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Classification

Higher order taxa

Eukaryota; Ascomycota; Eurotiomycetes; Eurotiales; Aspergillaceae; Monascus

Species

NCBI: [1]

Monascus purpureus

Description and significance

Monascus purpureus is an edible, xerophilic fungus known for its ability to grow on various agricultural crops, including rice and corn [7]. M. purpureus is most notable for its use in producing red yeast rice - a traditional fermented dish popular in East Asia - as well as for other myriad potential culinary and medicinal properties [7]. M. purpureus produces several secondary compounds including Monascus pigments (MP), that are used in the food industry as a natural alternative to artificial dyes and color enhancers. Current research focuses on the pigment’s stability in living systems under challenging conditions and changes such as acidic pH levels and increased temperatures [2].

The use of these pigments - particularly in the food industry - has garnered controversy due to the risk of contamination by citrinin, a secondary metabolite produced by M. purpureus, which has potential nephrotoxic (poisonous to the kidneys) properties [3]. Despite these concerns, MP’s potential anticancer properties are also being investigated27. Preliminary research has shown that M. purpureus can exhibit cancer-specific cytotoxicity, unlike some current cancer treatments [4].

Additionally, M. purpureus produces monacolins, a group of compounds that work to lower cholesterol and triglyceride levels in humans. A highly studied monacolin, monacolin K, appears to be chemically similar to a popular LDL (Low-Density Lipoprotein) cholesterol-lowering agent, Lovastatin, which introduces the potential of using red rice supplements in future treatment plans [3].. Despite the many potential medicinal and culinary properties M. purpureus from red yeast rice holds, there is debate about the safety and efficacy of using this species due to its potential nephrotoxic and myopathic-inducing properties.

Genome structure

The genome of M. purpureus has been fully sequenced. The genome is 23.44 Mbp long and, contains approximately 8,918 genes [5].. The genome’s guanine (G) and cytosine (C) content ranges between 49-52% [5].. The average gene length is 1815 base pairs (bp), with an average transcript length of 1626 bp5. The genome has an average exon length of 509 bp, accounting for the coding region, and the average intron length is 88 bp [5]. M. purpureus has protein-coding genes that are responsible for producing a wide range of proteins, including those involved in the production of secondary metabolites [6]. Notable secondary metabolites include monacolin K, citrinin, and M. purpureus pigments. Monacolin K is thought to be chemically similar to the LDL cholesterol-lowering agent, Lovastatin, and inhibits HMG- CoA reductase, the enzyme responsible for cholesterol production in the liver [6]. Monacolin K has been shown to effectively lower both total and LDL cholesterol level [6]. Citrinin is a mycotoxin (a substance that is produced by fungi that is toxic to humans) and is potentially carcinogenic (cancer-causing) and nephrotoxic (a substance that causes a decline in kidney function). Citrinin contamination is a concern in food safety and is one of the main issues limiting the use of M. purpureus fermentation products. Efforts are being made to develop M. purpureus strains that produce minimal or no citrinin while maintaining desirable metabolite production, such as Monacolin K [6]..

Cell structure

Monascus purpureus is a filamentous fungus that exhibits unique morphological characteristics. In the cell’s vegetative phase, it forms long, branch-like hyphae (or mycelia) that resemble strands, clumps, or pellets. The mycelium absorbs nutrients through apical growth[7]. Hyphal growth occurs at the tips, or apical regions, allowing the organism to spread into new areas. Each section of the hyphae is divided by septa, and each of these smaller parts houses multiple nuclei within each segment [7].

The cell wall of this fungus is enriched with chitin. The presence of chitin synthase enzymes maintains structural integrity of the fungal cells and influences both hyphal morphology and reproductive capacity through mycelial aggregation and spore formation [7]. Initially, M. purpureus presents as white mycelium but transitions to orange and red as it matures [7].

M. purpureus can reproduce both sexually and asexually. In sexual reproduction, it forms cleistothecia - spherical structures that contain ascospores, that facilitate the spread of the fungus, measuring approximately 5-6 microns wide. In asexual reproduction, M. purpureus generates spores called conidia, through specialized aerial hyphae [5].

5. Metabolic processes

Monascus purpureus is an acid-tolerant chemoheterotroph, deriving both energy and carbon from organic compounds such as glucose and starches - most commonly from rice - through fermentation [8], [9]. M. purpureus employs glycolysis to break down glucose into pyruvate, which produces ATP [8]. M. purpureus is both anaerobic and aerobic: in aerobic conditions, the pyruvate will enter the Krebs Cycle, but in anaerobic conditions, the fungus will utilize fermentation to generate ATP by converting pyruvate into products such as ethanol and other organic acids.

M. purpureus’s social and economic value comes from the beneficial secondary metabolites that it produces such as monascin, ankaflavin, monascorubrin, rubropunctatin, monascrorubramine, rubropunctamine, and most notably monacolin K, which act as a statin. The other compounds have various properties such as serving as antioxidants, the ability to lower cholesterol, and preventing diabetes in vulnerable populations [10]. M. purpureus pigments are mixtures of yellow, orange, and red pigments that are primarily composed of azaphilones, a naturally occurring chemical compound in fungi [7]. These pigments have been used for centuries as natural food colorants in Asian countries, but now have shown potential health benefits due to their antimicrobial (against both Gram-positive and Gram-negative bacteria), anticancer, as well as anti-inflammatory properties [7].

Ecology

Monascus purpureus is an edible and acid-tolerant fungus widely recognized for its role in the production of red fermented rice, as well as its adaptability and ability to thrive under both aerobic and anaerobic environments [11]. It grows particularly well in warm and humid environments, particularly in soil and decaying organic material across China and Japan. It is used in the preparation of a traditional dish called red fermented rice or red yeast rice [11].

The optimal growth conditions for M. purpureus include a temperature range of 25-45°C and a pH range of 2.5-8.0 [12]. It thrives in aerobic conditions with an oxygen partial pressure of 0.5 × 10^5 Pa, although it is also capable of anaerobic fermentation [13]. M. purpureus thrives in grain habitats, or environments rich in starch and sugar. Under these conditions, the fungus produces high pigment yields, using glucose and maltose as primary carbon sources [13].

Pathology

Although Monascus purpureus is widely recognized for its beneficial role in food fermentation across Asian countries, it remains prohibited in Europe and America. This is due to possible pathogenic properties of its secondary metabolites, specifically the mycotoxin, citrinin [14]. This compound can be produced during fermentation. Citrinin has a predicted LD50 of 105mg/kg citrinin per kilogram body mass in humans, meaning half of the individuals exposed will die to a dose of that quantity [14], [6]. Due to its nephrotoxic and hepatotoxic characteristics, it can pose significant health risks to both humans and animals when consumed at high levels [14], [6]. This concern has led to the monitoring of citrinin levels in red yeast rice and the establishment of maximum tolerance limits for citrinin in food among Asian countries that utilize M. purpureus [14], [2]. Despite the potential toxicity of citrinin, M. purpureus is not classified as pathogenic so long as the exposure level to its mycotoxins remains within safe limits., although the exact value varies by country [2]. When proper precautions are taken, the benefits of using M. purpureus in the fermentation process often outweigh the risks associated with citrinin. Citrinin formation can be overcome through various methods, including genetic modification of the producing strain or selection of citrinin-non-producing strains of the fungus. A recent study showed that up to 92% of citrinin content can be removed using ethanol-phosphate extraction while retaining 80% of the monacolin K [15]. Despite the considerable economic benefits of Monascus pigment, its commercial exploitation in the Western world remains limited largely due to a lack of awareness and reluctance from food public agencies. However, in recent years, several patents have been registered in countries such as France, Germany, and the United States that focus on the use of Monascus pigments as natural food pigment [16].

Current Research

Monascus purpureus is known to produce several beneficial pigments and other byproducts such as monascin, ankaflavin, monascorubrin, rubropunctatin, monascrorubramine, rubropunctamine, and most notably monacolin K [17], [18], [19], [20], [21], [22], [23]. Current research on this organism is focused in three main areas: medicinal product usage, controlling product and pigment production, and the exploration of alternative benefits to humans. Medicinal research has been centered around the safety of the existing well-understood compounds (monascin, ankaflavin, monascorubrin, rubropunctatin, monascrorubramine, rubropunctamine, monacolin K, etc.) Some Monascus compounds have anticancer properties which are of interest. Some antioxidant compounds from M. purpureus work by increasing the effect of other cancer drugs, while somewhat attenuating the effect on normal tissues [18], [21], [27]. Others work through independent cytotoxicity against cancer cells, with one, a novel compound called Monaspin B being able to cause apoptosis in leukemia cancer cells [18], [21], [28]. Ankaflavin, another Monascus pigment, also has antitumorigenic properties, inhibiting the ability of some cancers to metastasize [18]. There is also interest in more recently discovered compounds such as Monascuspirolide, which has been shown to have photoprotective effects [19], [24], [25], [26]. Once sufficient research has been conducted on the safety and efficacy of M. purpureus products, they may be commercialized to leverage their beneficial attributes such as their anticancer properties, ability to lower cholesterol, transform inorganic selenium into organic selenium, and prevent diabetes in vulnerable populations [21], [27], [28], [20], [23], [18].

There is a growing interest in how to control the production of pigments produced by M. purpureus and optimize the culture protocol of M. purpureus [30], [31], [32], [33]. The growth of M. purpureus and the production of its various metabolites can be affected by changes in temperature, humidity, pH, oxygen concentration, the addition of specific chemicals such as fatty acid sodium octanoate or a surfactant, substrate, nitrogen source, carbon source, cocultivation with other species, gene knockouts and manipulation and disruption of biosynthesis pathways, electrolytic stimulation, certain wavelengths of light, and even magnetic fields [18], [28], [30], [33], [34].

These changes can alter the rate of pigment production, the rate of release of these pigments, the ratio/distribution of pigments synthesized, as well as the growth rate of M. purpureus [18]. For instance, high temperatures typically reduce pigment production, although it may increase the production of yellow pigments specifically [18]. pH also has a major effect, with low pH favoring Yellow pigment synthesis, and high pH favoring red pigments [18]. Changes in chemical additives and growth materials can also have an impact [18]. The addition of a surfactant in submerged fermentation, for example, can increase the permeability of the cell wall, resulting in increased release of intracellular pigment [18]. Research suggests that rice is one of the best substrates for solid-state fermentation, but jackfruit seeds or cassava bagasse may be cheaper [18]. Nitrogen is required for water-soluble pigment production, but increasing nitrogen concentration can decrease the production of red pigments [18]. Carbon source is also important, glucose and its oligosaccharides are good carbon sources for growth and production, but high concentrations of glucose can reduce ethanol production, pigment synthesis, and growth rate [18].

There has also been research into other methods of affecting M. purpureus pigment synthesis and production. One method, cocultivation, that is, growing M. purpureus with another species, specifically Aspergillus oryzae can produce novel compounds (Monaspins A and B) due to the production of enzymes from one species catalyzing the reaction of metabolites produced by the other [28]. Knowing that morphology can also affect production, increasing the production of yellow pigments, some researchers have deleted genes that code for chitin, a major structural component, to control the morphology, reducing citrinin production as well as pigment production [30]. One study found that electrically stimulating M. purpureus can disrupt certain amination reactions, causing a relative increase in yellow pigment production [33]. Other studies have found that both light and magnetic fields can affect pigment production and Monascus growth, with low-frequency magnetic fields increasing the production of pigments while reducing citrinin production [34]. Despite all the research on optimizing and tailoring production, one challenge in the industrialization of M. purpureus is finding ways to prevent the excessive production of citrinin.

Current research is focused on boosting the production of beneficial compounds such as monacolin K while minimizing or eliminating citrinin production.15, 31Some methods have already been discussed, such as knocking out chitin synthesis genes, other methods include finding strains with naturally low levels of citrinin production.30,31 In the absence of existing strains, some studies seek to induce mutagenesis to mutate a strain lacking citrinin production genes [15].

Research has also centered around the discovery of more M. purpureus products, either those that have yet to be identified, products from using different materials to culture M. purpureus, or products made from new or different strains of M. purpureus [31]. This work includes the relatively novel method of cocultivation, when products are only produced when M. purpureus is grown alongside another microorganism, typically another fungus. This research has led to the discovery of an anti-leukemia compound produced by growing a strain of M. purpureus together with Aspergillus oryzae (A. oryzae) [28].