-spectrum antimicrobial activity, producing the antibiotic penicillin at a concentration of 358 µg/mL. Genome sequencing and assembly revealed a genome size of 33.19 Mb with a GC content of 48.84%, containing 6959 coding genes. Comparative analysis with eight terrestrial strains identified 88 unique genes primarily associated with penicillin and aflatoxins biosynthesis, carbohydrate degradation, viral resistance, and three secondary metabolism gene clusters. Furthermore, significant expansions in gene families related to DNA repair were observed, likely linked to the strain's adaptation to its environmental niche. CONCLUSIONS: Our findings provide insights into the genomic and biological characteristics of P. chrysogenum adaptation to extreme anaerobic subseafloor sedimentary environments, such as high temperature and pressure.

Penicillium chrysogenum: Beyond the penicillin.

Almost one century after the Sir Alexander Fleming's fortuitous discovery of penicillin and the identification of the fungal producer as Penicillium notatum, later Penicillium chrysogenum (currently reidentified as Penicillium rubens), the molecular mechanisms behind the massive production of penicillin titers by industrial strains could be considered almost fully characterized. However, this filamentous fungus is not only circumscribed to penicillin, and instead, it seems to be full of surprises, thereby producing important metabolites and providing expanded biotechnological applications. This review, in addition to summarizing the classical role of P. chrysogenum as penicillin producer, highlights its ability to generate an array of additional bioactive secondary metabolites and enzymes, together with the use of this microorganism in relevant biotechnological processes, such as bioremediation, biocontrol, production of bioactive nanoparticles and compounds with pharmaceutical interest, revalorization of agricultural and food-derived wastes or the enhancement of food industrial processes and the agricultural production.

Secondary metabolites from marine fungus Penicillium chrysogenum VH17 and their antimicrobial and cytotoxic potential.

One new compound, methyl 3-((1-((2-carbamoylphenyl)amino)-1-oxopropan-2-yl)amino)-3-oxopropanoate (1), along with nine known secondary metabolites (2-10) were isolated and elucidated chemical structures from the methanol extract of the marine-derived fungus Penicillium chrysogenum VH17. Subsequent bioassays showed the antimicrobial and cytotoxic potential of the isolated compounds. All compounds 1-10 displayed antimicrobial effects against at least one tested reference microorganism with MIC values ranging from 32 to 256 µg mL-1. Furthermore, compound 4 exhibited significant cytotoxicity against all tested cell lines HepG2, A549, and MCF7 with IC50 values of 29.43 ± 1.37, 33.02 ± 1.53, and 36.72 ± 1.88 µM, respectively, whereas compound 3 exhibited weak cytotoxicity against MCF7 and HepG2 cell lines with IC50 values of 87.17 ± 6.31 and 97.32 ± 5.66 µM, respectively.

A comprehensive review on the medicinally valuable endosymbiotic fungi Penicillium chrysogenum.

Recently, it has been shown that metabolites derived from endosymbiotic fungi attracted high attention, since plenty of them have promising pharmaceutical applications. The variation of metabolic pathways in fungi is considered an optimistic source for lead compounds. Among these classes are terpenoids, alkaloids, polyketides, and steroids, which have proved several pharmacological activities, including antitumor, antimicrobial, anti-inflammatory, and antiviral actions. This review concludes the major isolated compounds from different strains of Penicillium chrysogenum during the period 2013-2023, together with their reported pharmacological activities. From literature surveys, 277 compounds have been identified from P. chrysogenum, which has been isolated as an endosymbiotic fungus from different host organisms, with specific attention paid to those showing marked biological activities that could be useful in the pharmaceutical industry in the future. This review represents documentation for a valuable reference for promising pharmaceutical applications or further needed studies on P. chrysogenum.

Penicillium chrysogenum as a fungal factory for feruloyl esterases.

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus.

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MICS of 1-8 µg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.

Biosynthetic process and strain improvement approaches for industrial penicillin production.

Penicillins and cephalosporins are the most important class of beta (ß) lactam antibiotics, accounting for 65% total antibiotic market. Penicillins are produced by Penicillium rubens (popularly known as P. chrysogenum) were used to synthesize the active pharmaceutical intermediate (API), 6-aminopenicillinic acid (6-APA) employed in semisynthetic antibiotic production. The wild strains produce a negligible amount of penicillin (Pen). High antibiotic titre-producing P. chrysogenum strains are necessitating for industrial Pen production to meet global demand at lower prices. Classical strain improvement (CSI) approaches such as random mutagenesis, medium engineering, and fermentation are the cornerstones for high-titer Pen production. Since, Sir Alexander Fleming Discovery of Pen, great efforts are expanded to develop at a commercial scale antibiotics producing strains. Breakthroughs in genetic engineering, heterologous expression and CRISPR/Cas9 genome editing tools opened a new window for Pen production at a commercial scale to assure health crisis. The current state of knowledge, limitations of CSI and genetic engineering approaches to Pen production are discussed in this review.

Metabolic Profiling and In Vitro Assessment of the Biological Activities of the Ethyl Acetate Extract of Penicillium chrysogenum "Endozoic of Cliona sp. Marine Sponge" from the Red Sea (Egypt).

Marine sponge-derived endozoic fungi have been gaining increasing importance as promising sources of numerous and unique bioactive compounds. This study investigates the phytochemical profile and biological activities of the ethyl acetate extract of Penicillium chrysogenum derived from Cliona sp. sponge. Thirty-six compounds were tentatively identified from P. chrysogenum ethyl acetate extract along with the kojic acid (KA) isolation. The UPLC-ESI-MS/MS positive ionization mode was used to analyze and identify the extract constituents while 1D and 2D NMR spectroscopy were used for kojic acid (KA) structure confirmation. The antimicrobial, antioxidant, and cytotoxic activities were assessed in vitro. Both the extract and kojic acid showed potent antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC 250 ± 0.82 µg/mL. Interestingly, the extract showed strong antifungal activity against Candida albicans and Cryptococcus neoformans with MIC 93.75 ± 0.55 and 19.53 ± 0.48 µg/mL, respectively. Furthermore, KA showed the same potency against Fusarium oxysporum and Cryptococcus neoformans with MIC 39.06 ± 0.85 and 39.06 ± 0.98 µg/mL, respectively. Ultimately, KA showed strong antioxidant activity with IC50 33.7 ± 0.8 µg/mL. Moreover, the extract and KA showed strong cytotoxic activity against colon carcinoma (with IC50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively) and human larynx carcinoma (with equal IC50 30.8 ± 1.3 and ± 2.1 µg/mL, respectively), respectively. The current study represents the first insights into the phytochemical profile and biological properties of P. chrysoenum ethyl acetate extract, which could be a promising source of valuable secondary metabolites with potent biological potentials.

Antimicrobial Activity of Dihydroisocoumarin Isolated from Wadi Lajab Sediment-Derived Fungus Penicillium chrysogenum: In Vitro and In Silico Study.

Antibiotic resistance is considered a major health concern globally. It is a fact that the clinical need for new antibiotics was not achieved until now. One of the most commonly prescribed classes of antibiotics is ß-Lactam antibiotics. However, most bacteria have developed resistance against ß-Lactams by producing enzymes ß-Lactamase or penicillinase. The discovery of new ß-Lactamase inhibitors as new antibiotics or antibiotic adjuvants is essential to avoid future catastrophic pandemics. In this study, five dihydroisocoumarin: 6-methoxy mellein (1); 5,6-dihydroxymellein (2); 6-hydroxymellein (3); 4-chloro-6-hydroxymellein (4) and 4-chloro-5,6-di-hydroxymellein (5) were isolated from Wadi Lajab sediment-derived fungus Penicillium chrysogenum, located 15 km northwest of Jazan, KSA. The elucidation of the chemical structures of the isolated compounds was performed by analysis of their NMR, MS. Compounds 1-5 were tested for antibacterial activities against Gram-positive and Gram-negative bacteria. All of the compounds exhibited selective antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Bacillus licheniformis except compound 3. The chloro-dihydroisocoumarin derivative, compound 4, showed potential antimicrobial activities against all of the tested strains with the MIC value between 0.8-5.3 µg/mL followed by compound 5, which exhibited a moderate inhibitory effect. Molecular docking data showed good affinity with the isolated compounds to ß-Lactamase enzymes of bacteria; NDM-1, CTX-M, OXA-48. This work provides an effective strategy for compounds to inhibit bacterial growth or overcome bacterial resistance.

Production, Bioprocessing and Anti-Proliferative Activity of Camptothecin from Penicillium chrysogenum, "An Endozoic of Marine Sponge, Cliona sp.", as a Metabolically Stable Camptothecin Producing Isolate.

Exploring the metabolic potency of fungi as camptothecin producers raises the hope of their usage as an industrial source of camptothecin, due to their short-life span and the feasibility of metabolic engineering. However, the tiny yield and loss of camptothecin productivity of fungi during storage and sub-culturing are challenges that counteract this approach. Marine fungi could be a novel source for camptothecin production, with higher yield and reliable metabolic sustainability. The marine fungal isolate Penicillium chrysogenum EFBL # OL597937.1 derived from the sponge "Cliona sp." has been morphologically identified and molecularly confirmed, based on the Internal Transcribed Spacer sequence, exhibiting the highest yield of camptothecin (110 µg/L). The molecular structure and chemical identity of P. chrysogenum derived camptothecin has been resolved by HPLC, FTIR and LC-MS/MS analyses, giving the same spectroscopic profiles and mass fragmentation patterns as authentic camptothecin. The extracted camptothecin displayed a strong anti-proliferative activity towards HEP-2 and HCT-116 (IC50 values 0.33-0.35 µM). The yield of camptothecin was maximized by nutritional optimization of P. chrysogenum with a Plackett-Burman design, and the productivity of camptothecin increased by 1.8 fold (200 µg/L), compared to control fungal cultures. Upon storage at 4 °C as slope culture for 8 months, the productivity of camptothecin for P. chrysogenum was reduced by 40% compared to the initial culture. Visual fading of the mycelial pigmentation of P. chrysogenum was observed during fungal storage, matched with loss of camptothecin productivity. Methylene chloride extracts of Cliona sp. had the potency to completely restore the camptothecin productivity of P. chrysogenum, ensuring the partial dependence of the expression of the camptothecin biosynthetic machinery of P. chrysogenum on the chemical signals derived from the sponge, or the associated microbial flora. This is the first report describing the feasibility of P. chrysogenum, endozoic of Cliona sp., for camptothecin production, along with reliable metabolic biosynthetic stability, which could be a new platform for scaling-up camptothecin production.

Tolerance of three fungal species to lithium and cobalt: Implications for bioleaching of spent rechargeable Li-ion batteries.

AIMS: This paper aims to quantify the growth and organic acid production of Aspergillus niger, Penicillium chrysogenum and Penicillium simplicissimum when these fungi are exposed to varying levels of lithium (Li) and cobalt (Co). The study also tests whether pre-exposing the fungi to these metals enables the fungi to develop tolerance to Li or Co. METHODS AND RESULTS: When cultures of A. niger, P. chrysogenum or P. simplicissimum were exposed to 250 mg l-1 of Li or Co, biomass production and excretion of organic acids were significantly inhibited after 5 days of growth compared to cultures grown in the absence of these metals. Pre-exposing cultures of A. niger to 250 mg l-1 of Li or Co for 20 days significantly increased biomass production when the fungus was subsequently sub-cultured into 250 or 500 mg l-1 of Li or Co. However, pre-exposure of P. chrysogenum or P. simplicissimum to 250 mg l-1 of Li or Co for 20 days did not increase biomass production. CONCLUSIONS: Aspergillus niger, but not the Penicillium species, developed tolerance to Li and to Co during the 20-day pre-exposure period. Therefore, processes that utilize fungal bioleaching with A. niger to mobilize and recover valuable metals such as Li or Co should consider a pre-exposure step for fungi to improve their tolerance to metal toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: Fungi may have the ability to extract valuable metals such as Li and Co from spent rechargeable batteries. However, the toxicity of the extracted metals can inhibit fungal growth and organic acid production. Pre-exposure to metals may alleviate toxicity for some fungal species. This knowledge can be used to improve the design of bioleaching protocols, increasing the potential for fungal bioleaching to become an economical and environmentally friendly method of recovering Li and Co from spent batteries.

Exploring the quorum sensing inhibition of isolated chrysin from Penicillium chrysogenum DXY-1.

We recently and for the first time reported that ethyl acetate extracts isolated from Penicillium chrysogenum DXY-1 exhibited anti-quorum sensing (anti-QS) activity. Herein, another active molecule in the extracts was identified as chrysin by NMR and MS. A 20 µg/mL dose of chrysin inhibited violacein production regulated by QS in C. violaceum CV026 by 31.6%. A 40 µg/mL dose of chrysin suppressed pyocyanin production, elastase activity, proteolytic activity, and biofilm formation regulated by QS in P. aeruginosa PA01 by 41.4%, 13.8%, 8.3%, and 42.4%, respectively. And chrysin could inhibit the swarming activity of P. aeruginosa PA01. Further, molecular docking and CD analysis were used to address the mechanism of chrysin's activity in C. violaceum. Molecular docking results revealed that chrysin suppresses QS system by competing with the natural signal molecule C6HSL for binding to the same pocket of CviR receptor. At the same time, CD results also showed that chrysin could change the secondary structure composition of CviR, which greatly prevented the binding of C6HSL/CviR, and further playing its role on inhibiting bacterial QS system. All these data demonstate that chrysin may be used as a potential QS inhibitor to tackle increasing drug resistance.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

Optimal process design space to ensure maximum viability and productivity in Penicillium chrysogenum pellets during fed-batch cultivations through morphological and physiological control.

BACKGROUND: Biomass growth of Pencillium chrysogenum is characterised by a distinct pellet morphology consisting of compact hyphal agglomerates. Fungal pellets are advantageous in industrial process control due to rheological advantages but lead to biomass degradation due to diffusional limitations of oxygen and substrate in the pellet's core. Several fermentation parameters are known to affect key pellet characteristics regarding morphology, viability and productivity. Pellet morphology and size are affected by agitation. Biomass viability and productivity are tightly interlinked with substrate uptake and dissolved oxygen concentration. RESULTS: The goal of this study was to study the impact of the fermentation parameters power input, dissolved oxygen content and specific substrate uptake rate on morphology, biomass viability and productivity. A design of experiments (DoE) approach was conducted and corresponding responses were analysed using novel morphological descriptors analysed by a previously established flow cytometry method. Results clearly display inverse correlations between power input and pellet size, specific morphological parameters related to pellet density can be increased in direct proportion to power input. Biomass viability and productivity are negatively affected by high specific substrate uptake rates. CONCLUSIONS: Based upon multiple linear regression, it was possible to obtain an optimal design space for enhanced viability and productivity at beneficial morphological conditions. We could maintain a high number of pellets with favourable morphology at a power input of 1500 W/m3. A sound compromise between viability and high productivity is possible at a specific glucose uptake rate of 0.043 g/g/h at dissolved oxygen levels of 40% minimum.

Study of metabolism and identification of productive regions in filamentous fungi via spatially resolved time-of-flight secondary ion mass spectrometry.

Filamentous fungi are well-established production hosts that feature a strong interconnection between morphology, physiology, and productivity. For penicillin production in Penicillium chrysogenum, industrial processes frequently favor a pellet morphology comprising compact hyphal agglomerates. Inherently these tightly packed entanglements lead to inactive, degrading sections within the pellet's core because of limitations. Optimal process design requires detailed knowledge of the nature of the limitations and localization of productive zones in the biomass, which is generally obtainable through modeling and complex analytical methods such as oxygen microelectrode and histological investigations. Methods that combine physiological and morphological insight are crucial yet scarce for filamentous fungi. In this study, we used time-of-flight secondary ion mass spectrometry in combination with oxygen and glucose tracer substrates, requiring little effort for sample preparation and measurement. Our method is capable of analyzing oxygen and substrate uptake in various morphological structures by the use of 18O as a tracer. In parallel, we can assess productive biomass regions through identification of penicillin mass fragments to simultaneously study oxygen diffusion, substrate incorporation, and productive biomass sections.

Protein tyrosine phosphatase 1B (PTP1B) inhibitorsfrom the deep-sea fungus Penicillium chrysogenum SCSIO 07007.

Three new compounds, including two new 3,4,6-trisubstituted α-pyrone derivatives, chrysopyrones A and B (1 and 2), and one new indolyl diketopiperazine derivative, penilline C (3), along with twelve known compounds (4-15), were isolated and identified from the fungus Penicillium chrysogenum SCSIO 07007, separated from deep-sea hydrothermal vent environment sample collected from the Western Atlantic. Their structures and absolute configurations were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. All of the isolated compounds (1-15) were evaluated for their cytotoxic, antibacterial activities and enzyme inhibitory activities against acetylcholinesterase (AChE), α-glycosidase, and protein tyrosine phosphatase 1B (PTP1B). Among them, new compounds chrysopyrones A and B (1 and 2) displayed obvious inhibitory activities against PTP1B with IC50 values of 9.32 and 27.8 µg/mL, respectively. Furthermore, molecular docking was performed to investigate the inside perspective of the action in PTP1B enzyme.

Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.

Cytotoxic Secondary Metabolites Isolated from the Marine Alga-Associated Fungus Penicillium chrysogenum LD-201810.

A new pentaketide derivative, penilactonol A (1), and two new hydroxyphenylacetic acid derivatives, (2'R)-stachyline B (2) and (2'R)-westerdijkin A (3), together with five known metabolites, bisabolane-type sesquiterpenoids 4-6 and meroterpenoids 7 and 8, were isolated from the solid culture of a marine alga-associated fungus Penicillium chrysogenum LD-201810. Their structures were elucidated based on extensive spectroscopic analyses, including 1D/2D NMR and high resolution electrospray ionization mass spectra (HRESIMS). The absolute configurations of the stereogenic carbons in 1 were determined by the (Mo2(OAc)4)-induced circular dichroism (CD) and comparison of the calculated and experimental electronic circular dichroism (ECD) spectra, while the absolute configuration of the stereogenic carbon in 2 was established using single-crystal X-ray diffraction analysis. Compounds 2 and 3 adapt the 2'R-configuration as compared to known hydroxyphenylacetic acid-derived and O-prenylated natural products. The cytotoxicity of 1-8 against human carcinoma cell lines (A549, BT-549, HeLa, HepG2, MCF-7, and THP-1) was evaluated. Compound 3 exhibited cytotoxicity to the HepG2 cell line with an IC50 value of 22.0 µM. Furthermore, 5 showed considerable activities against A549 and THP-1 cell lines with IC50 values of 21.2 and 18.2 µM, respectively.

Insight into the Genome of Diverse Penicillium chrysogenum Strains: Specific Genes, Cluster Duplications and DNA Fragment Translocations.

BACKGROUND: There are eighteen species within the Penicillium genus section chrysogena, including the original penicillin producers Penicillium notatum (Fleming strain) and Penicillium chrysogenum NRRL 1951. Other wild type isolates of the Penicillium genus are relevant for the production of useful proteins and primary or secondary metabolites. The aim of this article is to characterize strain specific genes and those genes which are involved in secondary metabolite biosynthesis, particularly the mutations that have been introduced during the ß-lactams strain improvement programs. RESULTS: The available genomes of several classical and novel P. chrysogenum strains have been compared. The first genome sequenced was that of the reference strain P. chrysogenum Wis54-1255, which derives from the wild type P. chrysogenum NRRL 1951; its genome size is 32.19 Mb and it encodes 12,943 proteins. Four chromosomes were resolved in P. chrysogenum and P. notatum by pulse field gel electrophoresis. The genomes of three industrial strains have a similar size but contain gene duplications and truncations; the penicillin gene cluster copy number ranges from one in the wild type to twelve in the P. chrysogenum ASP-E1 industrial strain and is organized in head to tail tandem repeats. The genomes of two new strains, P. chrysogenum KF-25, a producer of antifungal proteins isolated from a soil sample, and P. chrysogenum HKF2, a strain with carbohydrate-converting activities isolated from a sludge treatment plant, showed strain specific genes. CONCLUSIONS: The overall comparison of all available P. chrysogenum genomes indicates that there are a significant number of strain-specific genes, mutations of structural and regulatory genes, gene cluster duplications and DNA fragment translocations. This information provides important leads to improve the biosynthesis of enzymes, antifungal agents, prebiotics or different types of secondary metabolites.