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The extremotolerant red yeast Rhodotorula mucilaginosa displays resilience to diverse environmental stressors, including cold, osmolarity, salinity, and oligotrophic conditions. Particularly, this yeast exhibits a remarkable ability to accumulate lipids and carotenoids in response to stress conditions. However, research into lipid biosynthesis has been hampered by limited genetic tools and a scarcity of studies on adaptive responses to nutrient stressors stimulating lipogenesis. This study investigated the impact of nitrogen stress on the adaptive response in Antarctic yeast R. mucilaginosa M94C9. Varied nitrogen availability reveals a nitrogen-dependent modulation of biomass and lipid droplet production, accompanied by significant ultrastructural changes to withstand nitrogen starvation. In silico analysis identifies open reading frames of genes encoding key lipogenesis enzymes, including acetyl-CoA carboxylase (Acc1), fatty acid synthases 1 and 2 (Fas1/Fas2), and acyl-CoA diacylglycerol O-acyltransferase 1 (Dga1). Further investigation into the expression profiles of RmACC1, RmFAS1, RmFAS2, and RmDGA1 genes under nitrogen stress revealed that the prolonged up-regulation of the RmDGA1 gene is a molecular indicator of lipogenesis. Subsequent fatty acid profiling unveiled an accumulation of oleic and palmitic acids under nitrogen limitation during the stationary phase. This investigation enhances our understanding of nitrogen stress adaptation and lipid biosynthesis, offering valuable insights into R. mucilaginosa M94C9 for potential industrial applications in the future. [ABSTRACT FROM AUTHOR]

"Green-covering and red-heart" Guanyin Tuqu (GRTQ), as a type of special fermentation starter, is characterized by the "green-covering" formed on the surface of Guanyin Tuqu (SQ) and the "red-heart" in the center of Guanyin Tuqu (CQ). However, the mechanisms that promote temporal succession in the GRTQ microbial ecology and the formation of "green-covering and red-heart" characteristics remain unclear. Herein, we correlated the temporal profiles of microbial community succession with the main environmental variables (temperature, moisture, and acidity) and spatial position (center and surface) in GRTQ throughout fermentation. According to the results of high-throughput sequencing and culture-dependent methods, the microbial communities in the CQ and SQ demonstrated functional complementarity. For instance, the bacterial richness index of the CQ was greater than that of SQ, and the fungal richness index of the SQ was greater than that of CQ at the later stage of fermentation. Furthermore, Saccharomycopsis, Saccharomyces, Aspergillus, Monascus, Lactobacillus, Bacillus, Rhodanobacter, and Chitinophaga were identified as the dominant microorganisms in the center, while the surface was represented by Saccharomycopsis, Aspergillus, Monascus, Lactobacillus, Acetobacter, and Weissella. By revealing the physiological characteristics of core microorganisms at different spatial positions of GRTQ, such as Aspergillus clavatus and Monascus purpureus, as well as their interactions with environmental factors, we elucidated the color formation mechanism behind the phenomenon of "green" outside and "red" inside. This study provides fundamental information support for optimizing the production process of GRTQ. [ABSTRACT FROM AUTHOR]

Toxic fungal species produce hazardous substances known as mycotoxins. Consumption of mycotoxin contaminated feed and food causes a variety of dangerous diseases and can even lead to death of animals and humans, raising global concerns for adverse health effects. To date, several strategies have been developed to counteract with mycotoxin contamination. Red yeast as a novel biological dietary agent is a promising strategy to eliminate mycotoxicity in living organisms. Poultry are most susceptible animals to mycotoxin contamination, as they are fed a mixture of grains and are at higher risk of co-exposure to multiple toxic fungal substances. Therefore, this study investigated the genetic mechanism underlying long-term feeding with red yeast supplementation in interaction with multiple mycotoxins using transcriptome profiling (RNA_Seq) in the liver of laying hens. The results showed a high number of significantly differentially expressed genes in liver of chicken fed with a diet contaminated with mycotoxins, whereas the number of Significantly expressed genes was considerably reduced when the diet was supplemented with red yeast. The expression of genes involved in the phase I (CYP1A1, CYP1A2) and phase II (GSTA2, GSTA3, MGST1) detoxification process was downregulated in animals fed with mycotoxins contaminated diet, indicating suppression of the detoxification mechanisms. However, genes involved in antioxidant defense (GSTO1), apoptosis process (DUSP8), and tumor suppressor (KIAA1324, FBXO47, NME6) were upregulated in mycotoxins-exposed animals, suggesting activation of the antioxidant defense in response to mycotoxicity. Similarly, none of the detoxification genes were upregulated in hens fed with red yeast supplemented diet. However, neither genes involved in antioxidant defense nor tumor suppressor genes were expressed in the animals exposed to the red yeast supplemented feed, suggesting decreases the adsorption of biologically active mycotoxins in the liver of laying hens. We conclude that red yeast can act as a mycotoxin binder to decrease the adsorption of mycotoxins in the liver of laying hens and can be used as an effective strategy in the poultry feed industry to eliminate the adverse effects of mycotoxins for animals and increase food safety for human consumers. [ABSTRACT FROM AUTHOR]

Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu. Monascus purpureus YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to α/β fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The π-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu. [ABSTRACT FROM AUTHOR]

“Green-covering” Tuqu (TQ), as one of Xiaoqu, is a special fermentative starter (also known as Jiuqu in Chinese) that originated in southern China and is characterized by a layer of green mold covering (Aspergillus clavatus) the surface and (sometimes) with a red heart. It plays a vital role in producing light-aroma-type Baijiu (LATB). However, to date, the microbiota that causes red heart of TQ remain largely unexplored, and it is still unclear how these microbiota influence on the quality of LATB. In this study, two types of TQ, one with a red heart (RH) and another with a non-red heart (NRH), were investigated by high throughput sequencing (HTS) and directional screening of culture-dependent methods. The obtained results revealed the differences in the microbial communities of different TQ and led to the isolation of two species of Monascus. Interestingly, the results of high performance liquid chromatography (HPLC) detection showed that citrinin was not detected, indicating that Monascus isolated from TQ was no safety risk, and the contents of gamma-aminobutyric acid in the fermented grains of RH were higher than that of NRH during the fermentation. Selecting the superior autochthonous Monascus (M1) isolated from the TQ to reinoculate into the TQ-making process, established a stable method for producing the experimental “red heart” Tuqu (ERH), which confirmed that the cause of “red heart” was the growth of Monascus strains. After the lab-scale production test, ERH increased ethyl ester production and reduced higher alcohols production. In addition, Monascus had an inhibitory effect on the growth of Saccharomyces and Aspergillus. This study provides the safe, health-beneficial, and superior fermentation strains and strategies for improving the quality of TQ and LATB. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A red yeast isolated from orange and grape soil and identified by the 26S rDNA sequence analysis revealed that it was Rhodotorula mucilaginosa and named TZR2014. Its biomass and carotenoid production reached a maximum when using the fermentation medium with pH 6.0, containing 5% glucose, 1% peptone, and 1.5% yeast powder. TZR2014 was resistant to 55°C for 15min, 0.2% pig bile salts for 4 h, and artificial gastric and intestinal fluids. A total of thirty 28-day weaned pigs were divided into three groups, and the piglets were fed a basal diet (CON), a basal diet and orally administered 1ml 1.0 × 1010 CFU/ml Candida utilis DSM 2361 three times (C. utilis), or a basal diet and orally administered 1ml 1.0 × 1010 CFU/mL TZR2014 three times daily (R. mucilaginosa) for 4 weeks. Compared with the piglets in the CON group, those in the C. utilis or R. mucilaginosa group reported an increased average daily weight gain and average daily feed intake (P < 0.05) and a decreased feed/gain (P < 0.05). The diarrhea rate of piglets in the R. mucilaginosa group was lower than that in the CON and C. utilis groups (P < 0.05). Compared with that in the CON and C. utilis groups, the R. mucilaginosa group reported an increased ileum villus height (P < 0.05), serum concentration of total antioxidant content, total superoxide dismutase, and glutathione peroxidase and pepsin and lipase activities in the intestinal content, while it reported a decreased serum concentration of malondialdehyde and pH of the intestinal tract (P < 0.05). The relative abundances of Proteobacteria and Megasphaera of caecum in the R. mucilaginosa group were lower than those in the CON and C. utilis groups (P < 0.05). The relative abundances of Prevotella, Ruminococcaceae, Succinivibrio, Rikenellaceae RC9 gut group, and Roseburia of caecum in the R. mucilaginosa group were higher than those in the CON and C. utilis groups (P < 0.05). R. mucilaginosa TZR2014 can produce carotenoids and adapts to the animal's gastrointestinal environment. Oral R. mucilaginosa TZR2014 improved growth performance, enhanced antioxidant capacity, strengthened gastrointestinal digestion, and maintained the intestinal microbiological balance of piglets. [ABSTRACT FROM AUTHOR]

Amino acid metabolism could exert regulatory effects on Monascus pigments (MPs) biosynthesis. In this work, MPs biosynthesis regulated by methionine and S-adenosylmethionine (SAM) was investigated in Monascus purpureus RP2. The results indicated that the addition of methionine in fermentation significantly reduced MPs production by 60–70%, and it induced a higher expression of SAM synthetase Mon2A2272 and consequently led to SAM accumulation. However, the addition of SAM in fermentation promoted MPs production by a maximum of 35%, while over-expression of the gene Mon2A2272 led to a decrease in MPs yield, suggesting that SAM synthetase and SAM were likely to play different regulatory roles in MPs biosynthesis. Furthermore, the gene transcription profile indicated that SAM synthetase expression led to a higher expression of the transcriptional regulatory protein of the MPs biosynthesis gene cluster, while the addition of SAM gave rise to a higher expression of MPs biosynthesis activator and the global regulator LaeA, which probably accounted for changes in MPs production and the mycelium colony morphology of M. purpureus RP2 triggered by methionine and SAM. This work proposed a possible regulation mechanism of MPs biosynthesis by SAM metabolism from methionine. The findings provided a new perspective for a deep understanding of MPs biosynthesis regulation in M. purpureus. [ABSTRACT FROM AUTHOR]

Monascus pigments (MPs) have been used in the food industry for more than 2,000 years and are known for their safety, bold coloring, and physiological activity. MPs are mainly yellow (YMPs), orange (OMPs), and red (RMPs). In this study, a mutant strain Monascus purpureus H14 with high production of water-soluble YMPs (WSYMPs, λ max at 370 nm) was generated instead of primary YMPs (λ max at 420 nm), OMPs (λ max at 470 nm), and RMPs (λ max at 510 nm) produced by the parent strain M. purpureus LQ-6 through dual mutagenesis of atmospheric and room-temperature plasma and heavy ion beam irradiation (HIBI), producing 22.68 U/ml extracellular YMPs and 10.67 U/ml intracellular YMPs. WSYMP production was increased by 289.51% in optimal conditions after response surface methodology was applied in submerged fermentation. Application of combined immobilized fermentation and extractive fermentation improved productivity to 16.89 U/ml/day, 6.70 times greater than with conservative submerged fermentation. The produced WSYMPs exhibited good tone stability to environmental factors, but their pigment values were unstable to pH, light, and high concentrations of Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+. Furtherly, the produced exYMPs were identified as two yellow monascus pigment components (monascusone B and C21H27NO7S) by UHPLC-ESI-MS. This strategy may be extended to industrial production of premium WSYMPs using Monascus. [ABSTRACT FROM AUTHOR]

During potato chips manufacturing, large amounts of wastewater and potato powder wastes are produced. The wastewater obtained at washing after cutting the peeled potatoes into slices was analyzed, and a large quantity of organic compounds and minerals such as starch (1.69%), protein (1.5%), total carbohydrate (4.94%), reducing sugar (0.01%), ash (0.14%), crude fat (0.11%), Ca (28 mg/L), Mg (245 mg/L), Fe (45.5 mg/L), and Zn (6.5 mg/L) were recorded; these wastes could be considered as valuable by-products if used as a fermentation medium to increase the value of the subsequent products and to exceed the cost of reprocessing. In this study, we used wastewater and potato powder wastes as a growth medium for pigment and biomass production by Monascus purpureus (Went NRRL 1992). The response surface methodology was used to optimize total pigment and fungal biomass production. The influence of potato powder waste concentration, fermentation period, and peptone concentration on total pigment and biomass production was investigated using the Box-Behnken design method with 3-factors and 3-levels. The optimal production parameters were potato powder waste concentration of 7.81%, fermentation period of 12.82 days, and peptone concentration of 2.87%, which produced a maximum total pigment of 29.86 AU/ml that include, respectively, a maximum biomass weight of 0.126 g/ml and the yield of pigment of 236.98 AU/g biomass. The pigments produced were used as coloring agents for ice lolly. This study has revealed that the ice lolly preparations supplemented with these pigments received high acceptability. Finally, we recommend using wastewater and potato powder wastes for pigment and biomass production, which could reduce the cost of the pigment production process on an industrial scale in the future. [ABSTRACT FROM AUTHOR]

During potato chips manufacturing, large amounts of wastewater and potato powder wastes are produced. The wastewater obtained at washing after cutting the peeled potatoes into slices was analyzed, and a large quantity of organic compounds and minerals such as starch (1.69%), protein (1.5%), total carbohydrate (4.94%), reducing sugar (0.01%), ash (0.14%), crude fat (0.11%), Ca (28 mg/L), Mg (245 mg/L), Fe (45.5 mg/L), and Zn (6.5 mg/L) were recorded; these wastes could be considered as valuable by-products if used as a fermentation medium to increase the value of the subsequent products and to exceed the cost of reprocessing. In this study, we used wastewater and potato powder wastes as a growth medium for pigment and biomass production by Monascus purpureus (Went NRRL 1992). The response surface methodology was used to optimize total pigment and fungal biomass production. The influence of potato powder waste concentration, fermentation period, and peptone concentration on total pigment and biomass production was investigated using the Box-Behnken design method with 3-factors and 3-levels. The optimal production parameters were potato powder waste concentration of 7.81%, fermentation period of 12.82 days, and peptone concentration of 2.87%, which produced a maximum total pigment of 29.86 AU/ml that include, respectively, a maximum biomass weight of 0.126 g/ml and the yield of pigment of 236.98 AU/g biomass. The pigments produced were used as coloring agents for ice lolly. This study has revealed that the ice lolly preparations supplemented with these pigments received high acceptability. Finally, we recommend using wastewater and potato powder wastes for pigment and biomass production, which could reduce the cost of the pigment production process on an industrial scale in the future.

Amino acid metabolism could exert regulatory effects on Monascus pigments (MPs) biosynthesis. In this work, MPs biosynthesis regulated by methionine and S-adenosylmethionine (SAM) was investigated in Monascus purpureus RP2. The results indicated that the addition of methionine in fermentation significantly reduced MPs production by 60–70%, and it induced a higher expression of SAM synthetase Mon2A2272 and consequently led to SAM accumulation. However, the addition of SAM in fermentation promoted MPs production by a maximum of 35%, while over-expression of the gene Mon2A2272 led to a decrease in MPs yield, suggesting that SAM synthetase and SAM were likely to play different regulatory roles in MPs biosynthesis. Furthermore, the gene transcription profile indicated that SAM synthetase expression led to a higher expression of the transcriptional regulatory protein of the MPs biosynthesis gene cluster, while the addition of SAM gave rise to a higher expression of MPs biosynthesis activator and the global regulator LaeA, which probably accounted for changes in MPs production and the mycelium colony morphology of M. purpureus RP2 triggered by methionine and SAM. This work proposed a possible regulation mechanism of MPs biosynthesis by SAM metabolism from methionine. The findings provided a new perspective for a deep understanding of MPs biosynthesis regulation in M. purpureus.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route. [ABSTRACT FROM AUTHOR]

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis , total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl. [ABSTRACT FROM AUTHOR]

Karyotype analysis and chromosome localization of pigment and citrinin biosynthesis gene clusters of Monascus ruber M7. Keywords: red mold rice; Monascus species; secondary metabolism; development; ecological status EN red mold rice Monascus species secondary metabolism development ecological status N.PAG N.PAG 4 04/10/21 20210331 NES 210331 Introduction Red mold rice (RMR), the fermented product of I Monascus i strains, also called Hongqu, red fermented rice (RFR), and red yeast rice (RYR), has been used as a food coloring agent, food preservative, and traditional medicine for about 2,000 years in China (Chen et al., [10]). Usually, the production of MonAzPs was often accompanied by the contamination of mycotoxin (citrinin), meaning that MonAzPs high-producing strains usually have strong abilities to synthesize citrinin (Wang et al., [31]). I Monascus i species have great abilities to produce polyketides, such as well-known I Monascus i azaphilone pigments (MonAzPs), monacolin K (MK), and citrinin. [Extracted from the article]

Monacolin K is a secondary metabolite produced by Monascus with beneficial effects on health, including the ability to lower cholesterol. We previously showed that the yield of monacolin K was significantly improved when glutamic acid was added to the fermentation broth of Monascus purpureus M1. In this study, we analyzed M. purpureus in media with and without glutamic acid supplementation using a metabolomic profiling approach to identify key metabolites and metabolic pathway differences. A total of 817 differentially expressed metabolites were identified between the two fermentation broths on day 8 of fermentation. Pathway analysis of these metabolites using the KEGG database indicated overrepresentation of the citric acid cycle; biotin metabolism; and alanine, aspartate, and glutamate metabolic pathways. Six differentially expressed metabolites were found to be related to the citric acid cycle. The effect of citric acid as an exogenous additive on the synthesis of monacolin K was examined. These results provide technical support and a theoretical basis for further studies of the metabolic regulatory mechanisms underlying the beneficial effects of monacolin K and medium optimization, as well as genetic engineering of Monascus M1 for efficient monacolin K production. [ABSTRACT FROM AUTHOR]

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus-related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as “winter”) was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT, the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus. Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus. Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites.

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus-related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as “winter”) was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT, the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus. Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus. Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yin, Zhu, Zhang, Huang and Jia.)

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus- related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as "winter") was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT , the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus . Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus . Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites.