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The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of β-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.

This study aimed to explore the production of red pigment from Monascus purpureus in waste culture medium and its potential health benefits. Subsequently, the M . purpureus cultivated in a medium containing dairy sludge as waste, the extracted pigment was purified, and subjected to various analyses, including liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) to verify its purity, high-pressure liquid chromatography (HPLC) to measure the citrinin levels, microbial, and antioxidant activity. Finally, fermentation was conducted in a batch system using a fermenter. M . purpureus was grown in a medium composed of dairy sludge, monosodium glutamate, and glucose, resulting in a biomass yield of 26.15 g/L. After extraction and purification, the sample yielded 4.85 g of dry color. Analysis confirmed the purity of the pigment by LCMS and NMR and revealed low citrinin levels by HPLC. In the fermenter, the sample obtained from enriched culture conditions displayed the highest concentration of monascorubramine, maximum specific growth rate of 0.029/1/h, a cell yield (Y x/s ) of 0.29 g/g, and a production efficiency of 65% for M . purpureus . The produced pigment sample showed potential for use in the food industry due to its low citrinin content and high concentration of red pigment.

More and more people pay attention to citrinin produced by Monascus, which has nephrotoxic activity in mammals. It was reported that pksCT gene is responsible for citrinin biosynthesis in Monascus purpureus. In this paper, two DNA fragments in both ends of pksCT were amplified by genomic PCR from fourteen Monascus spp. strains. The PCR products were gained from all of the strains. It is suggested that pksCT gene was highly conserved in different citrinin-producing Monascus strains. A pksCT-replacement vector (pHD106) was constructed to disrupt pksCT with a hygromycin resistance gene as the selection marker, and was transformed into M. aurantiacus Li AS3.4384. Three transformants (M. aurantiacus PHDS18, PHDS26, PHDS31) were selected from transformant selective plates. The targeting fragment D was gained by genomic PCR from PHDS18 and PHDS26 except PHDS31. The expressing citrinin capacities of PHDS26 was decreased by about 98%, while PHDS18 was reserved the high capacity of producing citrinin, after 10 days of growth on YM medium. The results indicated that PHDS26 is a pksCT-disrupted strain. There are maybe other genes besides pksCT responsible for citrinin biosynthesis in M. aurantiacus. It is the effective way to solve the problem of citrinin in M. aurantiacus products by constructing replacement vectors to disrupt the genes responsible for citrinin biosynthesis to reduce the capacity of expressing citrinin.

Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin biosynthesis in Penicillium marneffei , a diffusible red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate than Monascus species. We also documented that the red pigment of P. marneffei is a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin and showed that this polyketide gene cluster and pathway are also responsible for biosynthesis of ankaflavin and citrinin, a mycotoxin with nephrotoxic activity in mammals. The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus . MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA , and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Absent, small, or homeotic discs 2 (Ash2), a histone H3K4 methyltransferase complex, has been implicated in the control of hyphal development and secondary metabolism in many kinds of filamentous fungi. We constructed an Ash2 deletion mutant (ΔAsh2) by using an Agrobacterium-mediated gene knockout method to investigate the function of the Ash2 gene in the mold Monascus purpureus. Lack of the Ash2 gene resulted in the formation of a lower colony phenotype with fluffy aerial hyphae that autolyzed as the colony grew on potato dextrose agar at 30°C. The production of pigments and the number of conidia were significantly lower in the ΔAsh2 than in the wild type. Citrinin production by the ΔAsh2 was not detected during 15 days of fermentation. Relative expression levels of secondary metabolite regulatory genes PigR and CTNR, secondary metabolite synthesizing genes PKSPT and CTN, key genes of mitogen-activated protein kinase pathway Spk1 and its downstream gene mam2, the conidium development control gene BrlA, and global regulatory genes LaeA and VeA were detected by the quantitative real-time PCR. These results indicate that the Ash2 gene is involved in conidial germination, pigment production, and citrinin production and plays a key role in development and secondary metabolism in M. purpureus.

Background Monascus mycelia and pigments are promising sources of food and medicine with their potential pharmaceutical values and health-improving functions. Using high cell density fermentation of Monascus spp. to achieve higher mycelium and yellow pigment production is worthy to be researched. In this study, the characteristics and productivity shifting of pigments in high cell density culture of Monascus anka GIM 3.592 were investigated. Results The high yield of Monascus mycelia up to 39.77 g/L dry cell weight (DCW), which was achieved by fed-batch fermentation with the feeding medium containing C, N, P and trace elements, was four times higher than that of conventional batch culture. But the total pigment production decreased by 14.6 %, which suggested non-coupled growth. Potential novel yellow pigments accumulated constantly at the late stage of the fed-batch culture, which resulted in a shift in pigment characteristics so that yellow pigments became the dominant pigments. Citrinin production was extremely low and independent of feeding ingredients. Conclusions This study provided a suitable fermentation strategy to produce functional Monascus mycelia with a high proportion of yellow pigments in high cell density culture. For the first time, it reported the pigment productivity and characteristics shifting in high cell density culture of Monascus .

The effect of light on Monascus and the underlying mechanism have received a great deal of interest for the industrial application of Monascus pigments. In this study, we have examined the effects of blue light on the culture morphology, mycelium growth, pigments, and citrinin yield of Monascus in liquid-state and oscillation fermentation, and explored the mechanism at a physiological level. It was found that blue light affected the colony morphology, the composition (chitin content), and permeability of the Monascus mycelium cell wall in static liquid culture, which indicates blue light benefits pigments secreting from aerial mycelium to culture medium. In liquid oscillation fermentation, the yields of Monascus pigments in fermentation broth (darkness 1741 U/g, blue light 2206 U/g) and mycelium (darkness 2442 U/g, blue light 1900 U/g) cultured under blue light and darkness are different. The total pigments produced per gram of Monascus mycelium under blue light was also higher (4663 U/g) than that in darkness (4352 U/g). However, the production of citrinin (88 μg/g) under blue light was evidently lower than that in darkness (150 μg/g). According to the degradation of citrinin caused by blue light and hydrogen peroxide, it can be concluded that blue light could degrade citrinin and inhibit the catalase activity of Monascus mycelium, subsequently suppressing the decomposition of hydrogen peroxide, which is the active species that degrades citrinin.

Citrinin is a toxic secondary metabolite of Penicillium citrinum and its contamination in many food items has been widely reported. However, research on the citrinin biosynthesis pathway and its regulation mechanism in P. citrinum is rarely reported. In this study, we investigated the effect of different carbon sources on citrinin production by P. citrinum and used transcriptome analysis to study the underlying molecular mechanism. Our results indicated that glucose, used as the sole carbon source, could significantly promote citrinin production by P. citrinum in Czapek’s broth medium compared with sucrose. A total of 19,967 unigenes were annotated by BLAST in Nr, Nt, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Transcriptome comparison between P. citrinum cultured with sucrose and glucose revealed 1085 differentially expressed unigenes. Among them, 610 were upregulated while 475 were downregulated under glucose as compared to sucrose. KEGG pathway and Gene ontology (GO) analysis indicated that many metabolic processes (e.g., carbohydrate, secondary metabolism, fatty acid and amino acid metabolism) were affected, and potentially interesting genes that encoded putative components of signal transduction, stress response and transcription factor were identified. These genes obviously had important impacts on their regulation in citrinin biosynthesis, which provides a better understanding of the molecular mechanism of citrinin biosynthesis by P. citrinum.

As traditional edible fungi, Monascus spp. have been widely used as folk medicine, food colorants, and fermentation starters in East Asian countries for more than a thousand years. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, raises suspicions about the safety of Monascus products. Citrinin biosynthesis in Monascus is known to occur via a polyketide pathway and a citrinin biosynthesis gene cluster, which include the characterized polyketide synthetase pksCT . A gene, orf6 , encodes a protein that shows significant similarity to glyoxalase and is located between ctnE and orf1. This study analyzed orf6 function, and successfully obtained an orf6 disruption strain (Δ orf6 ). Citrinin production was significantly greater (3.6-fold) in the Δ orf6 strain than in the wild-type Monascus purpureus YY-1, and RT-PCR analysis further revealed increased expression of numerous genes of the citrinin biosynthesis gene cluster in Δ orf6 . Therefore, orf6 proved to be a major inhibitor, directly involved in citrinin biosynthesis. Moreover, pigment production in Δ orf6 was reduced by approximately 30%, while the transcription levels of many genes involved in Monascus pigments (MPs) biosynthesis had increased. This dichotomy indicated that MPs and citrinin yields may be improved simultaneously; however, a portion of the pigments was consumed to protect the cells from oxidative damage in the Δ orf6 strain . An Δ orf6 revertant restored the citrinin and pigment yields to normal levels. This study makes a contribution to explore the citrinin biosynthesis pathway and provides some theoretical guidance to improving the safety of Monascus -related products.