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.


Introduction
The genus Penicillium includes four subgenera: Penicillium, Furcatum, Biverticillium, and Aspergilloides. Penicillium is the subgenus that has been examined the most from the genus of Penicillium fungi (Kumar et al. 2018).
It is an anamorphic ascomycete with over 354 species (Nielsen et al. 2017) and is also known for its diverse spectrum of bioactive secondary metabolites with potential pharmacological actions, including antifungal, antibacterial, immunosuppressant and cholesterol-lowering agents (Rabha and Jha 2018). Studies on Penicillium have shown that the species taxonomical classification idea, based not only on DNA sequences, but also on ecological, morphological, and exo-metabolome profiles, provides a more accurate and true classification (Barreto et al. 2011).
Marine-derived fungi from the Penicillium genus have gained attention as a beneficial source of new characteristic natural products with potential applications in industry, agriculture, and medicine (Kodoli et al. 2021). Marine Penicillium fungi have been found in sediments, mangroves, sponges, and algae, and have been shown to have high novelty for more than 390 new metabolites in the last decade, including alkaloids, polyketides, terpenes, and macrolides (Yang et al. 2021) that possess important biological activities such as anticancer, antimicrobial, anti-inflammatory and larvicidal actions with prospective applications in new drug development (Elkhawas et al. 2020;Singab et al. 2022). The Penicillium chrysogenum species, which belongs to the Penicillium subgenus, is particularly interesting due to its ability to produce penicillin and small antifungal proteins, making it beneficial for controlling fungal infections formed by other filamentous fungi (Martín 2020). This review concentrates on P. chrysogenum bioactive metabolites and their biological properties through the years 2013-2023. Various online databases were utilized, such as Web of Science, Marinlit, Communicated by Yusuf Akhter. and Scifinder. The purpose of this review is to highlight all the progress made during the previous decade concerning the potential application of the isolated biomolecules (277 compounds and miscellaneous as shown in Table 1) from different strains of P. chrysogenum. This review deals with the chemical nature along with the reported pharmacological activities of the stated secondary metabolites, in addition to several miscellaneous compounds (Figs. 1, 2 and 3).

K-Miscellaneous
The antimicrobial proteins (AMPs) named PAF and PAFB were secreted from the filamentous fungus P. chrysogenum Q176 (Huber et al. 2020).
The P. chrysogenum Q176 antifungal protein C (PAFC) was also characterized, and was different phylogenetically from the two Penicillium AMPs, PAF and PAFB .
An acetyl xylan esterase (PcAxe) was cloned from P. chrysogenum P33 and expressed in Pichia pastoris GS115. rPcAxe comprises a domain of carbohydrate esterase and 62 domains of a glycosyl hydrolase family (Yang et al. 2017).
Two RG I-degrading enzymes, termed endo-RG and exo-RG lyases were reported to be secreted by P. chrysogenum 31B. The enzyme precisely acts on rhamnose (Rha) at the non-reducing end of RG oligosaccharides, but had not shown any action on flavonoid glycosides (Matsumoto et al. 2017).
Only asexual reproduction is known to be used by P. chrysogenum. But further proof points to the possibility of sexual reproduction with an unidentified sexual stage. P. chrysogenum has recently been found to contain the mating-type (MAT) and pheromone signaling genes, which are associated with mating in other sexual fungus. In heterothallic ascomycete fungi, complementing MAT1-1 and MAT1-2 isolates are required for sex to occur. In contrast to the original Fleming strain, which has the opposite MAT1-2 locus, NRRL1951 (P. chrysogenum derivatives) has a MAT1-1 locus with a MAT1-1-1 gene encoding a putative alpha-box transcription factor. Sexual crosses can  (Böhm et al. 2013).

Anti-fungal
According to Al-Saleem et al. (2022), P. chrysogenum extract exhibited significant antifungal activity towards Candida albicans and Cryptococcus neoformans with MIC 93.75 ± 0.55 and 19.53 ± 0.48 µg/mL, respectively. Moreover, kojic acid (156) revealed the same potency towards Fusarium oxysporum and Cryptococcus neoformans with MIC 39.06 ± 0.85 and 39.06 ± 0.98 µg/mL, respectively. Holzknecht et al. (2020) reported that the antifungal protein C (PAFC) produced by P. chrysogenum Q176 was produced together with PAF and PAFB into the culture broth. Recombinant PAFC's functional characterization revealed a promising novel molecule for anti-Candida therapy. In pre-established biofilms of two strains of C. albicans, the planktonic cells were killed by the thermotolerant PAFC while the sessile cells' metabolic activity decreased. One of the strains was a fluconazole-resistant that displayed greater PAFC sensitivity than the fluconazole-sensitive one. The absence of hemolytic activity supports the further use of PAFC in clinical therapy. Huber et al. (2020) found that PAF and PAFB, the antimicrobial proteins (AMPs) secreted by the filamentous fungus P. chrysogenum Q176, are highly stable due to a compact disulfide-bond, β-fold structure. In micromolar doses, these two AMPs effectively prevented the growth of several fungi including: Aspergillus fumigatus, Trichophyton spp., Aspergillus niger, and Candida spp., along with the Neurospora crassa and Saccharomyces cerevisiae., which were vulnerable to both proteins since their growth diminished at 0.25-4 μM PAF or PAFB doses, respectively. Xu et al. (2020) reported that penicierythritol A (221) isolated from endophytic P. chrysogenum XNM-12, the marine algal-derived fungus, had a moderate antifungal potential towards the plant pathogenic fungus Alternaria alternata with MIC 8 μg/mL.
As elaborated by Sonderegger et al. (2017), PAF, a cysteine-rich, cationic antifungal protein that is mostly made up of 55 amino acids, was abundantly generated by the filamentous ascomycete P. chrysogenum. Botrytis cinerea and Aspergillus fumigatus, two opportunistic human and plant diseases, were discovered to be particularly inhibited, even though in vitro and in vivo tests had shown that they were inert against mammalian cells. Zhu et al. (2017) have identified penochalasin K (85) from the mangrove endophytic fungus P. chrysogenum V11 culture. In fact, it exhibited serious inhibitory actions towards Colletotrichum gloeosporioides and Rhizoctonia solani (MICs Values = 6.13, 12.26 μM, respectively), which were notably higher than carbendazim.

Fig. 3 (continued)
According to Lopes et al. (2013), P. chrysogenum IFL1 produced bioactive compounds that spread on agro-industrial residues, cheese whey, and grape trash. The cheese whey culture filtrate hindered the development of the fungus Fusarium oxysporum as well as the amoeba Acanthamoeba polyphaga.

Anti-viral
In vitro as well as in vivo studies by Huber et al. (2020) on PAF and PAFB, the two antimicrobial proteins (AMPs) secreted by the filamentous fungus P. chrysogenum Q176, displayed that they had antiviral activity without triggering any cytotoxic effects or hemolytic activity on mammalian cells. Experiments in human cervix cancer cells showed that they both reduced Human Coronavirus cytopathogenic effects. Apparently, it was the very first study on the antiviral ability of small, cysteine-rich and cationic proteins derived from fungi.
Hawas and Abou El-Kassem (2019) used a scale-up fermentation approach that yielded haenamindole (74), an uncommon diketopiperazine (DKP) alkaloid, from the endophytic fungus P. chrysogenum in a biomaltpeptone medium. This step was proceeded by cytotoxicity-guided fractionation. It showed low HCV protease potential with an IC 50 value of 76.3 μM.

Anti-inflammatory
According to Zhang et al. (2017), HPABA (265) is considered a potent anti-inflammatory compound obtained from the marine P. chrysogenum and was found to be an analogue of aspirin by structure.
According to Al-Saleem et al. (2022), cytotoxic activity towards the cell lines (HCT-1 and HEP-2) were investigated using MTT method. In HCT-116 colon carcinoma cells, the ethyl acetate extract of P. chrysogenum and Kojic acid (156) exhibited strong activity with IC 50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively, with respect to HEP-2 larynx carcinoma, the total extract and Kojic acid (

P. chrysogenum
Xanthocillin X (Bladt et al. 2013) All structures were identified using extensive spectroscopical techniques such as UV, 1D and 2D. NMR and HRESIMS spectra, and literature data were used to determine their structures and absolute configurations Compounds isolated and identified from P. chrysogenum in different host organisms (*) repeated Compounds Page 23 of 28 240 cell lines with same IC 50 30.8 ± 1.3 and 30.8 ± 1.2 µg/mL, respectively. These substantial antimicrobial and cytotoxic activities may be due to the presence of penicillin G (212), Kojic acid (156), sohirnone B (217), and camptothecin (56) as major constituents in the ethyl acetate extract of P. chrysogenum.
Hawas and Abou El-Kassem (2019) used a scale-up fermentation approach that yielded Haenamindole (74), an uncommon diketopiperazine (DKP) alkaloid of the endophytic fungus P. chrysogenum in biomaltpeptone media. This step was proceeded by cytotoxicity-guided fractionation within a group of up to 12 cancer cell lines. Results revealed substantial cytotoxicity of the metabolite, with definite selectivity for colon-38 carcinoma cells compared to human normal cells. Ory et al. (2019) stated that the marine-derived fungus, P. chrysogenum MMS5 extract, shows strong antiproliferative action on breast cancer cells (MCF-7 cell line) in a real-case investigation. Its antiproliferative activity was validated with an IC 50 of 0.10 μM on MCF-7 cells mainly due to the presence of high amounts of ergosterol.
A study by Zhen et al. (2018) showed that chrysoxanthones A-C (161-163) were obtained from the P. chrysogenum strain HLS111. In vitro cytotoxic activity against different human cell lines were estimated for these metabolites against renal carcinoma (A498), multiform glioblastoma (U87 MG), leukemia (HL60), non-small cell lung tumor (NCI-H1650), and colonic carcinoma (HT29), by the MTT method. The anticancer activities of compounds (161-163) diminished significantly compared to secalonic acid D which was previously isolated. The cytotoxic effect was reduced due to the presence of β-methyl-γ-lactone ring, meaning that the tricyclic nucleus was important to achieve bioactivity.
Chromatographic analysis performed by Zhao et al. (2018) on extracts of P. chrysogenum AD-1540, a marine algal-derived endophytic fungus which was separated from the red alga Grateloupia turuturu 's inner tissue, afforded chryxanthones A and B (161-162), two novel benzophenone derivatives. Their cytotoxic profile was evaluated towards six human cancer cell lines: MCF-7, A549, HeLa, BT-549, HepG2 and THP-1. Chryxanthone A (161) exhibited moderate activity towards BT-549 and HeLa cancer cell lines, with IC 50 values of 20.4 and 23.5 µM, respectively, whereas chryxanthone B (162) inhibited the growth of A549 cell line selectively with an IC 50 value of 20.4 µM.
The antimetastatic and antiangiogenic properties of the halotolerant fungus P. chrysogenum-1 hPc-1 isolated from Tuz Lake Turkey, were assessed by Dikmen et al. (2017). Its extract activity on human umbilical vein endothelial cells (HUVEC) and colorectal cancer cells (Caco-2) was assessed using the WST-1 technique and real-time cell analysis system-DP. According to the results, mRNA expression levels of the genes for VEGF A, VEGF B, COX-10, EGFR, ANGPT-1, and IL-8 were lower in HUVEC and Caco-2 cells compared to the standard.
According to Zhu et al. (2017), MTT technique revealed the cytotoxicity profile of the isolated metabolites from the mangrove endophytic fungus P. chrysogenum V11 against three diverse human cancer cell lines, lung adenocarcinoma epithelial cell line (A549), gastric cancer cell line (SGC-7901), and a breast cancer cell line (MDA-MB-435). Penochalasin K (85) showed significant broad-spectrum inhibitory actions towards all the assayed cell lines (IC 50 < 10 μM). Also, chaetoglobosin A (84), chaetoglobosin C (81) and penochalasin I (83) exhibited mild to noticeable inhibitory activities on the mentioned cell lines with IC 50 values range of 6.56-37.56 μM. Chen et al. (2017) revealed that bipenicilisorin (186), which was obtained from a marine-derived fungus P. chrysogenum SCSIO 41,001, displayed cytotoxic effects towards Huh-7, K562, and A549 cancer cell lines significantly with IC 50 values at 2.59, 6.78, and 6.94 μM, respectively, while penicitrinone F (144) displayed a modest inhibitory effect against EV71 with IC 50 14.50 μM. Huang et al. (2016) tested compounds obtained from the culture of P. chrysogenum V11 for their anticancer activity. Penochalasin I (83) showed significant activity against SGC-7901 and MDA-MB-435 cells (IC 50 < 10 μM), while cytoglobosin C (90) had high activity against A549 and SGC-7901 cells (IC 50 < 10 μM). Guo et al. (2016) stated that 5-Hydroxymethyl-2-furancarboxaldehyde (270) recovered from the ethyl acetate extract of P. chrysogenum HGQ6 fermentation broth had activity towards BGC823 cell with the IC 50 value of 0.19 mg/mL. Previous studies by Hou et al. (2016) on P. chrysogenum, grown from a Gorgonian Carijoa sp. found in the South China Sea, yielded a novel flavone penimethavone A (154), with a unique methyl group at ring-B which is rarely found. It was tested in vitro for cytotoxicity against cervical cancer (HeLa), rhabdomyosarcoma, non-small cell lung cancer (A549) and human laryngeal epithelial (Hep-2) cell lines. Results indicated that it had moderate selective cytotoxic activity against (HeLa) and rhabdomyosarcoma cell lines, with IC 50 values of 8.41 and 8.18 µΜ, respectively. Mady et al. (2016) yielded meleagrin (61), by bioguided chromatographic analysis of the dichloromethane extract of P. chrysogenum mycelia which could inhibit the development of human breast cancer cell lines, MDA-468, MDA-MB-231, SK BR-3, BT-474, MCF7, and MCF7-dox, while comparable therapeutic doses revealed no impact on the growth of the non-carcinogenic human mammary epithelial cells MCF10A and viability. Additionally, its therapy inhibited the HGF-induced cell migration and invasion in breast cancer cell lines in a dose-dependent behavior.