Production Of Monascus Pigments Research Articles

Preliminary exploration of the mechanisms underlying morphology engineering of Monascus purpureus during submerged fermentation via multi-omics approaches

Herein, multi-omics approaches were employed to investigate the relationship between mycelia morphology and biosynthesis of Monascus pigments (MPs) during submerged fermentation of Monascus purpureus. Through heavy ion beam irradiation, eleven mutants with diverse mycelial morphologies and MPs productions were generated. Genome resequencing analysis revealed that the autologous genes of these mutants were primarily enriched in pathways associated with histone ubiquitination and methylation. Among them, M. purpureus ZS exhibited a 2.49-fold increase in MPs production and distinct mycelia morphology compared to the parent strain, thus it was selected for transcriptomic and metabolomic analyses. The results revealed showed significant alterations in lysine metabolism, lipid metabolism, as well as cell wall and membrane components. Additionally, upregulated CoA biosynthesis along with type I polyketide structures and the genetic cluster responsible for MPs biosynthesis facilitated accumulation of MPs. These findings provide a theoretical foundation for morphological engineering of Monascus while offering novel strategies to achieve higher levels of MPs production during submerged fermentation.

MpDLDH gene is crucial for regulating growth and pigment biosynthesis through acetyl-CoA modulation in Monascus purpureus

Monascus spp. are traditional medicinal and edible filamentous fungi in Asian countries. Monascus pigments (MPs) are the most well-known beneficial secondary metabolites produced by Monascus and used as natural food colorants with a long history. However, the understanding of the MPs biosynthesis remains limited due to their complexity and diversity, and the known MPs biosynthetic gene cluster (BGC) is also incomplete. In this study, we re-predicted the MPs BGC based on the genome sequence of Monascus purpureus and found seven new genes that may be involved in MPs biosynthesis. Among them, MpDLDH encodes a putative dihydrolipoamide dehydrogenase, which is the E3 subunit of pyruvate dehydrogenase (PDH) multiunit complex for catalyzing pyruvate to acetyl-CoA. MpDLDH knockout significantly decreased the acetyl-CoA content, and resulted in small pale colony, dramatically reduced biomass, few conidia, and absence of cleistothecia. MpDLDH deletion seriously repressed MPs biosynthesis, but did not change its composition. In addition to the increase of intracellular MPs production and acetyl-CoA in the late growth stage on solid medium, MpDLDH overexpression had no obvious effect on the phenotypes. Since acetyl-CoA is the crucial substrate for both MPs biosynthesis and tricarboxylic acid cycle, it is believed that MpDLDH of MPs BGC plays a key role in regulating cell growth, development and pigment formation through participating in the assembly of PDH complex to modulate the production of acetyl-CoA. These findings may deepen our understanding of the regulatory mechanisms between MPs biosynthesis and cell growth in Monascus species.

Functional Verification of Transcription Factor comp54181_c0 in Monascus purpureus.

Monacolin K is a valuable secondary metabolite produced after a period of fermentation by Monascus purpureus; however, our current understanding of the regulatory mechanisms of its synthesis remains incomplete. This study conducted functional analysis on the key transcription factor, comp54181_c0, that is involved in the synthesis of monacolin K in Monascus. Mutant strains with either knockout or overexpression of comp54181_c0 were constructed using CRISPR/Cas9. A comparison between the knockout and overexpression strains revealed changes in fungal morphology and growth, with a significant increase in the production of Monascus pigments and monacolin K when comp54181_c0 was absent. Real-time fluorescence quantitative PCR analysis revealed that comp54181_c0 significantly influenced the transcription of key genes related to monacolin K biosynthesis in Monascus. In conclusion, our study elucidates the crucial role of comp54181_c0 in Monascus, enriches our understanding of fungal secondary metabolite development and regulation, and provides a foundation for the development and regulation of Monascus and monacolin K production.

Phospholipid biosynthesis regulation for improving pigment production by Monascus in response to ammonium chloride stress.

In the actual industrial production process, the efficient biosynthesis and secretion of Monascus pigments (MPs) tend to take place under abiotic stresses, which often result in an imbalance of cell homeostasis. The present study aimed to thoroughly describe the changes in lipid profiles in Monascus purpureus by absolute quantitative lipidomics and tandem mass tag-based quantitative proteomics. The results showed that ammonium chloride stress (15 g/L) increased MP production while inhibiting ergosterol biosynthesis, leading to an imbalance in membrane lipid homeostasis in Monascus. In response to the imbalance of lipid homeostasis, the regulation mechanism of phospholipids in Monascus was implemented, including the inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of the biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway rather than the Kennedy pathway. The inhibition of lysophospholipid biosynthesis was attributed to the upregulated expression of protein and its gene related to lysophospholipase NTE1, while maintenance of the PC/PE ratio was achieved by the upregulated expression of protein and its gene related to CTP: phosphoethanolamine cytidylyltransferase and phosphatidylethanolamine N-methyltransferase in the Kennedy pathway. These findings provide insights into the regulation mechanism of MP biosynthesis from new perspectives.IMPORTANCEMonascus is important in food microbiology as it produces natural colorants known as Monascus pigments (MPs). The industrial production of MPs has been achieved by liquid fermentation, in which the nitrogen source (especially ammonium chloride) is a key nutritional parameter. Previous studies have investigated the regulatory mechanisms of substance and energy metabolism, as well as the cross-protective mechanisms in Monascus in response to ammonium chloride stress. Our research in this work demonstrated that ammonium chloride stress also caused an imbalance of membrane lipid homeostasis in Monascus due to the inhibition of ergosterol biosynthesis. We found that the regulation mechanism of phospholipids in Monascus was implemented, including inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway. These findings further refine the regulatory mechanisms of MP production and secretion.

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.