Occurrence, Risk Implications, Prevention and Control of CIT in Monascus Cheese: A Review.

Monascus is a filamentous fungus that has been used in the food and pharmaceutical industries. When used as an auxiliary fermenting agent in the manufacturing of cheese, Monascus cheese is obtained. Citrinin (CIT) is a well-known hepatorenal toxin produced by Monascus that can harm the kidneys structurally and functionally and is frequently found in foods. However, CIT contamination in Monascus cheese is exacerbated by the metabolic ability of Monascus to product CIT, which is not lost during fermentation, and by the threat of contamination by Penicillium spp. that may be introduced during production and processing. Considering the safety of consumption and subsequent industrial development, the CIT contamination of Monascus cheese products needs to be addressed. This review aimed to examine its occurrence in Monascus cheese, risk implications, traditional control strategies, and new research advances in prevention and control to guide the application of biotechnology in the control of CIT contamination, providing more possibilities for the application of Monascus in the cheese industry.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Penicilloneines A and B, Quinolone-Citrinin Hybrids from a Starfish-Derived Penicillium sp.

Penicilloneines A (1) and B (2) are the first reported quinolone-citrinin hybrids. They were isolated from the starfish-derived fungus Penicillium sp. GGF16-1-2, and their structures were elucidated using spectroscopic, chemical, computational, and single-crystal X-ray diffraction methods. Penicilloneines A (1) and B (2) share a common 4-hydroxy-1-methyl-2(1H)-quinolone unit; however, they differ in terms of citrinin moieties, and these two units are linked via a methylene bridge. Penicilloneines A (1) and B (2) exhibited antifungal activities against Colletotrichum gloeosporioides, with lethal concentration 50 values of 0.02 and 1.51 µg/mL, respectively. A mechanistic study revealed that 1 could inhibit cell growth and promote cell vacuolization and consequent disruption of the fungal cell walls via upregulating nutrient-related hydrolase genes, including putative hydrolase, acetylcholinesterase, glycosyl hydrolase, leucine aminopeptidase, lipase, and beta-galactosidase, and downregulating their synthase genes 3-carboxymuconate cyclase, pyruvate decarboxylase, phosphoketolase, and oxalate decarboxylase.

Five previously undescribed citrinin derivatives from the endophytic fungus Penicillium citrinum GZWMJZ-836.

Penicillium citrinum GZWMJZ-836 is an endophytic fungus from Drynaria roosii Nakaike. Five previously undescribed citrinin derivatives (1-5) and six intermediates related to their biosynthesis (6-11) were obtained from the extract of this strain's solid fermentation using multiple column chromatography separations, including high-performance liquid chromatography. The structures of these compounds were determined through comprehensive spectroscopic analyses, primarily using NMR and HRESIMS data. The stereochemistry was mainly confirmed by ECD calculations, and the configurations of C-7' in compounds 4 and 5 were determined using 13C NMR calculations. Compounds 4-5 and 8 showed antibacterial activity against five strains, with minimum inhibitory concentration values ranging from 7.8 to 125 µM. Compounds 4 and 7 exhibited inhibitions against three plant pathogenic fungi, with IC50 values ranging from 66.6 to 152.1 µM. Additionally, a putative biosynthetic pathway for compounds 1-5 derived from citrinin was proposed.

Citrisorbicillinol, an undescribed hybrid sorbicillinoid with osteogenic activity from Penicillium citrinum ZY-2.

Citrisorbicillinol (1), along with six other known compounds (2-7), was isolated from an endphyte Penicillium citrinum ZY-2 of Plantago asiatica L. Citrisorbicillinol (1) was characterized as a skeletally unprecedented hybrid sorbicillinoid, and its unique framework is likely formed by intermolecular [4 + 2] cycloaddition between intermediates derived from citrinin and sorbicillinoid biosynthetic gene clusters. Compounds 1 and 2 demonstrated to promote osteoblastic differentiation in MC3T3-E1 cells, and to be osteogenic in the prednisolone induced osteoporotic zebrafish. Compounds 3-7 exhibited moderate cytotoxicity against four human cancer cell lines.

The marine Penicillium sp. GGF16-1-2 metabolite dicitrinone G inhibits pancreatic angiogenesis by regulating the activation of NLRP3 inflammasome.

Citrinin derivatives have been found to have various pharmacological activities, such as anti-inflammatory, anti-tumor, and antioxidant effects. Dicitrinone G (DG) was a new citrinin dimer isolated from marine-derived fungus Penicillium sp. GGF 16-1-2 which has potential activity. Here, we aim to investigate whether DG has anti-pancreatic cancer activity. In xenograft tumor model, 2 × 106 BXPC-3 cells were injected into the hind flank of NU/NU nude mice by subcutaneously for 2 weeks followed by treating with DG (0.25, 0.5, 1 mg/kg) and 5-FU (30 mg/kg) for 4 weeks. Tumor volume and weight were measured, and the expression of CD31, IL-18, NLRP3, and Caspase-1 in tumor tissue were detected. In vitro, HUVECs were treated with conditioned medium (CM) derived from BXPC-3 cells, the effects of DG on angiogenesis were detected by tube formation and western blot analysis. In vivo studies showed that the tumor growth and angiogenesis were greatly suppressed. The tumor weight inhibition rates of DG and 5-FU groups were about 42.36%, 38.94%, 43.80%, and 31.88%. Furthermore, the expression of CD31 and Caspase-1 were decreased. In vitro, CM derived from BXPC-3 cells which treated with DG could inhibit the tube formation and expression of pro-angiogenic NICD in HUVECs. Our study suggests that DG could suppress angiogenesis via the NLRP3/IL-18 pathway and may have the potential to inhibit tumor development.

Action of the fungal compound citrinin, a bioherbicide candidate, on photosystem II.

BACKGROUND: Bioherbicides are becoming more attractive as safe weed control tools towards sustainable agriculture. Natural products constitute an important source chemicals and chemical leads for discovery and development of novel pesticide target sites. Citrinin is a bioactive compound produced by fungi of the genera Penicillium and Aspergillus. However, its physiological-biochemical mechanism as a phytotoxin remains unclear. RESULTS: Citrinin causes visible leaf lesions on Ageratina adenophora similar to those produced by the commercial herbicide bromoxynil. Phytotoxicity bioassay tests using 24 plant species confirmed that citrinin has a broad activity spectrum and therefore has potential as a bioherbicide. Based on chlorophyll fluorescence studies, citrinin mainly blocks PSII electron flow beyond plastoquinone QA at the acceptor side, resulting in the inactivation of PSII reaction centers. Furthermore, molecular modeling of citrinin docking to the A. adenophora D1 protein suggests that it binds to the plastoquinone QB site by a hydrogen bond between the O1 hydroxy oxygen atom of citrinin and the histidine 215 of the D1 protein, the same way as classical phenolic PSII herbicides do. Finally, 32 new citrinin derivatives were designed and sorted according to free energies on the basis of the molecular model of an interaction between the citrinin molecule and the D1 protein. Five of the modeled compounds had much higher ligand binding affinity within the D1 protein compared with lead compound citrinin. CONCLUSION: Citrinin is a novel natural PSII inhibitor that has the potential to be developed into a bioherbicide or utilized as a lead compound for discovery of new derivatives with high herbicidal potency. © 2023 Society of Chemical Industry.

Exposure to Mycotoxin Citrinin Promotes Carcinogenic Potential of Human Renal Cells.

Mycotoxin citrinin (CTN), commonly found in food and health supplements, may induce chromosomal instability. In this study, human renal proximal tubule epithelial cells (hRPTECs) that were exposed to CTN (10 and 20 µM) over 3 days exhibited numerical chromosomal aberrations. Short-term (3 days) and long-term (30 days) exposures to CTN significantly promoted mitotic spindle abnormalities, wound healing, cell migration, and anchorage-independent growth in human embryonic kidney 293 (HEK293) cells. Short-term exposure to 10 and 20 µM CTN increased the number of migrated cells on day 10 by 1.7 and 1.9 times, respectively. The number of anchorage-independent colonies increased from 2.2 ± 1.3 to 7.8 ± 0.6 after short-term exposure to 20 µM CTN and from 2.0 ± 1.0 to 12.0 ± 1.2 after long-term exposure. The transcriptomic profiles of CTN-treated HEK293 were subjected to over-representative analysis (ORA), gene set enrichment analysis (GSEA), and Ingenuity pathway analysis (IPA). Short-term exposure to CTN promoted the RTK/KRAS/RAF/MAPK cascade, while long-term exposure altered the extracellular matrix organization. Both short- and long-term CTN exposure activated cancer and cell cycle-related signaling pathways. These results demonstrate the carcinogenic potential of CTN in human cells and provide valuable insights into the cancer risk associated with CTN.

Citriquinolinones A and B: Rare Isoquinolinone-Embedded Citrinin Analogues and Related Metabolites from the Deep-Sea-Derived Aspergillus versicolor 170217.

A systematic chemical investigation of the deep-sea-derived fungus Aspergillus versicolor 170217 resulted in the isolation of six new (1-6) and 45 known (7-51) compounds. The structures of the new compounds were established on the basis of exhaustive analysis of their spectroscopic data and theoretical-statistical approaches including GIAO-NMR, TDDFT-ECD/ORD calculations, DP4+ probability analysis, and biogenetic consideration. Citriquinolinones A (1) and B (2) feature a unique isoquinolinone-embedded citrinin scaffold, representing the first exemplars of a citrinin-isoquinolinone hybrid. Dicitrinones K-L (3-4) are two new dimeric citrinin analogues with a rare CH-CH3 bridge. Biologically, frangula-emodin (32) and diorcinol (17) displayed remarkable anti-food allergic activity with IC50 values of 7.9 ± 3.0 µM and 13.4 ± 1.2 µM, respectively, while diorcinol (17) and penicitrinol A (20) exhibited weak inhibitory activity against Vibrio parahemolyticus, with MIC values ranging from 128 to 256 µM.

Novel citrinin derivatives from fungus Penicillium sp. TW131-64 and their antimicrobial activities.

Six new citrinin derivatives (1, 2, 4, 10, 11, and 16), along with fourteen known analogues, were acquired from Penicillium sp. TW131-64, a marine-derived fungus strain. The chemical structures of new compounds were identified through adopting various spectroscopic methods in combination with X-ray diffraction technology and comparison of the experimental electronic circular dichroism (ECD) with calculated ones. Among them, compounds 1-4 were nitrogen-containing citrinin derivatives existing in enantiomers which were resolved by chiral chromatography. A putative biosynthetic pathway for compounds 1-4 was proposed. Additionally, the antimicrobial activities of these compounds were detected by the broth microdilution assays. Citrinin derivatives 1, 2, 4 and their corresponding enantiomers (1a, 2a, 4a, 1b, 2b, and 4b) exhibited potent antimicrobial activities towards Helicobacter pylori standard strains and multidrug-resistant strains (MIC values ranging from 0.25 to 8 µg/mL), which were comparable or even better than metronidazole. Moreover, compounds 1a and 1b also showed remarkable broad antimicrobial effects towards Staphylococcus aureus, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, vancomycin-resistant Enterococcus faecium (VRE), and Candida albicans. In summary, our studies demonstrated that citrinin enantiomers 1a-4a and 1b-4b, especially 1a and 1b, can be lead compounds in the research and development (R & D) of novel antimicrobial drugs. KEY POINTS: • 3 novel nitrogen-containing citrinin derivatives (1, 2, 4) were isolated. • citrinin derivatives 1-4 in enantiomers were resolved by chiral chromatography. • citrinin derivatives 1a and 1b showed broad and significant antimicrobial effects.

Neotricitrinols A-C, unprecedented citrinin trimers with anti-osteoporosis activity from the deep-sea-derived Penicillium citrinum W23.

Marine fungi are prolific source for the discovery of structurally diverse and bioactive molecules. In our search for new anti-osteoporosis compounds from deep-sea-derived fungi, we prioritized a fungus whose extract exhibited moderate activity and rich chemical diversity. The investigation of this strain afforded a class of citrinins, including three new citrinin trimers, neotricitrinols A-C (1-3), and three known dimeric/monomeric precursors (4-6). Neotricitrinols A-C (1-3) feature a unique octacyclic carbon scaffold among the few reported citrinin trimers with their absolute configurations established by spectroscopic analysis, theoretical-statistical approaches (GIAO-NMR, TDDFT-ECD/ORD calculations), DP4+ probability analysis as well as biogenetic consideration. A plausible biosynthetic pathway linking 1-3 from the common intermediate metabolite penicitrinol A (4) was proposed. Biologically, neotricitrinol B (2) showed potential anti-osteoporosis activity by promoting osteoblastogenesis and inhibiting adipogenic differentiation on primary bone mesenchymal stem cells, while displaying no cytotoxicity.

Machine Learning-Mediated Ultrasensitive Detection of Citrinin and Associated Mycotoxins in Real Food Samples Discerned from a Photoluminescent Carbon Dot Barcode Array.

Economically viable remote sensing of foodborne contaminants using minimalistic chemical reagents and simultaneous automation calls for a concrete integration of a chemical detection strategy with artificial intelligence. In a first of its kind, we report the ultrasensitive detection of citrinin and associated mycotoxins like aflatoxin B1 and ochratoxin A using an Alizarin Red S (ARS) and cystamine-derived carbon dot (CD) that aptly amalgamate with machine learning algorithms for automation. The photoluminescence response of the CD as a function of various solvents and pH is used to generate array channels that are further modulated in the presence of the mycotoxins whose digital images were acquired to determine pixelation, essentially creating a barcode. The barcode was fed to machine learning algorithms that actualize and intertwine convoluted databases, demonstrating Extreme Gradient Boosting (XGBoost) as the optimized model out of eight algorithms tested. Spiked samples of wheat, rice, gram, maize, coffee, and milk were used to evaluate the testing model where an exemplary accuracy of 100% even at 10 pmol of mycotoxin concentration was achieved. Most importantly, the coexistence of mycotoxins could also be detected through the CD array and XGBoost synergy hinting toward a broader scope of the developed methodology for smart detection of foodborne contaminants.

[Mycotoxins in spices consumed in Russia].

Spices and herbs have been used since ancient times as flavor and aroma enhancers, colorants, preservatives and traditional medicines. As many other plant products, they can be exposed to contaminants, ones of which are mycotoxins, secondary metabolites of fungi. Such contamination can occur during harvesting, processing and storage, distribution, retailing and consumer use. Although they are used and consumed in small quantities, but added to a wide variety of products, especially ready-to-eat products. So the assessment of their contamination with mycotoxins is very important. The aim of the study was to investigate the contamination of spices and herbs with mycotoxins of fungi of the genera Aspergillus, Penicillium, Fusarium and Alternaria, as well as to assess the mycotoxins intake per person when consuming these food groups. Material and methods. Concentration of mycotoxins in 155 samples of spices and herbs was determined by ultra high-performance liquid chromatography coupled to tandem mass-spectrometric detection (UHPLC-MS/MS). The list of mycotoxins included deoxynivalenol, aflatoxins, ochratoxin A, zearalenone, T-2 toxin, fumonisins, sterigmatocistin, HT-2 toxin, diacetoxyscirpenol, enniatins, beauvericin, neosolaniol, citreoviridin, mycophenolic acid, citrinin, tentoxin, altenuene, alternariol and its monomethyl ether. Results. Among the regulated in plant products mycotoxins in the studied samples there were found aflatoxins (B1 - in 19% of samples, from 0.4 to 48.2 µg/kg, B2 - 8%, from < limit of quantitation (LOQ) to 3.2 µg/kg, G1 - 2%, 0.75-21 µg/kg, G2 - 5%, 0.5- 12.5 µg/kg), ochratoxin A (15% samples, 0.8-14 µg/kg), fumonisin B1 (8%, 16.1-722.6 µg/kg), and fumonisin B2 (14%, < LOQ - 79.6 µg/kg). T-2 toxin and deoxynivalenol were found in 10% of samples (< LOQ - 6.5 µg/kg and < LOQ - 65.5 µg/kg respectively), zearalenone - in 4 samples (1.7-106.2 µg/kg), HT-2 toxin - in 8 samples (5.4-19.8 µg/kg). Among little-studied (emergent) mycotoxins in the spices and herbs samples there were found tentoxin (in 36% of samples, in an amount from 0.7 to 10.9 µg/kg), altenuene (in 8%, 14.5-161.5 µg/kg). 10% of the samples were contaminated with alternariol and its methyl ether (from less than LOQ to 12.8 and < LOQ to 55.7 µg/kg, respectively), 4% - with sterigmatocystin (0.4-7.8 µg/kg), 5% - mycophenolic acid (13.1-297 µg/kg), 2% of the samples were contaminated with citrinin and enniatin B (< LOQ - 27.7 and 0.1-1 µg/kg), in 9 samples (6%) beauvericin was detected (< LOQ - 1.7 µg/kg). Over 60% of samples were contaminated with more than one mycotoxin. The content of aflatoxin B1 exceeded the maximum permissible level set in the EU (5 µg/kg) in nine samples. Conclusion. To the best of our knowledge, the present study is the first in the Russian Federation to report results indicating to the contamination of spices and herbs with mycotoxins. High occurrence of aflatoxins, tentoxin, ochratoxin A and fumonisin B2 has been observed. In calculating the potential exposure of mycotoxins, the possibility of high levels of aflatoxin B1 intake have been shown to be possible, which could lead to a public health risk when consuming contaminated spices, herbs and foods containing them.

Anti-Klebsiella pneumoniae activity of secondary metabolism of Achromobacter from the intestine of Periplaneta americana.

BACKGROUND: Klebsiella pneumoniae is one of the main pathogens of clinical isolation and nosocomial infections, as K. pneumoniae show broad-spectrum resistance to ß-lactam and carbapenem antibiotics. It is emerging clinical need for a safe and effective drug to anti-K. pneumoniae. At present, Achromobacter mainly focused on its degradation of petroleum hydrocarbons, polycyclic aromatic hydrocarbons, assisting insects to decompose, degrade heavy metals and utilize organic matter, but there were few reports on the antibacterial activity of the secondary metabolites of Achromobacter. RESULTS: In this study, a strain WA5-4-31 from the intestinal tract of Periplaneta americana exhibited strong activity against K. Pneumoniae through preliminary screening. The strain was determined to be Achromobacter sp. through the morphological characteristics, genotyping and phylogenetic tree analysis, which is homologous to Achromobacter ruhlandii by 99%, its accession numbe in GenBank at National Center for Biotechnology Information (NCBI) is MN007235, and its deposit number was GDMCC NO.1.2520. Six compounds (Actinomycin D, Actinomycin X2, Collismycin A, Citrinin, Neoechinulin A and Cytochalasin E) were isolated and determined by activity tracking, chemical separation, nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Among them, Actinomycin D, Actinomycin X2, Collismycin A, Citrinin and Cytochalasin E showed a good effect on anti-K. pneumoniae, with MIC values of 16-64 µg/mL. CONCLUSIONS: The study reported Achromobacter, which was from the intestinal tract of Periplaneta americana with the activity against K. Pneumoniae, can produce antibacterial compounds for the first time. It lays the foundation for development of secondary metabolites of insect intestinal microorganisms.

Transportation of citrinin is regulated by the CtnC gene in the medicinal fungus Monascus purpureus. / 药用真菌紫色红曲霉通过CtnCåŸºå› è°ƒæŽ§æ¡”éœ‰ç´ çš„è½¬è¿.

Monascus is one of the most essential microbial resources in China, with thousands of years of history. Modern science has proved that Monascus can produce pigment, ergosterol, monacolin K, γ-aminobutyric acid, and other functionally active substances. Currently, Monascus is used to produce a variety of foods, health products, and pharmaceuticals, and its pigments are widely used as food additives. However, Monascus also makes a harmful polyketide component called citrinin in the fermentation process; citrinin has toxic effects on the kidneys such as teratogenicity, carcinogenicity, and mutagenicity (Gong et al., 2019). The presence of citrinin renders Monascus and its products potentially hazardous, which has led many countries to set limits and standards on citrinin content. For example, the citrinin limit is less than 0.04 mg/kg according to the Chinese document National Standard for Food Safety Food Additive Monascus (GB 1886.181-2016) (National Health and Family Planning Commission of the People's Republic of China, 2016), and the maximum level in food supplements based on rice fermented with Monascus purpureus is 100 µg/kg in the European Union (Commission of the European Union, 2019).

A two-year study on the occurrence and concentration of mycotoxins in corn varieties with different endosperm textures.

BACKGROUND: Mycotoxin monitoring in cereal grains has great importance in the food and feed industries. This study evaluated mycotoxin contamination in corns with different endosperm textures in 2 years of cultivation. Samples of dent, semi-dent, flint and semi-flint corns from field experiments were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). RESULTS: Occurrences of fumonisins B1 (FB1 ) and B2 (FB2 ) in 2020 were 45.72% (mean 270 µg kg-1 ) and 35.89% (94.97 µg kg-1 ), respectively, and 68.98% (446 µg kg-1 ) and 45.83% (152 µg kg-1 ) in 2021. Occurrence of aflatoxin B1 was 11.96% (0.16 µg kg-1 ) in 2020 and 11.11% (0.13 µg kg-1 ) in 2021. In 2020, deoxynivalenol (DON) and zearalenona (ZEA) presented occurrences of 1.28% and 1.70%, with means of 4.08 and 2.45 µg kg-1 , respectively. In 2021, results were 8.33% (31.00 µg kg-1 ) for DON and 8.79% (4.38 µg kg-1 ) for ZEA. Citrinin, diacetoxyscirpenol and fusarenon-X did not occur in 2020 but presented 1.66%, 0.83%, and 2.50% positive rates in 2021, respectively. In 2020, flint corn presented the lowest concentration of FB1 whereas dent corn presented the highest concentration of FB1 and FB2 (P < 0.05). In 2021, dent corn presented the highest means of FB1 , FB2 and diacetoxyscirpenol (P < 0.05). Dent and semi-dent presented the highest concentration of nivalenol (P < 0.05). CONCLUSION: The endosperm texture influenced mycotoxin contamination in corn grains, especially FB1 and FB2 , which had the highest concentration in dent corn in the 2 years of this study. © 2023 Society of Chemical Industry.

Peniciriols A and B, two new citrinin derivatives from an endophytic fungus Penicillum citrinum TJNZ-27.

Two undescribed citrinin derivatives, named peniciriols A-B (1-2), together with six known compounds were isolated from endophytic fungus Penicillum citrinum TJNZ-27. The structures of two new compounds were well established by the detail interpretation of NMR and HRESIMS data as well as ECD measurement powered by molecular calculation. Among them, compound 1 shared an unprecedented dimerized citrinin skeleton with the formation of an intriguing 9H-xanthene ring system, whereas compound 2 possess a highly substituted phenylacetic acid skeleton, which was rarely-occurring in natural secondary metabolites. Moreover, these novel compounds were tested for cytotoxic and antibacterial activities, whereas these novel compounds did not exhibit any noticeable cytotoxic or antibacterial activities.

An oxidoreductase gene CtnD involved in citrinin biosynthesis in Monascus purpureus verified by CRISPR/Cas9 gene editing and overexpression.

Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression.

Citrinin Is a Potential Quorum Sensing Inhibitor against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that infects patients by regulating virulence factors and biofilms through a quorum sensing (QS) system to protect itself from antibiotics and environmental stress. Therefore, the development of quorum sensing inhibitors (QSIs) is expected to become a new strategy for studying drug resistance to P. aeruginosa infections. Marine fungi are valuable resources for screening QSIs. A marine fungus, Penicillium sp. JH1, with anti-QS activity was isolated from the offshore waters of Qingdao (China), and citrinin, a novel QSI, was purified from secondary metabolites of this fungus. Citrinin could significantly inhibit the production of violacein in Chromobacterium violaceum CV12472 and the production of three virulence factors (elastase, rhamnolipid and pyocyanin) in P. aeruginosa PAO1. It could also inhibit the biofilm formation and motility of PAO1. In addition, citrinin downregulated the transcript levels of nine genes (lasI, rhlI, pqsA, lasR, rhlR, pqsR, lasB, rhlA and phzH) associated with QS. Molecular docking results showed that citrinin bound to PqsR and LasR with better affinity than the natural ligands. This study laid a foundation for the further study of the structure optimization and structure-activity relationship of citrinin.

Histone deacetylase MrHos3 negatively regulates the production of citrinin and pigments in Monascus ruber.

Monascus spp. can produce a variety of beneficial metabolites widely used in food and pharmaceutical industries. However, some Monascus species contain the complete gene cluster responsible for citrinin biosynthesis, which raises our concerns about the safety of their fermented products. In this study, the gene Mrhos3, encoding histone deacetylase (HDAC), was deleted to evaluate its effects on the production of mycotoxin (citrinin) and the edible pigments as well as the developmental process of Monascus ruber M7. The results showed that absence of Mrhos3 caused an enhancement of citrinin content by 105.1%, 82.4%, 111.9%, and 95.7% at the 5th, 7th, 9th, and 11th day, respectively. Furthermore, deletion of Mrhos3 increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. In addition, deletion of Mrhos3 led to an increase in total pigment content and six classic pigment components. Western blot results revealed that deletion of Mrhos3 could significantly elevate the acetylation level of H3K9, H4K12, H3K18, and total protein. This study provides an important insight into the effects of hos3 gene on the secondary metabolites production in filamentous fungi.