Characterization of the asexual developmental genes brlA and wetA in Monascus ruber M7.

Monascus spp. are widely used in the production of monacolin K and food- grade pigments in East Asia. In Aspergillus species, the three transcription factors BrlA â†’ AbaA â†’ WetA sequentially function as the central activators of asexual development (conidiation), leading to the formation of conidiophores. Unlike their close relative Aspergillus spp., Monascus spp. produce basipetospora-type asexual spores (conidia), and their genomes contain homologs of brlA and wetA but not abaA. In the present study, to investigate their roles in Monascus conidiation, MrbrlA and MrwetA were functionally characterized by gene knockout and overexpression in Monascus ruber M7. The results revealed that the deletion and overexpression of MrbrlA and/or MrwetA caused no apparent changes in the morphology, size, number, structure, or germination of conidia. However, deletion and overexpression of MrwetA severely repressed sexual development and affected the production of secondary metabolites. Taken together, these results suggest that the well-established central regulatory model of conidiation in Aspergillus is not applicable in their Monascus relatives. The results of the present study could enrich our understanding of the asexual development regulatory networks in filamentous fungi.

Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation.

Extreme environments, for example high-salt-stress condition, that can induce secondary metabolite biosynthesis in fungi are a promising and effective strategy for producing natural Monascus pigments used as food colourants and nutraceutical supplements. In this study, the relationship between the mycelial morphology and expression of pigment biosynthetic genes in high-salt-stress fermentation (HSF) with Monascus ruber CGMCC 10910 was investigated. The Monascus fungus grew well under HSF conditions with 35 g/l NaCl, and the intracellular yellow pigment yield in HSF was 40% higher than that in conventional batch fermentation (CBF). Moreover, the mycelial morphology was maintained in a better state, with a hyphal diameter of 5-6 µm in HSF, indicating good biocatalytic activity for pigment synthesis. The rate of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p < 0.05) changed, and the extracellular yellow pigments were transformed into each other, indicating that the pigment biosynthesis pathway was changed to promote yellow pigment accumulation in HSF. The pigment biosynthesis genes MpPKS5, MpFasB2, mppE, mppD and mppB were significantly (p < 0.05) up-regulated by approximately 58.4-106.1%, whereas the regulatory genes mppR1 and mppR2 were significantly (p < 0.05) down-regulated by approximately 23.2% and 59.0% in HSF. Notably, the mppE gene was highly correlated with (r > 0.95, p < 0.05) hyphal diameter. These findings indicated that the cultivation of the Monascus fungus under high-salt-stress conditions was beneficial for pigment biosynthesis by controlling the mycelial morphology to regulate gene expression. This study first described the relationship between the mycelial morphology and expression of pigment biosynthetic genes in Monascus during fermentation. KEY POINTS: • High-salt-stress fermentation (HSF) was first performed to improve Monascus pigment yield. • Pigment biosynthesis was enhanced by maintaining the mycelial morphology in an improved state in HSF. • Gene expression was up-/downregulated to promote yellow pigment accumulation in HSF. • The mycelial morphology was highly related to the expression of pigment biosynthetic genes in HSF.

Parameters of the fermentation of soybean flour by Monascus purpureus or Aspergillus oryzae on the production of bioactive compounds and antioxidant activity.

The objective of this work was to evaluate the effects the solid-state fermentation parameters of defatted soybean flour (DSF) by Monascus purpureus or Aspergillus oryzae on the bioactive compounds. Central composite rotatable design, multi-response optimization, and Pearson's correlation were used. The fermentation parameters as initial pH (X1), DSF-to-water ratio (X2), and incubation temperature (X3) were taken as independent variables. The function responses were isoflavone content, total phenolic content (TPC), and antioxidant activity. All fermentation parameters affected the isoflavone content when fermented by Monascus purpureus, whereas the TPC or antioxidant activities remained almost unchanged. For the fermentation by Aspergillus oryzae, all the function responses were influenced by X2 and X3 and were independent of the X1. Estimated optimum conditions were found as x1 = 6.0, x2 = 1:1, and x3 = 30 °C for both fungi. Achieving suitable fermentation parameters is essential to increase bioactive compounds in the DSF that makes it promising for food industrial applications.

Efficient Biosynthesis of Natural Yellow Pigments by Monascus purpureus in a Novel Integrated Fermentation System.

Because of the increasing demand for healthy and safe food, Monascus spp. have gained much attention as a sustainable source of natural food colorant. In this study, a novel integrated fermentation system consisting of surfactant and in situ extractant was established for efficiently producing yellow pigments by M. purpureus sjs-6. The maximum production of Monascus yellow pigment (669.2 U/mL) was obtained when 40% soybean oil (as extractant) was supplied at the beginning and 5 g/L Span-80 (as surfactant) was supplied at the 72nd h, which resulted in production 27.8-times of that of the control. Critical factors such as alleviating the product inhibition, increasing the membrane permeability, changing the hyphal morphology, and influencing the cell activity have been suggested as the underlying mechanisms. This system is of great significance for the bioprocess, which suffers product inhibition, and it can serve as a promising step for enhancing the yield of hydrophobic metabolites.

mrskn7, a putative response regulator gene of Monascus ruber M7, is involved in oxidative stress response, development, and mycotoxin production.

Skn7, a response regulator (RR), is associated with oxidative stress adaptation, hypo-osmotic stress response, fungicide sensitivity, cell wall biosynthesis, cell cycle regulation, sexual mating, and sporulation in many filamentous fungi and yeasts. In this study a Skn7-like protein gene mrskn7 (Monascus ruber skn7) was isolated, sequenced, and disrupted to investigate its function in M. ruber Bioinformatics predicted that the deduced protein encoded by mrskn7 contained the conserved DNA-binding and signal-receiver domains similar to the Skn7-like protein structure in other filamentous fungi. The Δmrskn7 strain produced fewer conidia and less mycotoxin, demonstrated increased sensitivity to peroxide but the same level of osmotic resistance to NaCl and glycerol with the wild-type. Additionally, cleistothecia observed at different time point showed a different morphology between the wild-type and the Δmrskn7 strain, suggesting the involvement of mrskn7 in sexual development of M. ruber These results indicated that mrskn7 plays important roles in asexual and sexual development, the production of mycotoxin as well as regulation of oxidative stress signal in M. ruber.

Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 µM) or DFC (0-10 µM) was co-treated with 6-OHDA (200 µM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.

Complete genome sequence and transcriptomics analyses reveal pigment biosynthesis and regulatory mechanisms in an industrial strain, Monascus purpureus YY-1.

Monascus has been used to produce natural colorants and food supplements for more than one thousand years, and approximately more than one billion people eat Monascus-fermented products during their daily life. In this study, using next-generation sequencing and optical mapping approaches, a 24.1-Mb complete genome of an industrial strain, Monascus purpureus YY-1, was obtained. This genome consists of eight chromosomes and 7,491 genes. Phylogenetic analysis at the genome level provides convincing evidence for the evolutionary position of M. purpureus. We provide the first comprehensive prediction of the biosynthetic pathway for Monascus pigment. Comparative genomic analyses show that the genome of M. purpureus is 13.6-40% smaller than those of closely related filamentous fungi and has undergone significant gene losses, most of which likely occurred during its specialized adaptation to starch-based foods. Comparative transcriptome analysis reveals that carbon starvation stress, resulting from the use of relatively low-quality carbon sources, contributes to the high yield of pigments by repressing central carbon metabolism and augmenting the acetyl-CoA pool. Our work provides important insights into the evolution of this economically important fungus and lays a foundation for future genetic manipulation and engineering of this strain.

Effects of koji-making with mixed strains on physicochemical and sensory properties of Chinese-type soy sauce.

BACKGROUND: Two kinds of soy sauces were prepared with Aspergillus oryzae koji (SSAO) and mixed koji (SSAOM, A. oryzae mouldstarter:Monascus purpureus mouldstarter = 1:2, w/w) respectively. The effects of mixed koji on the essential indices, oxygen radical absorption capacity, color indices, free amino acids and volatile compounds of soy sauce have been studied, followed by a sensory evaluation between SSAO and SSAOM. RESULTS: The contents of non-salt soluble solid, reducing sugar, total acid, total nitrogen and amino nitrogen in SSAOM increased by 21.50%, 9.88%, 15.35%, 5.98% and 41.43%, respectively, compared with the control SSAO, owing to the higher activities of acid protease and glucoamylase in the mixed koji. Moreover, SSAOM showed higher antioxidant activity, higher levels of free amino acids and much more attractive color. Meanwhile, flavor groups such as esters, aldehydes, pyrazines and sulfur-containing compounds in SSAOM were also improved. The contents of free amino acids and aroma compounds were consistent with the sensory evaluation. According to descriptive sensory analysis, SSAOM showed higher intensity for sweet and umami attributes; in addition, higher flowery, burnt, fruity and caramel-like attributes were perceived in SSAOM, while SSAO showed higher ethanolic and sour attributes. CONCLUSIONS: The investigated soy sauce prepared with mixed koji can be considered as an effective method to accelerate the fermentation process and improve the flavor of soy sauce.

Insights into Monascus biology at the genetic level.

The genus of Monascus was nominated by van Tieghem in 1884, but its fermented product-red mold rice (RMR), namely red yeast rice, has been used as folk medicines, food colorants, and fermentation starters for more than thousands of years in oriental countries. Nowadays, RMR is widely developed as food supplements around the world due to its functional compounds such as monacolin K (MK, also called lovastatin) and γ-aminobutyric acid. But the usage of RMR also incurs controversy resulting from contamination of citrinin (a kind of mycotoxin) produced by some Monascus strains. In the past decade, it has made great progress to Monascus spp. at the genetic level with the application of molecular biology techniques to restrain the citrinin production and increase the yields of MK and pigment in RMR, as well as aid Monascus classification and phylogenesis. Up to now, hundreds of papers about Monascus molecular biology (MMB) have been published in the international primary journals. However, to our knowledge, there is no MMB review issued until now. In this review, current understanding of Monascus spp. from the view of molecular biology will be covered and insights into research areas that need to be further investigated will also be discussed.

Preeclamptic cord blood hemolysis and the effect of Monascus purpureus and Saccharomyces cerevisiae in modulating preeclamptic stress.

BACKGROUND: Preeclampsia is associated with impaired antioxidant defense that results in materno- fetal complications. In addition to antioxidant deficiency, hemolytic disorder has also been observed in preeclamptic mother. METHODS: This study aims in analyzing the fetal complications using cord blood RBC (red blood cell); further the antihemolytic and antioxidant efficiency of two common probiotic yeasts Monascus purpureus and Saccharomyces cerevisiae in preeclamptic and normotensive RBCs were assessed. RESULTS: There was a significant decrease in the antioxidant status (p<0.05) with increased oxidative stress, nitrative stress (p<0.05) and hemolysis (p<0.001) in preeclamptic RBC comparatively. M. purpureus demonstrated a highly significant reactive oxygen radical scavenging activity (p<0.001) whereas S. cerevisiae exhibited a highly significant nitric oxide radical scavenging activity (p<0.001). It was noted that oxidative stress hemolysis was decreased with increased antioxidant level in cord blood RBC from both samples after incubation with both yeasts in a similar manner. The antihemolytic property of M. purpureus and S. cerevisiae suggests that S. cerevisiae functions efficiently with increasing stress. CONCLUSION: This study demonstrates for the first time that despite their differential scavenging activities, a diet rich in M. purpureus and S. cerevisiae could equally serve as a good natural supplement to alleviate the stress status in the preeclamptic fetus (Tab. 4, Fig. 1, Ref. 39).

Identification of Mga1, a G-protein alpha-subunit gene involved in regulating citrinin and pigment production in Monascus ruber M7.

The filamentous fungi Monascus spp., which have been used in traditional fermented food in Asia for centuries, are well-known producers of a group of bioactive metabolites that are widely used as food additives and nutraceutical supplements worldwide. However, its potential to produce the mycotoxin citrinin poses a threat to food safety. Here, a G-protein alpha-subunit-encoding gene, Mga1 (Monascus G-protein alpha-subunit 1), which encodes a protein showing a high degree of identity to Group I alpha-subunits of fungal heterotrimeric G-proteins, was cloned from Monascus ruber M7. An Mga1-disrupted strain was obtained by homologous recombination. The disruptant produced approximately nine times more citrinin and 71% more pigments than the wild-type strain M7, indicating that the G-protein alpha-subunit encoded by Mga1 is involved in a signal transduction pathway regulating citrinin and pigment biosynthesis in M. ruber M7.

Effect of red mold rice on antifatigue and exercise-related changes in lipid peroxidation in endurance exercise.

This study evaluated the effect of red mold rice supplementation on antifatigue and exercise-related changes in lipid peroxidation of male adult Wistar rats through swimming exercise. Thirty 16-week-old rats were studied by dividing them into three groups (ten for each group). Other than the control group (CD), the other two groups were divided into a high-dose (HD) treatment group (5 g red mold rice/kg body weight for the HD group), and a low-dose (LD) group (1 g red mold rice/kg body weight for the LD group). Swimming endurance tests were conducted after 28 days of red mold rice supplementation, and the result showed that the treatment group showed a higher exercise time (CD, 78.0+/-6.4; LD, 104.2+/-9.6; and HD, 129.4+/-10.9 min; p<0.05) and a higher blood glucose concentration (CD, 76.67+/-8.08; LD, 111.34+/-8.50; and HD, 117.67+/-11.06 mg/dl; p<0.05) than the CD. Moreover, the blood lactate (CD, 45.00+/-0.90; LD, 31.41+/-1.80; and HD, 28.89+/-1.62 mg/dl; p<0.05), blood urea nitrogen (CD, 21.87+/-0.75; LD, 20.33+/-0.83; and HD, 20.53+/-1.09 mg/dl; p<0.05), and hemoglobin (CD, 14.20+/-0.21; LD, 13.70+/-0.55; and HD, 13.28+/-0.35 g/dl; p<0.05) were also significantly lower than those of the CD. Besides, the result suggested that the red mold rice supplementation may decrease the contribution of exercise-induced oxidative stress and improve the physiological condition of the rats.

Efficacy and safety of Monascus purpureus Went rice in subjects with hyperlipidemia.

OBJECTIVE: The purpose of this study was to assess the lipid-lowering effect of Monascus purpureus Went rice on serum lipids in patients with hyperlipidemia, and to assess its safety by reporting adverse events and clinical laboratory measurements. DESIGN AND METHODS: This was a randomized, double-blind, placebo-controlled study. In all, 79 patients (aged 23-65 years) with a mean baseline low-density lipoprotein cholesterol (LDL-C) level of 5.28 mmol/l (203.9 mg/dl) received a twice daily dose of placebo or Monascus purpureus Went rice (600 mg) for 8 weeks. RESULTS: At week 8, Monascus purpureus Went rice therapy reduced LDL-C by 27.7%, total cholesterol by 21.5%, triglycerides by 15.8% and apolipoprotein B by 26.0%. High-density lipoprotein cholesterol and apolipoprotein A-I levels were increased by 0.9 and 3.4% respectively (not significant). No patient in the Monascus purpureus Went rice treatment group had an alanine aminotransferase (ALT), aspartate aminotransferase (AST) or creatine phosphokinase (CPK) measurement that was > or = 3 times the upper limit of normal at week 4 and week 8. CONCLUSION: Monascus purpureus Went rice significantly reduced LDL-C, total cholesterol, triglycerides and apolipoprotein B levels, and was well tolerated in patients with hyperlipidemia. However, this study only provides data from an 8-week trial and long-term safety and efficacy data are needed.

Effect of red mold rice supplements on serum and egg yolk cholesterol levels of laying hens.

Monacolin K is a secondary metabolite of Monascus species and reduces cholesterol levels. This research focuses on the effect of adding red mold rice to hens' diet on cholesterol level in egg yolk and on cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in serum. Forty-eight Hy-line laying hens of 48 weeks of age were studied by dividing them into four groups. Except for the control group, the feed for three other groups contained 2.0, 5.0, and 8.0% red mold rice (monacolin K concentrations were 0.0145, 0.035, and 0.056%, respectively). The experiment lasted 6 weeks. During this period, egg weight and egg production were recorded every day, and cholesterol, triglyceride, HDL, and LDL in serum were measured weekly as well. The result showed that the cholesterol in eggs produced by experimental groups was lower than that of the control group (0%, 194.14 +/- 8.30; 2%, 167.17 +/- 4.34; 5%, 168.93 +/- 9.38; 8%, 183.02 +/- 7.63 mg/egg; p < 0.05), and the triglyceride (0%, 1494 +/- 178; 2%, 1280 +/- 174; 5%, 1189 +/- 248; 8%, 1381 +/- 218 mg/dL; p < 0.05) and LDL levels (0%, 36.81 +/- 5.53; 2%, 32.25 +/- 7.93; 5%, 30.06 +/- 4.39; 8%, 28.81 +/- 4.16 mg/dL; p < 0.05) were also significantly lowered in the experimental groups. However, the HDL level did not show significant change for either control or experimental groups (0%, 36.06 +/- 3.96; 2%, 36.25 +/- 5.39; 5%, 33.13 +/- 3.68; 8%, 31.44 +/- 4.29 mg/dL; p > 0.05). Besides, the addition of red mold rice also helps to inhibit production of malondialdehyde (MDA) in serum lipid oxidation (0%, 27.42 +/- 0.53; 2%, 25.62 +/- 0.76; 5%, 24.35 +/- 0.59; 8%, 23.63 +/- 0.48 microM; p < 0.05).