Enhancing extracellular monascus pigment production in submerged fermentation with engineered microbial consortia.

In this study, we investigated the impact of microbial interactions on Monascus pigment (MP) production. We established diverse microbial consortia involving Monascus purpureus and Lactobacillus fermentum. The addition of Lactobacillus fermentum (4% at 48 h) to the submerged fermentation of M. purpureus resulted in a significantly higher MP production compared to that achieved using the single-fermentation system. Co-cultivation with immobilized L. fermentum led to a remarkable increase of 59.18% in extracellular MP production, while mixed fermentation with free L. fermentum caused a significant decrease of 66.93% in intracellular MPs, contrasting with a marginal increase of 4.52% observed during co-cultivation with immobilized L. fermentum and the control group respectively. The findings indicate an evident enhancement in cell membrane permeability of M. purpureus when co-cultivated with immobilized L. fementum. Moreover, integrated transcriptomic and metabolomic analyses were conducted to elucidate the regulatory mechanisms underlying MP biosynthesis and secretion following inoculation with immobilized L. fementum, with specific emphasis on glycolysis, steroid biosynthesis, fatty acid biosynthesis, and energy metabolism.

Monaspin B, a Novel Cyclohexyl-furan from Cocultivation of Monascus purpureus and Aspergillus oryzae, Exhibits Potent Antileukemic Activity.

Natural products are a rich resource for the discovery of innovative drugs. Microbial cocultivation enables discovery of novel natural products through tandem enzymatic catalysis between different fungi. In this study, Monascus purpureus, as a food fermentation strain capable of producing abundant natural products, was chosen as an example of a cocultivation pair strain. Cocultivation screening revealed that M. purpureus and Aspergillus oryzae led to the production of two novel cyclohexyl-furans, Monaspins A and B. Optimization of the cocultivation mode and media enhanced the production of Monaspins A and B to 1.2 and 0.8 mg/L, respectively. Monaspins A and B were structurally elucidated by HR-ESI-MS and NMR. Furthermore, Monaspin B displayed potent antiproliferative activity against the leukemic HL-60 cell line by inducing apoptosis, with a half-maximal inhibitory concentration (IC50) of 160 nM. Moreover, in a mouse leukemia model, Monaspin B exhibited a promising in vivo antileukemic effect by reducing white blood cell, lymphocyte, and neutrophil counts. Collectively, these results indicate that Monaspin B is a promising candidate agent for leukemia therapy.

Bidirectional fermentation of Monascus and Mulberry leaves enhances GABA and pigment contents: establishment of strategy, studies of bioactivity and mechanistic.

Bidirectional fermentation is a technology that utilizes fungi to ferment medicinal edible substrates, with synergistic and complementary advantages. In this work, a fermentation strategy was established to produce a high yield of γ-aminobutyric acid (GABA) and Monascus pigments (MPs) using Monascus and mulberry leaves (MLs). Firstly, the basic fermentation parameters were determined using single-factor experiments, followed by Plackett-Burman (PB) experimental design to identify MLs, glucose, peptone, and temperature as significant influencing factors. The fermentation parameters were optimized using an artificial neural network (ANN). Finally, the effects of bidirectional fermentation of MLs and Monascus were investigated by bioactivity analysis, microstructure observation, and RT-qPCR. The outcomes showed that the bidirectional fermentation significantly increased the bioactive content and promoted the secondary metabolism of Monascus. The established fermentation conditions were 44.2 g/L of MLs, 57 g/L of glucose, 15 g/L of peptone, 1 g/L of MgSO4, 2 g/L of KH2PO4, 8% (v/v) of inoculum, 180 rpm, initial pH 6, 32 °C and 8 days. The content of GABA reached 13.95 g/L and the color value of MPs reached 408.07 U/mL. This study demonstrated the feasibility of bidirectional fermentation of MLs and Monascus, providing a new idea for the application of MLs and Monascus.

Role of histone H3K4 methyltransferase in regulating Monascus pigments production by red light-coupled magnetic field.

Light, magnetic field, and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of Monascus purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). Twenty micromolar 5-AzaC significantly promoted the growth, development, and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light, and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on the growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development, and MPs synthesis of M. purpureus by the RLCMF.

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.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

BACKGROUND: Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. RESULTS: The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. CONCLUSION: The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

An R-R-type MYB transcription factor promotes non-climacteric pepper fruit carotenoid pigment biosynthesis.

Carotenoids are major accessory pigments in the chloroplast, and they also act as phytohormones and volatile compound precursors to influence plant development and confer characteristic colours, affecting both the aesthetic and nutritional value of fruits. Carotenoid pigmentation in ripening fruits is highly dependent on developmental trajectories. Transcription factors incorporate developmental and phytohormone signalling to regulate the biosynthesis process. By contrast to the well-established pathways regulating ripening-related carotenoid biosynthesis in climacteric fruit, carotenoid regulation in non-climacteric fruit is poorly understood. Capsanthin is the primary carotenoid of non-climacteric pepper (Capsicum) fruit; its biosynthesis is tightly associated with fruit ripening, and it confers red pigmentation to the ripening fruit. In the present study, using a coexpression analysis, we identified an R-R-type MYB transcription factor, DIVARICATA1, and demonstrated its role in capsanthin biosynthesis. DIVARICATA1 encodes a nucleus-localised protein that functions primarily as a transcriptional activator. Functional analyses showed that DIVARICATA1 positively regulates carotenoid biosynthetic gene (CBG) transcript levels and capsanthin levels by directly binding to and activating CBG promoter transcription. Furthermore, an association analysis revealed a significant positive association between DIVARICATA1 transcription level and capsanthin content. ABA promotes capsanthin biosynthesis in a DIVARICATA1-dependent manner. Comparative transcriptomic analysis of DIVARICATA1 in Solanaceae plants showed that its function likely differs among species. Moreover, the pepper DIVARICATA1 gene could be regulated by the ripening regulator MADS-RIN. The present study illustrates the transcriptional regulation of capsanthin biosynthesis and offers a target for breeding peppers with high red colour intensity.

New insights on low-temperature storage regulating garlic greening and the accumulation of pigment precursors via glutathione metabolism and energy cycles.

To explore regulation mechanism of temperature on garlic greening and pigment precursors' accumulation, greening capacities, pigment precursors and critical metabolites, enzyme and genes involved in glutathione and NADPH metabolism of garlic stored at five temperatures (4, 8, 16, 24 and 30 ℃) were analyzed. Results showed that garlic pre-stored at 4, 8 and 16 ℃ were more likely to green than ones at 24 and 30 ℃ after pickling. After 25 days, more S-1-propenyl-l-cysteine sulfoxide (1-PeCSO) were detected in garlic stored at 4, 8 and 16 ℃ (753.60, 921.85 and 756.75 mAU, respectively) than that at 24 and 30 ℃ (394.35 and 290.70 mAU). Pigment precursors' accumulation in garlic was mainly realized by glutathione and NADPH metabolism under low-temperature storage, through enhancements of activities or expressions for GR (GSR), GST (GST), γ-GT (GGT1, GGT2), 6PGDH (PGD) and ICDHc (IDH1). This study enriched the mechanism of garlic greening.

Supplementary effect of whey components on the monascin productivity of Monascus sp.

BACKGROUND: Monascus sp. has been used in fermented foods for centuries. It can synthesize yellow, red, and orange pigments as secondary metabolites. Here, we focused on yellow pigment monascin, responsible for anti-inflammation and antidiabetic effects, and investigated whether whey could be a suitable substrate with or without rice powder for monascin production using M. purpureus AHU 9085, M. pilosus NBRC 4520 and M. ruber NBRC 32318. RESULTS: The growth and monascin production of the three Monascus strains were dependent on three liquid media consisting of whey and/or rice. All strains showed the best growth in a rice and whey mixed medium, in which M. ruber NBRC 32318 exhibited the highest total monascin production. Subsequent investigation of the effects of whey components indicated that a mineral cocktail in whey was particularly effective in stimulating the monascin production efficiency of M. ruber NBRC 32318. However, this recipe exhibited less stimulation, or even inhibition, for M. pilosus NBRC 4520 and M. purpureus AHU 9085, respectively. In terms of total monascin production, rice with whey provided the highest amount due to growth promotion along with relatively high production efficiency. CONCLUSION: The effect of whey on growth and monascin production was strongly dependent on the Monascus strains. Even a mineral cocktail in whey could regulate monascin productivity in a strain-specific manner. Further studies are needed to elucidate the mechanism behind the diverse responses by the minerals in the production of monascin from Monascus. © 2023 Society of Chemical Industry.

Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber.

AIMS: In this study, Mrhst4, encoding a member of NAD+-dependent histone deacetylase (HDAC), was deleted to evaluate its regulation on the production of Monascus azaphilone pigments (MonAzPs) and mycotoxin, as well as the developmental process in Monascusruber. METHODS AND RESULTS: Agrobacterium tumefaciens-mediated transformation was applied in this study to generate the Mrhst4 null strain. Mrhst4-deleted strain did not display obvious differences in the sexual and asexual reproduction, colonial morphology, and micro-morphology. UV-Vis scan and UPLC detection showed that disruption of Mrhst4 significantly increased the MonAzPs yields, and citrinin content was dramatically enhanced during the tested period. RT-qPCR results showed that the absence of Mrhst4 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. The Western blot assay suggested that deletion of Mrhst4 could significantly elevate the acetylation levels of H3K4, H3K9, H3K18, H3K56, and H4K12, but attenuated the lysine acetylation modification of H4Pan, H4K8, and H4K16. CONCLUSION: MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production.

Natural edible pigments: A comprehensive review of resource, chemical classification, biosynthesis pathway, separated methods and application.

Natural edible pigments, high safety and low toxicity, usually possess various nutritional and pharmacological effects, and they have huge practical application value in the market. However, until now, there is no systematic review about the resources, chemical classifications and application about them. Moreover, the extracted methods and biosynthesis pathways which are very important informations for obtaining high-yield and high-purity natural edible pigments from natural resources are still lacking. Therefore, It is necessary to make a comprehensive review of natural edible pigments. In this work, we systematically summarize the resources, chemical classifications, biosynthesis pathways, extraction and separation methods, as well as application of natural edible pigments for the first time. Our work will provide reference data and give the inspiration for further industrial application of natural edible pigments.

A mutant of Monascus purpureus obtained by carbon ion beam irradiation yielded yellow pigments using various nitrogen sources.

The industrial production of monascus yellow pigments (MYPs) has not yet been done due to the lack of high-quality industrial Monascus strains. In this work, we employed carbon ion beam (12C6+) irradiation to screen Monascus strains that produce high-quality extracellular MYPs (extr-MYPs). One genetically stable M. purpureus mutant of BWY-5 with extr-MYPs accumulation was obtained under 12C6+ irradiation (80 MeV/u, 200 Gy). M. purpureus BWY-5 could use various nitrogen sources to produce single pH stable extr-MYPs (around 80 AU at 370 nm). Moreover, citrinin was not detected by HPLC method. Transcriptomics of the MYP production strain suggested that Carbon ion beam irradiation led to deletion (MpigF, MpigG and MpigH), downregulation (CtnE, CtnH and CtnI) and upregulation (MpigM) of genes related with biosynthesis of MOPs and MRPs, citrinin, and extr-MYPs, respectively. The results showed that M. purpureus BWY-5 has the potential to be used as an industrial Monascus strain and platform for extr-MYPs production and monascus polyketide synthetic pathway studies, respectively.

Regulated synthesis and metabolism of Monascus pigments in a unique environment.

Microbial fermentation in extreme environments is the focus of research, which provides new insights for the production and application of Monascus pigments. In this paper, the regulation of Monascus pigments metabolism by optimizing the conditions, such as osmotic pressure, high sugar stress, light, extreme temperature, high-water content, low-frequency magnetic field and ultrasonics, is reviewed and summarized in four parts: the mycelium growth of Monascus spp., metabolic pathways, gene expression and composition characteristics of Monascus pigments. The relationship between mycelial morphology, gene expression and pigments production during fermentation under unique environments is discussed. Based on the changes in metabolic pathways and composition characteristics, the regulatory mechanism of Monascus pigments under unique conditions is proposed. Moreover, the fermentation strategy and application prospects of Monascus pigments in unique environments are also discussed. This work will provide a theoretical basis and practical guidance for the optimized production of Monascus pigments.

Quinoid Pigments of Sea Urchins Scaphechinus mirabilis and Strongylocentrotus intermedius: Biological Activity and Potential Applications.

This review presents literature data: the history of the discovery of quinoid compounds, their biosynthesis and biological activity. Special attention is paid to the description of the quinoid pigments of the sea urchins Scaphechinus mirabilis (from the family Scutellidae) and Strongylocentrotus intermedius (from the family Strongylocentrotidae). The marine environment is considered one of the most important sources of natural bioactive compounds with extremely rich biodiversity. Primary- and some secondary-mouthed animals contain very high concentrations of new biologically active substances, many of which are of significant potential interest for medical purposes. The quinone pigments are products of the secondary metabolism of marine animals, can have complex structures and become the basis for the development of new natural products in echinoids that are modulators of chemical interactions and possible active ingredients in medicinal preparations. More than 5000 chemical compounds with high pharmacological potential have been isolated and described from marine organisms. There are three well known ways of naphthoquinone biosynthesis-polyketide, shikimate and mevalonate. The polyketide pathway is the biosynthesis pathway of various quinones. The shikimate pathway is the main pathway in the biosynthesis of naphthoquinones. It should be noted that all quinoid compounds in plants and animals can be synthesized by various ways of biosynthesis.

A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes.

A number of biopigment applications in various industrial sectors are gaining importance due to the growing consumer interest in their natural origin. Thus, this work was conducted to valorize endophytic fungi as an efficient production platform for natural pigments. A promising strain isolated from leaves of Origanum majorana was identified as Monascus ruber SRZ112 produced several types of pigments. The nature of the pigments, mainly rubropunctamine, monascin, ankaflavin, rubropunctatin, and monascorubrin in the fungal extract was studied by LC/ESI-MS/MS analyses. As a first step towards developing an efficient production of red pigments, the suitability of seven types of agro-industrial waste was evaluated. The highest yield of red pigments was obtained using potato peel moistened with mineral salt broth as a culture medium. To increase yield of red pigments, favourable culture conditions including incubation temperature, incubation period, pH of moistening agent, inoculum concentration, substrate weight and moisture level were evaluated. Additionally, yield of red pigments was intensified after the exposure of M. ruber SRZ112 spores to 1.00 KGy gamma rays. The final yield was improved by a 22.12-fold increase from 23.55 to 3351.87 AU g-1. The anticancer and antioxidant properties of the pigment's extract from the fungal culture were also studied. The obtained data indicated activity of the extract against human breast cancer cell lines with no significant cytotoxicity against normal cell lines. The extract also showed a free radical scavenging potential. This is the first report, to our knowledge, on the isolation of the endophytic M. ruber SRZ112 strain with the successful production of natural pigments under solid-state fermentation using potato peel as a substrate.

Biosynthesis and polyketide oxidation of Monascus red pigments in an integrated fermentation system with microparticles and surfactants.

Monascus red pigments are widely used in the food industry, mainly as intracellular red pigments. The low yields of extracellular red pigments (ERPs) make them unsuitable for large-scale industrial production. Herein, a novel integrated fermentation system (IFS) consisting of sodium starch octenyl succinate and Triton X-100 was explored for increasing yield, resulting in an ERP yield of 126.7 U/mL, 82.6% higher production than controls (69.4 U/mL). Major ERP components in control fermentations were monascopyridine A and monascopyridine B, but dehydro derivatives, rubropunctamine and monascorubramine, predominated in the test fermentations, presumably due to polyketide oxidation induced by Triton X-100. Improvement of hyphal morphology, membrane permeability, respiratory activity, and gene expression for red pigment biosynthesis is likely to be critical to increase yield and change the compositions. This study provides an effective strategy to accelerate the biosynthesis and secretion of Monascus pigments.

Ascomycota as a source of natural colorants.

In the last few decades, there has been a great demand for natural colorants. Synthetic colorants are known to be easy to produce, are less expensive, and remain stable when subjected to chemical and physical factors. In addition, only small amounts are required to color any material, and unwanted flavors and aromas are not incorporated into the product. Natural colorants present in food, in addition to providing color, also have biological properties and effects that aid in the prevention and cure of many diseases. The main classes of colorants produced by phylum Ascomycota include polyketides and carotenoids. A promising producer of colorants should be able to assimilate a variety of sources of carbon and nitrogen and also exhibit relative stability. The strain should not be pathogenic, and its product should not be toxic. Production processes should also provide the expected color with a good yield through simple extraction methods. Research that seeks new sources of these compounds should continue to seek products of biotechnological origin in order to be competitive with products of synthetic and plant origin. In this review, we will focus on the recent studies on the main producing species, classes, and metabolic pathways of colorants produced by this phylum, historical background, impact of synthetic colorants on human health and the environment, social demand for natural colorants and also an in-depth approach to bioprocesses (influences on production, optimization of bioprocess, extraction, and identification), and limitations and perspectives for the use of fungal-based dyes.

Characterization of a silent azaphilone biosynthesis gene cluster in Aspergillus terreus NIH 2624.

Filamentous fungal secondary metabolites are an important source of bioactive components. Genome sequencing ofAspergillus terreusrevealed many silent secondary metabolite biosynthetic gene clusters presumed to be involved in producing secondary metabolites. Activation of silent gene clusters through overexpressing a pathway-specific regulator is an effective avenue for discovering novel fungal secondary metabolites. Replacement of the native promoter of the pathway-specific activator with the inducible Tet-on system to activate thetazpathway led to the discovery of a series of azaphilone secondary metabolites, among which azaterrilone A (1) was purified and identified for the first time. Genetic deletion of core PKS genes and transcriptional analysis further characterized thetazgene cluster to consist of 16 genes with the NR-PKS and the HR-PKS collaborating in a convergent mode. Based on the putative gene functions and the characterized compounds structural information, a biosynthetic pathway of azaterrilone A (1) was proposed.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: The Mrada3 gene plays a vital role in the sporulation development and secondary metabolism of Monascus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Mrada3 is first identified as an essential regulator for sexual development in Monascus species, enriching the regulatory knowledge of sexual development in filamentous fungi.

The Absence of the AtSYT1 Function Elevates the Adverse Effect of Salt Stress on Photosynthesis in Arabidopsis.

Arabidopsis thaliana SYNAPTOTAGMIN 1 (AtSYT1) was shown to be involved in responses to different environmental and biotic stresses. We investigated gas exchange and chlorophyll a fluorescence in Arabidopsis wild-type (WT, ecotype Col-0) and atsyt1 mutant plants irrigated for 48 h with 150 mM NaCl. We found that salt stress significantly decreases net photosynthetic assimilation, effective photochemical quantum yield of photosystem II (ΦPSII), stomatal conductance and transpiration rate in both genotypes. Salt stress has a more severe impact on atsyt1 plants with increasing effect at higher illumination. Dark respiration, photochemical quenching (qP), non-photochemical quenching and ΦPSII measured at 750 µmol m-2 s-1 photosynthetic photon flux density were significantly affected by salt in both genotypes. However, differences between mutant and WT plants were recorded only for qP and ΦPSII. Decreased photosynthetic efficiency in atsyt1 under salt stress was accompanied by reduced chlorophyll and carotenoid and increased flavonol content in atsyt1 leaves. No differences in the abundance of key proteins participating in photosynthesis (except PsaC and PsbQ) and chlorophyll biosynthesis were found regardless of genotype or salt treatment. Microscopic analysis showed that irrigating plants with salt caused a partial closure of the stomata, and this effect was more pronounced in the mutant than in WT plants. The localization pattern of AtSYT1 was also altered by salt stress.