The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)-assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites. KEY POINTS: • MpSTE1 is the first characterized regulator for tightly regulating hyphal branching • Overexpression of mpSTE1 significantly improves secondary metabolite production • A high-throughput image analysis tool was developed for counting hyphal branching.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

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.

Unveiling the potential of red koji polysaccharides: biosynthesis, extraction, and multifaceted biological activities.

Red koji polysaccharides, derived from the fermentation of Monascus, have been recognized for their health-enhancing properties. This article reviews their structural characteristics, biosynthesis pathways, and biological activities. It emphasizes the need for sustainable practices in fermentation and the optimization of extraction methods for scalable production. The significance of exploring the molecular mechanisms involved in their biosynthesis is also highlighted to enhance yield and efficiency. Research indicates that red koji polysaccharides possess diverse biological functions, beneficial for pharmaceutical applications due to their health benefits and minimal toxicity. The review points out the necessity for more detailed studies on key enzymes and genes in biosynthesis to improve production methods. It also identifies the current challenges in production scalability and extraction efficiency. Furthermore, while these polysaccharides show potential in pharmaceuticals, their clinical efficacy and mechanism of action in human subjects require further investigation. The review briefly explores potential structural modifications to improve their biological activities. The review concludes that red koji polysaccharides hold significant untapped potential, particularly in drug formulation. Future research should focus on overcoming current production and application challenges, including conducting clinical trials to validate their efficacy and exploring structural modifications for enhanced therapeutic benefits. This comprehensive understanding of red koji polysaccharides paves the way for their expanded application in the pharmaceutical industry. © 2024 Society of Chemical Industry.

The response pattern of the microbial community structure and metabolic profile of jiupei to Bacillus subtilis JP1 addition during baijiu fermentation.

BACKGROUND: Baijiu brewing is a complex and multifaceted multimicrobial co-fermentation process, in which various microorganisms interact to form an interdependent micro-ecosystem, subsequently influencing metabolic activities and compound production. Among these microorganisms, Bacillus, an important bacterial genus in the liquor brewing process, remains unclear in its role in shaping the brewing microbial community and its functional metabolism. RESULTS: A baijiu fermentation system was constructed using B. subtilis JP1 isolated from native jiupei (grain mixture) combined with daqu (a saccharifying agent) and huangshui (a fermentation byproduct). Based on high-throughput amplicon sequencing analysis, it was evident that B. subtilis JP1 significantly influences bacterial microbial diversity and fungal community structure in baijiu fermentation. Of these, Aspergillus and Monascus emerge as the most markedly altered microbial genera in the jiupei community. Based on co-occurrence networks and bidirectional orthogonal partial least squares discriminant analysis models, it was demonstrated that the addition of B. subtilis JP1 intensified microbial interactions in jiupei fermentation, consequently enhancing the production of volatile flavor compounds such as heptanoic acid, butyl hexanoate and 3-methylthiopropanol in jiupei. CONCLUSION: B. subtilis JP1 significantly alters the microbial community structure of jiupei, enhancing aroma formation during fermentation. These findings will contribute to a broader application in solid-state fermentation. © 2024 Society of Chemical Industry.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

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

Linoleic acid functions as a quorum-sensing molecule in Monascus purpureus-Saccharomyces cerevisiae co-culture.

When Monascus purpureus was co-cultured with Saccharomyces cerevisiae, we noted significant changes in the secondary metabolism and morphological development of Monascus. In yeast co-culture, although the pH was not different from that of a control, the Monascus mycelial biomass increased during fermentation, and the Monacolin K yield was significantly enhanced (up to 58.87% higher). However, pigment production did not increase. Co-culture with S. cerevisiae significantly increased the expression levels of genes related to Monacolin K production (mokA-mokI), especially mokE, mokF, and mokG. Linoleic acid, that has been implicated in playing a regulating role in the secondary metabolism and morphology of Monascus, was hypothesized to be the effector. Linoleic acid was detected in the co-culture, and its levels changed during fermentation. Addition of linoleic acid increased Monacolin K production and caused similar morphological changes in Monascus spores and mycelia. Exogenous linoleic acid also significantly upregulated the transcription levels of all nine genes involved in the biosynthesis of Monacolin K (up to 69.50% higher), consistent with the enhanced Monacolin K yield. Taken together, our results showed the effect of S. cerevisiae co-culture on M. purpureus and suggested linoleic acid as a specific quorum-sensing molecule in Saccharomyces-Monascus co-culture.

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.

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

AIMS: Extreme environment of microbial fermentation is the focus of research, which provides new thinking for the production and application of Monascus pigments (MPs). In this work, the high-sugar synergistic high-salt stress fermentation (HSSF) of MPs was investigated. METHODS AND RESULTS: The Monascus fungus grew well under HSSF conditions with 35 g L-1 NaCl and 150 g 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, 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, and 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. CONCLUSIONS: 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.

Histone deacetylase MrHos3 negatively regulates the production of citrinin and pigments in Monascus ruber.

Monascus spp. can produce a variety of beneficial metabolites widely used in food and pharmaceutical industries. However, some Monascus species contain the complete gene cluster responsible for citrinin biosynthesis, which raises our concerns about the safety of their fermented products. In this study, the gene Mrhos3, encoding histone deacetylase (HDAC), was deleted to evaluate its effects on the production of mycotoxin (citrinin) and the edible pigments as well as the developmental process of Monascus ruber M7. The results showed that absence of Mrhos3 caused an enhancement of citrinin content by 105.1%, 82.4%, 111.9%, and 95.7% at the 5th, 7th, 9th, and 11th day, respectively. Furthermore, deletion of Mrhos3 increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. In addition, deletion of Mrhos3 led to an increase in total pigment content and six classic pigment components. Western blot results revealed that deletion of Mrhos3 could significantly elevate the acetylation level of H3K9, H4K12, H3K18, and total protein. This study provides an important insight into the effects of hos3 gene on the secondary metabolites production in filamentous fungi.

Overexpression of MrEsa1 accelerated growth, increased ascospores yield, and the polyketide production in Monascus ruber.

Esa1 has been proven to be an important histone acetyltransferase involved in the regulation of growth and metabolism. Monascus spp. with nearly 2000 years of edible history in East Asian countries can produce a variety of polyketides. It is unknown whether Esa1 plays a regulatory role in Monascus spp. In this study, we isolated the homology of histone acetyltransferase Esa1 (named MrEsa1) and constructed a mresa1-overexpressed strain. Western blot experiments showed that MrEsa1 hyperacetylated at K4 and K9 of the H3 subunit in Monascus ruber. Overexpression of mresa1 led to the larger colony diameter and increased dry cell mass; meanwhile, the conidia germination rate was significantly accelerated in the mresa1-overexpressed strain before 4 h, and the number of ascospores in the mresa1-overexpressed strain was significantly higher than that in WT. In addition, the Monascus azaphilone pigments (MonAzPs) and citrinin production of the mresa1-overexpressed strain were 1.7 and 2.4 times more than those of WT, respectively. Reverse transcription-quantitative polymerase chain reaction experiment suggested that mrpigB, mrpigH, mrpigJ, and mrpigK, involved in MonAzPs synthesis, and pksCT, mrl3, and mrl7, involved in citrinin synthesis, were upregulated in mresa1-overexpressed strain. This study provides important insights into the effect of MrEsa1 on the developmental process and the production of secondary metabolites in Monascus spp.

Optimization of L-glutaminase production by Monascus ruber URM 8542 isolated from ice cream industrial effluent.

L-glutaminase is a hydrolytic enzyme with wide biotechnological applications. Mostly, these enzymes are employed in the feed industry for flavor enhancement and acrylamide mitigation. Also, L-glutaminase may have antiviral and antineoplastic effects making it a good choice for pharmaceutical applications. In this study, the strain Monascus ruber URM 8542 was identified through classical and molecular taxonomy using partial sequencing of ß-tubulin and calmodulin genes. Subsequently, the optimal culture conditions were evaluated by submerged fermentation (L-glutamine 10 g.L- 1) for L-glutaminase excretion. The isolate was identified as M. ruber URM 8542 which showed significant extracellular enzyme production with a yield of 11.4 times in relation to the specific activity of intracellular L-glutaminase. Regarding the optimization experiments, several factors such as L-glutamine concentration, temperature, and pH were compared using a full factorial design (23). The concentrations greater than 1% proved to be significantly better for glutaminase production (R2 = 0.9077). Additionally, the L-glutaminase was optimally active at pH 7.0 and 30 ºC. The L-glutaminase was remarkably stable across an alkaline pH range (7.0-8.0) and had a thermal stability ranging from 30 ºC to 60 ºC for 1 h. Taken together, these findings suggest that the L-glutaminase produced by M. ruber is a promising candidate for pharmacological application, although further studies need to be performed. To the best of our knowledge, this is the first report of L-glutaminase production by Monascus ruber.

Investigation of citrinin and monacolin K gene clusters variation among pigment producer Monascus species.

The filamentous fungi Monascus spp. have been widely used in the production of food colorants. However, the presence of mycotoxin citrinin and the antihypercholestrolemia agent monacolin K in Monascus-fermented products (MFPs) has raised food safety concerns. Here we de novo-sequenced the genomes of 26 Monascus species and proposed an unprecedented classification system, consist of sections A, B and C, according to the biosynthetic gene clusters (BGCs) distribution and phylogeny results. Based on the absence of citrinin gene cluster, section B species were genetically incapable of synthesizing citrinin. A distinguished section A strain named Monascus sanguineus was believed to be a promising food-pigment-producer particularly owing to the simultaneous inactivation of citrinin and monacolin K clusters. Interestingly, gene losses within Monascus secondary metabolism gene clusters were broadly discovered, which may convey a selective advantage in nutrients and energy competition to support the strong pigment-producing ability. Overall, our sectional delimitation system will reshape the industrial strategies for this economically important fungus.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.

Natural pigment from Monascus: The production and therapeutic significance.

OBJECTIVE: The present review highlights the advantages of using natural colorant over the synthetic one. We have discussed the fermentation parameters that can enhance the productivity of Monascus pigment on agricultural wastes. BACKGROUND: Food industry is looking for natural colours because these can enhance the esthetic value, attractiveness, and acceptability of food while remaining nontoxic. Many synthetic food colours (Azorubine Carmoisine, quinoline) have been prohibited due to their toxicity and carcinogenicity. Increasing consumer awareness towards the food safety has forced the manufacturing industries to look for suitable alternatives. In addition to safety, natural colorants have been found to have nutritional and therapeutic significance. Among the natural colorants, microbial pigments can be considered as a viable option because of scalability, easier production, no seasonal dependence, cheaper raw materials and easier extraction. Fungi such as Monascus have a long history of safety and therefore can be used for production of biopigments. METHOD: The present review summarizes the predicted biosynthetic pathways and pigment gene clusters in Monascus purpureus. RESULTS: The challenges faced during the pilot-scale production of Monascus biopigment and taming it by us of low-cost agro-industrial substrates for solid state fermentation has been suggested. CONCLUSION: Keeping in mind, therapeutic properties of Monascus pigments and their derivatives, they have huge potential for industrial and pharmaceutical application. APPLICATION: Though the natural pigments have wide scope in the food industry. However, stabilization of pigment is the greatest challenge and attempts are being made to overcome this by complexion with hydrocolloids or metals and by microencapsulation.

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.

Effects of mokF gene deletion and overexpression on the Monacolin K metabolism yields of Monascus purpureus.

Monascus purpureus is a fungus known for producing various physiologically active secondary metabolites. Of these, Monacolin K, a compound with hypocholesterolemic effects, is controlled by the biosynthetic gene mokF. Here, mokF deletion and overexpression strains (F2 and C3, respectively) were constructed using genetic engineering and compared with the M. purpureus wild strain (M1). The results showed that Monacolin K production was reduced by 50.86% in F2 and increased by 74.19% in C3. Of the three strains, C3 showed the highest production of Monacolin K and the most abnormal morphology. In addition, mokF influenced the expression level of mokA-mokI and might play an important role in regulating the biosynthesis of secondary metabolites in M. purpureus. Overall, our study verified the function of mokF in M. purpureus using gene deletion and overexpression technology. KEY POINTS: • The deletion and overexpression strains of mokF gene were successfully constructed. • The deletion or overexpression of mokF gene directly affected Monacolin K production. •The mokF gene had little effect on Monascus pigments and cell biomass.

Utilization of low-cost agricultural by-product rice husk for Monascus pigments production via submerged batch-fermentation.

BACKGROUND: Monascus pigments (MPs) produced by the genus Monascus, have been utilized for more than 2000 years in the food industry. In the present study, by submerged batch-fermentation (SBF), we were able to obtain a mutant strain with a high tolerance of inhibitory compounds generated from rice husk hydrolysate, allowing the production of MPs. RESULTS: The mutant strain, M. Purpureus M523 with high rice husk hydrolysate tolerance was obtained using the atmospheric and room temperature plasma (ARTP) screening system, producing 39.48 U mL-1 extracellular total MPs (yellow and orange MPs), using non-detoxified rice husk diluted sulfuric acid hydrolysate (RHSAH) as the carbon source in SBF. Extracellular MPs (exMPs) production was enhanced to 72.1 and 80.7 U mL-1 in supplemented SBF (SSBF) and immobilized fermentation (IF) using non-detoxified RHSAH, with productivities of 0.16 and 0.37 U mL-1  h-1 , respectively. In addition, our findings revealed that despite having a high RHSAH tolerance, the mutant strain was unable to degrade phenolic compounds. Furthermore, we discovered that inhibitory compounds, including furfural (Fur) and 5'-hydroxymethyl furfural (5'-HMF), not only inhibit MP biosynthesis, but also regulate the conversion of pigment components. CONCLUSION: The low-cost agricultural by-product, rice husk, can serve as an efficient substitute for MP production with high productivity via IF by Monascus spp. © 2021 Society of Chemical Industry.

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

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

Volatile Organic Compound-Mediated Antifungal Activity of Pichia spp. and Its Effect on the Metabolic Profiles of Fermentation Communities.

Volatile organic compounds (VOCs) are chemicals responsible for antagonistic activity between microorganisms. The impact of VOCs on microbial community succession of fermentation is not well understood. In this study, Pichia spp. were evaluated for VOC production as a part of antifungal activity during baijiu fermentation. The results showed that the abundance of Pichia in the defect group (agglomerated fermented grains) was lower than that in control group, and a negative interaction between Pichia and Monascus was determined (P < 0.05). In addition, the disruption of fungi was significantly related to the differences of metabolic profiles in fermented grains. To determine production of VOCs from Pichia and its effect on Monascus purpureus, a double-dish system was assessed, and the incidence of M. purpureus reduction was 39.22% after 7 days. As to antifungal volatile compounds, 2-phenylethanol was identified to have an antifungal effect on M. purpureus through contact and noncontact. To further confirm the antifungal activity of 2-phenylethanol, scanning electron microscopy showed that 2-phenylethanol widely and significantly inhibited conidium germination and mycelial growth of filamentous fungi. Metatranscriptomic analysis revealed that the Ehrlich pathway is the metabolic path of 2-phenylethanol in Pichia and identified potential antifungal mechanisms, including protein synthesis and DNA damage. This study demonstrated the role of volatile compound-mediated microbial interaction in microbiome assembly and discovered a plausible scenario in which Pichia antagonized fungal blooms. The results may improve the niche establishment and growth of the functional yeast that enhances the flavor of baijiu.IMPORTANCE Fermentation of food occurs within communities of interacting species. The importance of microbial interactions in shaping microbial structure and metabolic performance to optimize the traditional fermentation process has long been emphasized, but the interaction mechanisms remain unclear. This study applied metabolome analysis and amplicon sequencing along with metatranscriptomic analysis to examine the volatile organic compound-mediated antifungal activity of Pichia and its effect on the metabolism of ethanol during baijiu fermentation, potentially enhancing the establishment of the fermentation niche and improving ethanol metabolism.