Fungus Monascus Research Articles

Enhancing bioprocessing of red pigment from immobilized culture of gamma rays mutant of the endophytic fungus Monascus ruber SRZ112

Considerable attention has been paid to exploring the biotechnological applications of several Monascus sp. for pigment production. In this study, our focus is on enhancing the bioprocessing of red pigment (RP) derived from the endophytic fungus Monascus ruber SRZ112. To achieve this, we developed a stable mutant strain with improved productivity through gamma irradiation. This mutant was then employed in the immobilization technique using various entrapment carriers. Subsequently, we optimized the culture medium for maximal RP production using the Response Surface Methodology. Finally, these immobilized cultures were successfully utilized for RP production using a semi-continuous mode of fermentation. After eight cycles of fermentation, the highest RP yield by immobilized mycelia reached 309.17 CV mL−1, a significant increase compared to the original titer. Importantly, this study marks the first report on the successful production of Monascus RP in a semi-continuous mode using gamma rays’ mutant strain, offering prospects for commercial production.

Quality Control and Safety Assessment of Online-Purchased Food Supplements Containing Red Yeast Rice (RYR).

Dietary supplements containing red yeast rice (RYR), a fermentation product of the fungus Monascus purpureus grown on white rice, remain popular in Europe as proclaimed cholesterol-lowering aids. The cholesterol-lowering effects are due to the occurrence of monacolin K, which is often present as a mixture of monacolin K lactone (MK) and as monacolin K hydroxy acid (MKA). MK is structurally similar to the cholesterol-lowering medicine lovastatin. Recently, due to safety concerns linked to the use of statins, the European Commission prohibited RYR supplements with a maximum serving exceeding 3 mg of total monacolins per day. Moreover, the amount of the mycotoxin citrinin, potentially produced by M. purpureus, was also reduced to 100 µg/kg. Evidently, manufacturers that offer their products on the European market, including the online market, must also be compliant with these limits in order to guarantee the safety of their products. Therefore, thirty-five different RYR supplements, purchased from an EU-bound e-commerce platform or from registered online pharmacies, were screened for their compliance to the European legislation for citrinin content and the amount of total monacolin K. This was conducted by means of a newly developed LC-MS/MS methodology that was validated according to ISO 17025. Moreover, these supplements were also screened for possible adulteration and any contamination by micro-organisms and/or mycotoxins. It was found that at least four of the thirty-five RYR supplements (≈11%) might have reason for concern for the safety of the consumer either due to high total monacolin K concentrations exceeding the European predefined limits for total monacolins or severe bacterial contamination. Moreover, three samples (≈9%) were likely adulterated, and the labeling of six of the seventeen samples (≈35%) originating from an EU-based e-commerce platform was not compliant, as either the mandatory warning was missing or incomplete or the total amount of monacolins was not mentioned.

Screening Antibacterial Photodynamic Effect of Monascus Red Yeast Rice (Hong-Qu) and Mycelium Extracts

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts’ exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60–80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains.Key points• A protocol of high-quality protoplasts for CGES has been developed in M. pilosus.• The GRF of CGES was about five times that of ATMT in M. pilosus.• The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Constituents Isolated from the Fungus Monascus sp.

Constituents Isolated from the Fungus Monascus sp.

Metabolic Regulation of Two pksCT Gene Transcripts in Monascus ruber Impacts Citrinin Biosynthesis.

Citrinin (CIT), a secondary metabolite produced by the filamentous fungi Monascus species, exhibits nephrotoxic, hepatotoxic, and carcinogenic effects in mammals, remarkably restricting the utilization of Monascus-derived products. CIT synthesis is mediated through the pksCT gene and modified by multiple genetic factors. Here, the regulatory effects of two pksCT transcripts, pksCTα, and pksCTβ, generated via pre-mRNA alternative splicing (AS), were investigated using hairpin RNA (ihpRNA) interference, and their impact on CIT biosynthesis and the underlying mechanisms were assessed through chemical biology and transcriptome analyses. The CIT yield in ihpRNA-pksCTα and ihpRNA-pksCT (α + β) transformants decreased from 7.2 μg/mL in the wild-type strain to 3.8 μg/mL and 0.08 μg/mL, respectively. Notably, several genes in the CIT biosynthetic gene cluster, specifically mrl3, mrl5, mrr1, and mrr5 in the ihpRNA-pksCT (α + β) transformant, were downregulated. Transcriptome results revealed that silencing pksCT has a great impact on carbohydrate metabolism, amino acid metabolism, lipid metabolism, and AS events. The key enzymes in the citrate cycle (TCA cycle) and glycolysis were significantly inhibited in the transformants, leading to a decrease in the production of biosynthetic precursors, such as acetyl-coenzyme-A (acetyl-coA) and malonyl-coenzyme-A (malonyl-coA). Furthermore, the reduction of CIT has a regulatory effect on lipid metabolism via redirecting acetyl-coA from CIT biosynthesis towards lipid biosynthesis. These findings offer insights into the mechanisms underlying CIT biosynthesis and AS in Monascus, thus providing a foundation for future research.

Precursor-directed production of water-soluble red Monascus pigments with high thermal stability via azaphilic addition reaction-based semi-synthesis

Red Monascus pigments (MPs) are a large group of polyketides from the fungus Monascus which have been widely used as food colorants. In this study, a variety of red MPs congeners were prepared to explore promising water-soluble candidates for application in liquid food formulations. The results showed that by combining the two-stage, low-pH fermentation strategy with a downstream purification step of fractional crystallization, precursors of red MPs, namely monascorubrin and rubropunctatin, were obtained with a purity of 91.9%. Then, via the azaphilic addition reaction, 18 types of red MPs congeners carrying different amino acid moieties (MPs-aa) were semi-synthesized. Compared to rubropunctamine and monascorubramine, the water solubility, pH and thermal stability of MPs-aa were improved greatly. MPs-His, MPs-Phe, MPs-Tyr and MPs-Trp were identified to be the most resistant to pasteurization. These findings provide water-soluble red MPs candidates with high thermal stability and an attractive approach for their large scale production.

Transportation of citrinin is regulated by the CtnC gene in the medicinal fungus Monascus purpureus.

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).

Histone Acetyltransferase Rtt109 Regulates Development, Morphogenesis, and Citrinin Biosynthesis in Monascus purpureus.

Histone acetyltransferase (HAT) has been reported to be pivotal for various physiological processes in many fungi. However, the functions that HAT Rtt109 perform in edible fungi Monascus and the underlying mechanism remains unclear. Here, we identified the rtt109 gene in Monascus, constructed the rtt109 knockout strain (Δrtt109) and its complementary strain (Δrtt109:com) by CRISPR/Cas9 methods, and functionally characterized the roles that Rtt109 play in Monascus. Deletion of rtt109 significantly reduced conidia formation and colony growth, whereas, it increased the yield of Monascus pigments (MPs) and citrinin (CTN). Further real-time quantitative PCR (RT-qPCR) analysis indicated that Rtt109 remarkably affected the transcriptional expression of key genes related to development, morphogenesis, and secondary metabolism of Monascus. Together, our results revealed the critical roles of HAT Rtt109 in Monascus, and enriched our current knowledge of the development and regulation of secondary metabolism in fungi, throwing light on restraining or eliminating citrinin in the development and industrial applications of Monascus.

Characterization of the Red Biochromes Produced by the Endophytic Fungus Monascus purpureus CPEF02 with Antimicrobial and Antioxidant Activities

Food acceptability and appeal are significantly influenced by colour. Harmful effects associated with synthetic colorants are well established, and research is currently focused on developing natural, synthetic chemical-free substitutes from fungal sources, with broad applications in food, medicine, textiles and agriculture. Additionally, the market’s dearth of natural red colour substitutes requires the creation of novel red pigment alternatives from secure and scalable sources. The goal of the current research was to establish new endophytic marine fungi that are naturally occurring bio-sources of the red pigment. Based on its profuse extracellular red pigment-producing capacity, the fungus CPEF02 was selected and identified as Monascus purpureus CPEF02 via internal transcribed spacer (ITS) sequences and phylogenetic analysis. The chemical moieties of the pigmented extracts were identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The optimal culture conditions for maximum pigment production were investigated by surveying various media compositions. The methanolic fungal colourant extract was shown to have substantial antibacterial and antifungal activities against anthropogenic pathogens, Staphylococcus aureus (MTCC 1430), methicillin-resistant Staphylococcus aureus (ATCCBAA811), Salmonella typhimurium (MTCC 3241) and Vibrio cholerae (N16961) at a 100 µg/mL concentration and at a 1 mg/mL concentration for Alternaria solani (ITCC 4632) and Rhizoctonia solani (AG1-IA). This extract also exhibited antioxidant activity against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical with an IC50 of 14.42 µg/mL and a Trolox equivalent antioxidant capacity of 0.571 µM Trolox/µg of the methanolic colourant extract. The findings suggested that M. purpureus’s pigment could be a source of an industrially useful natural red colourant.

Secondary Metabolites with Antifungal Activities from Mangrove Derived Fungus Monascus purpureus WMD2424

The mold Monascus, also called red yeast rice, anka, or koji, has been used as the natural food coloring agent and food additives for more than 1000 years in Asian countries. It has also been used in Chinese herbology and traditional Chinese medicine due to its easing digestion and antiseptic effects. However, under different culture conditions, the ingredients in Monascus-fermented products may be changed. Therefore, an in-depth understanding of the ingredients, as well as the bioactivities of Monascus-derived natural products, is important. Here, through the thorough investigation into the chemical constituents of M. purpureus wmd2424, five previously undescribed compounds, monascuspurins A-E (1-5), were isolated from the EtOAc extract of mangrove-derived fungus Monascus purpureus wmd2424 cultured in RGY medium. All the constituents were confirmed via HRESIMS and 1D- and 2D-NMR spectroscopy. Their antifungal activity was also evaluated. Our results showed that four constituents (compounds 3-5) possessed mild antifungal activity against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of the type strain Monascus purpureus wmd2424 has never been studied.

Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering

Monascus pigments have various food industry applications and are pharmacologically active. Genome sequencing-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been implemented to increase pigment production in Monascus. To increase pigment production in M. ruber KACC46666, the CRISPR/Cas9 system was used to introduce mutations in two negative regulator genes (MpigI and MpigI′), among other genes involved in the Monascus pigment biosynthesis pathway. Dual single-guide RNAs were constructed to inactivate MpigI and MpigI′. After CRISPR/Cas9 inactivation, yellow, orange, and red pigment expression in the resulting △MpigI16-7 strain (among several Cas9-mediated mutants studied) was 2.5-, 12.4-, and 18.5-fold, respectively, higher than that in the wild-type strain. This study provides valuable information regarding CRISPR-guided metabolic engineering for natural colorant production.

Inhibitory effect of Monascus purpureus pigment extracts against fungi and mechanism of action

The fungus Monascus produces several secondary metabolites of different pigment hues. These pigments have shown various biological activities. In this study, Monascus purpureus pigment extracts were tested (in vitro) against Penicillium expansum MTCC 4900, Rhizopus stolinfer MTCC 10595, and Aspergillus niger MTCC 8652 for antifungal activity. The UV–visible spectrum of M. purpureus fermented rice extracts showed λmax at 395, 425, and 500 nm. This indicated the solubility of yellow, orange, and red pigments in polar-based solvent extraction. The M. purpureus pigment extracts inhibited the radial growth and conidial germination of the test fungi. The fungi treated with pigment extract stained with DiBAC (a vital stain) emitted green fluorescence under a fluorescent microscope. These results indicated that the pigment extracts have affected the membrane potential of the treated fungi. Hence, the fungicidal activity of the pigment extracts is due to the disruption of the cell membrane. The HPLC analysis of the pigment revealed the presence of two major peaks. The UV–visible spectrum corresponding to the HPLC peak at 12-min retention time revealed the presence of orange pigment rubropunctatin. Apparently the rubropunctatin present in the extracts exhibited fungicidal activity. Further studies are warranted to assess the applications of M. purpureus pigments in preventing and treating fungus-related diseases.

Use of common carp waste for pigment production by Monascus purpureus

Fish processing worldwide generates a large amount of waste that has no further use. In this work, we tested the possible biotechnological use of two waste materials from common carp – waste scales and residues from filleting process. Both sources were subjected to alkaline hydrolysis and subsequently used as a source of nitrogen, phosphorus and other elements for the cultivation of the fungus Monascus purpureus, which is known for the production of pigments with potential in pharmaceutics and other sectors of industry. After 14 day-long cultivation of Monascus purpureus in a medium containing hydrolysate as the sole source of all nutrients except carbon, the extraction of pigments and their further analysis were carried out. It was shown that a simple as well as inexpensive alkaline hydrolysis can be used to prepare a complex culture medium suitable for pigment production using fish waste. Moreover, there is a big potential that this medium would also be suitable for many more biotechnological applications.

Application of Cellulase for Contributing Phenolic Release and Conversion in Oats (Avena sativa L.) During Microbial Fermentation.

In this work, Monascus fermentation and cellulase hydrolysis (MCF) of oats (Avena sativa L.) to release and convert phenolic fraction was investigated. Results showed the fungus Monascus grew well with a biomass of 27.03mg/g glucosamine equivalent in MCF, following the destruction of oat cellular structures. SDS-PAGE revealed lots of enzymes were regulated with the α-amylase and FPase activity achieved 139.25 U/g and 1.84 U/g in MCF, respectively. Compared with unfermented oats, content of the total phenolic fractions was increased by 19.2 times in MCF, suggesting a phenolic release process occurred during fermentation. Moreover, the soluble-free chlorogenic acid upregulated to 510.00mg/kg whereas the insoluble-bound ferulic acid downregulated to 193.36mg/kg in MCF, indicating a transformation process of chlorogenic acid from ferulic acid in oats was enhanced. Based on this, a possible pathway of phenolic release and conversion in oats during fermentation with Monascus spp. was revealed. This study was helpful to enrich the theory of microbial metabolism and transformation in grain materials.

Physiological Parameters, Intestinal Microbial Population and Internal Organ Weight of Broilers Supplemented with the Fungus Monascus Purpureus

Physiological Parameters, Intestinal Microbial Population and Internal Organ Weight of Broilers Supplemented with the Fungus Monascus Purpureus

Monascus sp. Pigment Potency as Simple Preservative in Food Ingredients

Everyday foodstuffs such as tofu, chicken, and fish are ingredients that have a short shelf life if stored outdoors or in the open air. The fungus Monascus sp. is a fungus whose pigment is often used in giving color to food. This study aims to determine the potential of Monascus sp pigment isolated from commercial red rice (Angkak) as a preservative in tofu, chicken, and fish at room temperature. The methods used included isolation of the fungus Monascus sp from commercial red rice (Angkak), macroscopic and microscopic morphological characterization, solid fermentation using rice, harvesting pigments with water solvent, and pigment resistance tests on tofu, chicken meat, and fish. The results obtained from the application of the crude extract of the Monascus sp. pigment in fish survived for two days, chicken meat lasted up to three days, and tofu lasted four days. Each experiment had different results during the organoleptic test and compared to a positive control, namely turmeric, proving that turmeric was still better at resisting food spoilage. This research needs to be investigated further regarding the potential if the pure extract of the Monascus sp pigment is used.

Mrada3 is required for sexual reproduction and secondary metabolite production in industrial fungi Monascus strain.

Monascus spp. are valuable industrial fungi for producing beneficial compounds. Because sporulation is often coupled with the production of secondary metabolites, the current study was performed to investigate how Mrada3 regulated asexual and sexual development and the production of edible pigments and mycotoxin. The functional characteristics of Mrada3 were identified by gene deletion and overexpression in Monascus ruber M7 (the wild-type, WT). The results revealed that the ΔMrada3 strain aborted sexual development, but it produced many more conidia than WT. RNA-seq data showed that the deletion of Mrada3 altered the expression levels of partial genes involved in sexual and asexual development. In addition, the deletion of Mrada3 also resulted in slower growth, lower pigment production and increased citrinin yield during the late period. For the Mrada3-overexpressed strain, the number of ascospores and pigment content were significantly higher than those of WT, but citrinin was slightly lower than that of WT. The Mrada3 gene plays a vital role in the sporulation development and secondary metabolism of Monascus species. Mrada3 is first identified as an essential regulator for sexual development in Monascus species, enriching the regulatory knowledge of sexual development in filamentous fungi.

Comparative transcriptome analysis of Monascus purpureus at different fermentation times revealed candidate genes involved in exopolysaccharide biosynthesis

Exopolysaccharides (EPS), metabolites of the medicinal edible fungus Monascus purpureus, have antioxidant, immunomodulatory, and anti-inflammatory effects. However, the biosynthetic mechanism of EPS from M. purpureus is still unclear, which hinders its utilization. In this study, the fermentation conditions of M. purpureus were optimized and comparative transcriptomic analysis was performed to understand the mechanisms and effects of fermentation on EPS synthesis. The optimal medium composition was 40 g/L mannose, 4 g/L yeast powder, 1 g/L MgSO4·7H2O, 0.8 g/L KH2PO4, 1.6 g/L K2HPO4·3H2O, and 2 mL/L Tween 80, and the optimal cultivation conditions were an inoculum of 7 %, culture temperature 30 °C, initial pH 6.0, and 180 rpm for 4 d. A total of 8095 unigenes were obtained, and 17 key enzymes for EPS synthesis were identified. Interestingly, 12 carbohydrate metabolism subcategories were enriched in the group with 4 days of fermentation compared to 2 days, with most of the differentially expressed genes (DEGs) being upregulated, but only nine carbohydrate metabolism subcategories were enriched with longer fermentation time, with all DEGs being downregulated. This study provides a theoretical basis for enhancing the EPS content and reveals the dynamics of EPS synthesis in M. purpureus, providing important targets for future EPS molecular modifications and gene knockdown studies.

Investigation of citrinin and monacolin K gene clusters variation among pigment producer Monascus species

The filamentous fungi Monascus spp. have been widely used in the production of food colorants. However, the presence of mycotoxin citrinin and the antihypercholestrolemia agent monacolin K in Monascus-fermented products (MFPs) has raised food safety concerns. Here we de novo-sequenced the genomes of 26 Monascus species and proposed an unprecedented classification system, consist of sections A, B and C, according to the biosynthetic gene clusters (BGCs) distribution and phylogeny results. Based on the absence of citrinin gene cluster, section B species were genetically incapable of synthesizing citrinin. A distinguished section A strain named Monascus sanguineus was believed to be a promising food-pigment-producer particularly owing to the simultaneous inactivation of citrinin and monacolin K clusters. Interestingly, gene losses within Monascus secondary metabolism gene clusters were broadly discovered, which may convey a selective advantage in nutrients and energy competition to support the strong pigment-producing ability. Overall, our sectional delimitation system will reshape the industrial strategies for this economically important fungus.