Production Of Monascus Pigments Research Articles

Mrvam7, a conserved SNARE gene involved in vacuolar fusion, is required for development and secondary metabolism in Monascus ruber M7

Vam7 (vacuolar morphogenesis 7), a Qc-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that mediates vacuolar fusion, is involved in vacuolar morphogenesis, vegetative growth, sexual and asexual reproduction, and compartmentalized biosynthesis of secondary metabolites in various filamentous fungi. In this study, the homolog of vam7 gene, mrvam7, in Monascus ruber M7 was functionally characterized through gene modification and transcriptome analysis. The results showed that the mrvam7 complementation and overexpression strains exhibited phenotypic similarities to M. ruber M7, whereas the mrvam7 null strain exhibited fragmented vacuoles, slowed growth and diminished sexual and asexual sporulation. Furthermore, the mrvam7 knockout strain displayed a notable increase in citrinin production alongside a significant decrease in Monascus pigments production. Transcriptome data revealed that the deletion of mrvam7 altered the expression levels of several genes involved in sexual and asexual reproduction, carbon and nitrogen source utilization, and secondary metabolites biosynthetic gene clusters. Collectively, these findings underscore the critical role of mrvam7-mediated vacuolar fusion in growth, development, and the compartmentalized biosynthesis and distribution of secondary metabolites in M ruber M7. This study provides novel insights into the mechanisms underlying of compartmentalized secondary metabolites biosynthesis in Monascus species.

The effects of environmental factors on the synthesis of water-soluble Monascus red pigments via submerged fermentation: a review.

Monascus pigments (MPs) have been used as natural food pigments for many years. There is a high demand for Monascus red pigments (MRPs) to enhance color and for antibacterial and cancer prevention therapies in food and medicine. Most MRPs are not water soluble, and the yield of water-soluble MRPs is naturally low. On the other hand, water-soluble MRP is more cost effective for application in industrial mass production. Therefore, it is important to improve the yield of water-soluble MRPs. Environmental factors have a significant influence on the synthesis of water-soluble MRPs, which is crucial for the development of industrial production of water-soluble MRPs. This review introduces the biosynthetic pathways of water-soluble MRPs and summarizes the effects of environmental factors on the yield of water-soluble MRPs. Acetyl coenzyme A (acetyl-CoA) is a precursor for MPs synthesis. Carbon and nitrogen sources and the carbon/nitrogen ratio can impact MP production by regulating the metabolic pathway of acetyl-CoA. Optimization of fermentation conditions to change the morphology of Monascus can stimulate the synthesis of MPs. The appropriate choice of nitrogen sources and pH values can promote the synthesis of MRPs from MPs. Additives such as metal ions and non-ionic surfactants can affect the fluidity of Monascus cell membrane and promote the transformation of MRPs into water-soluble MRPs. This review will lay the foundation for the industrial production of water-soluble MRPs. © 2024 Society of Chemical Industry.

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.

COMPASS core subunits MpSet1 and MpSwd3 regulate Monascus pigments synthesis in Monascus purpureus.

In eukaryotes, methylation of histone H3 at lysine 4 (H3K4me) catalyzed by the complex of proteins associated with Set1 (COMPASS) is crucial for the transcriptional regulation of genes and the development of organisms. In Monascus, the functions of COMPASS in establishing H3K4me remain unclear. This study first identified the conserved COMPASS core subunits MpSet1 and MpSwd3 in Monascus purpureus and confirmed their roles in establishing H3K4me2/3. Loss of MpSet1 and MpSwd3 resulted in slower growth and development and inhibited the formation of cleistothecia, ascospores, and conidia. The loss of these core subunits also decreased the production of extracellular and intracellular Monascus pigments (MPs) by 94.2%, 93.5%, 82.7%, and 82.5%, respectively. In addition, RNA high-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) showed that the loss of MpSet1 and MpSwd3 altered the expression of 2646 and 2659 genes, respectively, and repressed the transcription of MPs synthesis-related genes. In addition, the ΔMpset1 and ΔMpswd3 strains demonstrated increased sensitivity to cell wall stress with the downregulation of chitin synthase-coding genes. These results indicated that the COMPASS core subunits MpSet1 and MpSwd3 help establish H3K4me2/3 for growth and development, spore formation, and pigment synthesis in Monascus. These core subunits also assist in maintaining cell wall integrity.

Mannitol improves Monascus pigment biosynthesis with rice bran as a substrate in Monascus purpureus.

To reduce the production cost of Monascus pigments (MPs), the utilization of rice bran (RB), an agricultural waste product, as a substrate in submerged fermentation was conducted in this study. To improve MP production, different nutritional ingredients including mannitol (Man), NH4NO3 (AN), ZnSO4 (Zn), and optimization (Opti), which was a synthesis of the three above ones, were added in rice bran (RB) medium. The yields of MPs, pigment constituents, and growth and development of Monascus purpureus M9 were investigated in this study. Man had the maximum color value of 3,532 U/g, which was 18.69 times more than that of RB and reached up to 76.65% of the value of rice (Rice) fermentation. Man significantly increased the production of two orange pigments, monascorubrin and rubropunctatin, of which the yields were 69.49 and 95.36% of the counterpart of Rice. The biomass and colony diameter of Opti presented the maximum value among different groups. AN and RB induced more asexual spore formation, whereas Opti and Man promoted sexual spore production. Comparative transcriptomic analysis showed that different nutritional ingredients led to changes in pigment production, promoting the growth and development of M. purpureus M9 through the regulation of related gene expression. Man and Opti improved MP production by regulating the primary metabolism, including the Embden-Meyerhof pathway (EMP), the pentose phosphate (PP) pathway, the tricarboxylic (TCA) cycle, fatty acid degradation (FAD), fatty acid biosynthesis (FAB), amino acid metabolism (AAM), and fructose and mannose metabolism (FMM), to provide the precursors (acetyl-CoA and malonyl-CoA) for MP biosynthesis. This study presents a low-cost method for increasing MP production and explains the molecular mechanisms of different nutritional ingredients for enhancing MP biosynthesis.

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.

Selective production, relationship and controversy between Monascus pigments and citrinin

Monascus pigments (MPs) are a series of high-quality natural edible pigments produced by Monascus spp. , which possess a variety of biological functions such as antioxidation, antibacterial, regulating cholesterol, anti-inflammatory, anti-cancer and antitumor. MPs have been widely used in food, medicine, cosmetics, chemical and dyes industries as functionally active ingredients. However, a mycotoxin named citrinin produced by some strains of Monascus restricts the application of MPs. It is proposed that there was a confusing relationship between the biosynthesis of MPs and citrinin. Therefore, a comprehensive understanding of the relationship can be used to develop strategies aimed at a selective production of MPs. In the present review, the discovery of MPs and citrinin, and common-used strategies for regulating their production are sorted. The possible links and controversies on synthetic pathways and productions between MPs and citrinin are also proposed and discussed. We hope this review contributes to a comprehensive understanding of the relationship between the biosynthesis of MPs and citrinin.

Comparative analysis of different rice substrates for solid-state fermentation by a citrinin-free Monascus purpureus mutant strain with high pigment production

Monascus pigment (MP) is a safe and natural microbial coloring agent. MP and red yeast rice (RYR) play an important role in the food industry. In this study, a citrinin-free Monascus puprureus MS-12 with high MP production was generated using dual mutagenesis of atmospheric and room-temperature plasma and heavy ion beam irradiation. The fermentation characteristics of eight rice substrates for solid-state fermentation (SSF) were compared to produce a premium RYR. Indica rice was the optimal fermentation substrate (MP = 4526 U/g in RYR), followed by japonica rice (MP = 3556 U/g). The MP value was <1000 U/g using other substrates (red, brown and black rice) for fermentation. RT-qPCR results demonstrated that the expression levels of MP synthesis- and the glycolysis-related genes of the mutant strain were upregulated compared with those of the parental strain, with MpigA exhibiting a 1.70-fold increase, while the relative expression levels of citrinin synthesis-related genes were downregulated; citE was downregulated 7.84-fold. In comparison to M. purpureus MS-12 mRNA levels using indica rice as the fermentation substrate during SSF, genes for MP, citrinin biosynthesis, and glycolysis resulted in the downregulation of other substrates. Our results show that colored and glutinous rice are unsuitable for RYR preparation via SSF. The maximum MP value was obtained using indical rice as the fermentation substrate, resulting from the upregulated expression levels of the pigment biosynthesis gene cluster and glycolytic pathway.

Regulation of the pigment production by changing cellmorphology and gene expression of Monascus ruber in high-sugar synergistic high-salt stress fermentation.

Extreme environment of microbial fermentation is the focus of research, which provides new thinking for the production and application of Monascus pigment. In this work, the high-sugar synergistic high-salt stress fermentation (HSSF) of Monascus pigment was investigated. The Monascus fungus grew well under HSSF conditions with 35g L-1 NaCl and 150g L-1 glucose, and the extracellular yellow pigment and intracellular orange pigment yield in HSSF was 98% and 43% higher than that in conventional fermentation (CF), respectively. Moreover, the mycelial morphology was maintained in a better status with more branches and complete surface structure, indicating good biocatalytic activity for pigment synthesis. Four extracellular yellow pigments (Y1, Y2, Y3, Y4) were transformed into each other, and ratio of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p<0.05) changed. Moreover, the ratio of unsaturated fatty acids to saturated fatty acids (unsaturated/saturated) was significantly (p<0.05) increased, indicating that the metabolism and secretion of intracellular and extracellular pigment might be regulated in HSSF. The pigment biosynthesis genes mppB, mppC, mppD, MpPKS5, and MpFasB2 were up-regulated, whereas the genes mppR1, mppR2, and mppE were down-regulated, suggesting that the gene expression to regulate pigment biosynthesis might be a dynamic change process in HSSF. The HSSF system of MPs is successfully performed to improve the pigment yields. Mycelial morphology is varied to enhanced pigment secretion and gene expression is dynamically regulated to promote pigment accumulation in HSSF.

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.

Comparative Transcriptomic Analysis of Key Genes Involved in Citrinin Biosynthesis in Monascus purpureus.

Monascus pigments (MPs) display many beneficial biological activities and have been widely utilized as natural food-grade colorants in the food processing industry. The presence of the mycotoxin citrinin (CIT) seriously restricts the application of MPs, but the gene regulation mechanisms governing CIT biosynthesis remain unclear. We performed a RNA-Seq-based comparative transcriptomic analysis of representative high MPs-producing Monascus purpureus strains with extremely high vs. low CIT yields. In addition, we performed qRT-PCR to detect the expression of genes related to CIT biosynthesis, confirming the reliability of the RNA-Seq data. The results revealed that there were 2518 differentially expressed genes (DEGs; 1141 downregulated and 1377 upregulated in the low CIT producer strain). Many upregulated DEGs were associated with energy metabolism and carbohydrate metabolism, with these changes potentially making more biosynthetic precursors available for MPs biosynthesis. Several potentially interesting genes that encode transcription factors were also identified amongst the DEGs. The transcriptomic results also showed that citB, citD, citE, citC and perhaps MpigI were key candidate genes to limit CIT biosynthesis. Our studies provide useful information on metabolic adaptations to MPs and CIT biosynthesis in M. purpureus, and provide targets for the fermentation industry towards the engineering of safer MPs production.

Clean Monascus pigments production from Chinese rice wine wastes through submerged fermentation

The potential for Monascus pigments (MPs) production in a mixture of Chinese rice wine lees, soaked rice wastewater and broken rice was explored. MPs production reached 120.1 U/mL with optimal solid-liquid ratio of culture medium 1:10 and 20 g/L broken rice addition at shake flask level. We further explored the effects of stirring speed on MPs production in 7.5 L agitated bioreactor. The stirring speed had a great impact on cell morphology and the maximum MPs concentration and yield were 128.4 U/mL and 8174 U/g respectively at 400 rpm. The soluble chemical oxygen demand for the soaked rice wastewater decreased by 78.7%, which was mainly due to the intake of carbohydrates, organic acids and amino acids. Furthermore, a simultaneous fermentation process and production system with two-stage stirring speed control strategy was further developed in the 50 L pilot scale, in which the MPs production reached 158.9 U/mL. Meanwhile, Chinese rice wine wastes were fully utilized without addition of organic solvents during the process and no generation of new waste. Our results showed that Monascus was capable of converting Chinese rice wine wastes into natural pigments by submerged fermentation, which could provide an economic and environmentally friendly approach to reduce the cost of wastes treatment as well as high-value utilization.

A Zn(II)(2)Cys(6) transcription factor MPsGeI suppresses pigment biosynthesis in Monascus

High-quality natural edible pigments known as monascus pigments (MPs) are widely used in food, medicine, and chemical industries as active functional ingredients. At the transcriptional level, the expression of MPs genes are tightly controlled, limiting their productivity and color value. Hitherto our understanding of the regulation of expression of MPs genes has been rather limited. Here, we describe a pathway-specific Zn(II)(2)Cys(6) transcription factor involved in the MPs biosynthetic cluster named MPsGeI, which encodes a 813-amino-acid protein with six introns. Expression of all MPs biosynthetic genes and accumulation of MPs were remarkably increased in ΔMPsGeI strain, and MPs production was significantly reduced in MPsGeI over-expressing strain. Results clearly demonstrated that MPsGeI negatively regulates MPs accumulation via transcriptional regulation of MPs biosynthetic genes, and plays a central repressive role in MPs' biosynthesis. Transcriptomic analyses revealed that MPsGeI disruptant regulated higher concentrations of precursors flowing to pigment and resulted in accumulation of a large amount of red MPs in hyphae. This work offers an efficient method for increasing MPs's productivity and color value and provides novel insights into the regulatory mechanisms of fungal cellular processes, which will assist to enhance MPs production and application.

Mphy gene reveals the involvement of light source in the regulation of pigment metabolism in Monascusruber

The growth, development and pigment metabolism of Monascus are regulated by light. However, the function of the photoreceptor gene in the Monascus genome has not yet been clearly verified. In this paper, the gene sequence Mphy, which is homologous to plant phytochrome, was firstly found from Monascus ruber C100 by comparing it with the genome. Then, taking M. ruber C100 as the main research object, by knocking out and overexpressing the Mphy gene, the involvement of Mphy in the regulation of light on the growth and development of Monascus and the metabolism of Monascus pigments (MPs) were preliminarily explored. Compared with the original strain C100, the Mphy knockout strain (ΔMphy) and the overexpression strain (OE-Mphy) grew slowly and produced low spores. At the same time, under different light conditions, red light can promote the growth and sporulation of Monascus, while blue light can inhibit it. MPs are positively regulated by the Mphy gene, that is, the deletion of the Mphy gene reduces the production of MPs, while the overexpression of the gene increases the accumulation of the pigment, especially under red light conditions. These results indicate that the homologous genes of phytochromes are involved in the process of light-regulated growth, development and pigment metabolism in Monascus. Based on the above results, we found that under red light conditions, Mphy controls the process of pigment metabolism by affecting key enzymes in TCA metabolism and key signaling pathways such as the MAPK signaling pathway.

The acyl-CoA-binding protein 2 exhibited the highest affinity for palmitoyl-CoA and promoted Monascus pigment production

PurposeThe present study aimed to explore the binding ability of acyl-CoA binding protein 2 to fatty acid acyl-CoA esters and its effect on Monascus pigment production in M. ruber CICC41233.MethodsThe Mracbp2 gene from M. ruber CICC41233 was cloned with a total DNA and cDNA as the templates through the polymerase chain reaction. The cDNA of the Mracbp2 gene fragment was ligated to expression vector pGEX-6P-1 to construct pGEX-MrACBP2, which was expressed in Escherichia coli BL21 to obtain the fusion protein GST-MrACBP2 and then measure the binding ability of fatty acid acyl-CoA esters. Additionally, the DNA of the Mracbp2 gene fragment was ligated to expression vector pNeo0380 to construct pNeo0380-MrACBP2, which was homologously over-expressed in M. ruber CICC41233 to evaluate Monascus pigment production and fatty acid.ResultsThe cloned Mracbp2 gene of the DNA and cDNA sequence was 1525 bp and 1329 bp in length, respectively. The microscale thermophoresis binding assay revealed that the purified GST-MrACBP2 had the highest affinity for palmitoyl-CoA (Kd =70.57 nM). Further, the Mracbp2 gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP-E was isolated. In the Monascus pigments fermentation, the expression level of the Mracbp2 gene was increased by 1.74-fold after 2 days and 2.38-fold after 6 days. The palmitic acid content and biomass in M. ruber ACBP2-E were significantly lower than that in M. ruber CICC41233 on 2 days and 6 days. However, compared with M. ruber CICC41233, the yields of total pigment, ethanol-soluble pigment, and water-soluble pigment in M. ruber ACBP2-E increased by 63.61%, 71.61%, and 29.70%, respectively.ConclusionsThe purified fusion protein GST-MrACBP2 exhibited the highest affinity for palmitoyl-CoA. The Mracbp2 gene was overexpressed in M. ruber CICC41233, which resulted in a decrease in palmitic acid and an increase in Monascus pigments. Overall, the effect of MrACBP2 on the synthesis of fatty acid and Monascus pigment was explored. This paper explored the effect of MrACBP2 on the fatty acid synthesis and the synthesis of Monascus pigment. The results indicated the regulation of fatty acid synthesis could affect Monascus pigment synthesis, providing a novel strategy for improving the yield of Monascus pigment.

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.

Solid-state fermentation for the production of Monascus pigments from soybean meals

The aim of the present study was to investigate feasibility of soybean meal powder as a nitrogen substrate for the production of pigments by Monascus purpureus ATCC 16362 in solid-state fermentation using soybean meals as nitrogen sources to replace yeast extracts. Central composite design was used to assess the optimum level of soybean meal replacement (0–100%), ZnSO4.7H2O concentration (0–0.02 g/L) and thermal stress time of spore suspension at 70°C (50–90 s). Red Monascus pigment and biomass productions were assessed as dependent responses. A pigment yield of 7.06 AU/g dry fermented substrate was achieved using soybean meal powder with optimized process parameters such as a 50% initial moisture content and incubation at 30 °C for 21 days. Addition of Triton X-100 to solid-state fermentation media significantly increased red Monascus pigments (p < 0.05). Stability of the red Monascus pigments was assessed under storage time (90 days). At high temperatures of storage, degradation constant of the red pigments increased in the presence of light during the shelf-life. This study recommends use of Monascus purpureus for the efficient production of red pigments using low-cost substrates in solid-state fermentation by food industries.

Comparative Transcriptomic and Metabolomic Analyses Reveal the Regulatory Effect and Mechanism of Tea Extracts on the Biosynthesis of Monascus Pigments

Monascus pigments (MPs) are natural edible pigments with high safety and strong function, which have been widely used in food and health products. In this study, different types of tea extracts (rich in polyphenols) were used to regulate the biosynthesis of MPs. The results showed that 15% ethanol extract of pu-erh tea (T11) could significantly increase MPs production in liquid fermentation of Monaco’s purpureus M3. Comparative transcriptomic and metabolomic analyses combined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to further explore the regulatory mechanism of T11 on the biosynthesis of MPs. Comparative transcriptomic analysis showed that there were 1503 differentially expressed genes (DEGs) between the Con group and the T11 group, which were mainly distributed in carbohydrate metabolism, amino acid metabolism, energy metabolism, lipid metabolism, metabolism of terpenoids and polyketides, etc. A total of 115 differential metabolites (DMs) identified by metabolomics between the Con and T11 groups were mainly enriched in glutathione metabolism, starch and sucrose metabolism, alanine, aspartic acid and glutamate metabolism and glycine, serine and threonine metabolism, etc. The results of metabolomics were basically consistent with those of gene transcriptomics, indicating that the regulatory effect of T11 on the biosynthesis of MPs is mainly achieved through affecting the primary metabolic pathway, providing sufficient energy and more biosynthetic precursors for secondary metabolism. In this study, tea extracts with low economic value and easy access were used as promoters of MPs biosynthesis, which may be conducive to the application of MPs in large-scale industrial production. At the same time, a more systematic understanding of the molecular regulatory mechanism of Monascus metabolism was obtained through multi-omics analysis.

Construction of gene modification system with highly efficient and markerless for Monascus ruber M7.

Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in Monascus spp.. However, the resistance selection genes that can have been used for genetic modification in Monascus spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M. ruber M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔmrpyrGΔmrlig4 and ΔmrpyrGΔmrlig4::mrpyrG, in which we used the endogenous gene mrpyrG from M. ruber M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted mrlig4 related to non-homologous end joining in M. ruber M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M. ruber M7. However, the U addition also has a certain effect on the orange and red pigments yield of M. ruber M7, which needs our further study. Finally, we applied the system to delete multiple genes from M. ruber M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔmrpyrGΔmrlig4 was about 18 times of that of M. ruber M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in Monascus spp., and also lays a foundation for investigating the effects of multi-genes modification on Monascus spp..

Diverse Effects of Amino Acids on Monascus Pigments Biosynthesis in Monascus purpureus.

Amino acids could act as nitrogen sources, amido group donors, or bioactive molecules in fungi fermentation, and consequently, play important roles in Monascus pigments (MPs) biosynthesis. But the understanding of the effects of various amino acids on MPs biosynthesis is still incomprehensive. In this work, 20 free amino acids were added to the fermentation medium to evaluate their effects on MPs biosynthesis in Monascus purpureus RP2. Six amino acids, namely, histidine (HIS), lysine (LYS), tyrosine (TYR), phenylalanine (PHE), methionine (MET), and cysteine (CYS), were selected as the valuable ones as they exerted significant effects on the production yield and even on the biosynthesis metabolic curves of MPs. Moreover, the dose-dependent and synergistic effects of valuable amino acids on MPs biosynthesis were observed by tests of serial concentrations and different combinations. In addition, it revealed that HIS and MET were the prominent amino acids with dominant and universal influences on MPs biosynthesis. The analog compounds of HIS (amitrole) and MET [calcium 2-hydroxy-4-(methylthio)] were added to the fermentation medium, and the results further confirmed the extraordinary effects of HIS and MET and their analogs on MPs biosynthesis. Furthermore, the gene transcription profile indicated that a differential expression pattern was observed in the polyketide synthase (PKS) cluster responsible for MPs biosynthesis in response to HIS and MET, revealing that they could oppositely regulate MPs biosynthesis in different ways. These findings would benefit the understanding of MPs biosynthesis regulation mechanism in M. purpureus and contribute to the industrial production of MPs by fermentation.