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.

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.

Disruption of UDP-galactopyranose mutase expression: A novel strategy for regulation of galactomannan biosynthesis and monascus pigments secretion in Monascus purpureus M9.

The impact of the cell wall structure of Monascus purpureus M9 on the secretion of extracellular monascus pigments (exMPs) was investigated. To modify the cell wall structure, UDP-galactopyranose mutase (GlfA) was knocked out using Agrobacterium-mediated transformation method, leading to a significant reduction in the Galf-based polysaccharide within the cell wall. Changes in mycelium morphology, sporogenesis, and the expression of relevant genes in M9 were also observed following the mutation. Regarding MPs secretion, a notable increase was observed in six types of exMPs (R1, R2, Y1, Y2, O1 and O2). Specifically, these exMPs exhibited enhancement of 1.33, 1.59, 0.8, 2.45, 2.89 and 4.03 times, respectively, compared to the wild-type strain. These findings suggest that the alteration of the cell wall structure could selectively influence the secretion of MPs in M9. The underlying mechanisms were also discussed. This research contributes new insights into the regulation of the synthesis and secretion of MPs in Monascus spp..

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.

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.

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

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.

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.

An oxidoreductase gene CtnD involved in citrinin biosynthesis in Monascus purpureus verified by CRISPR/Cas9 gene editing and overexpression.

Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression.

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.

Terminal carboxylation of branched carbon chain contributing to acidic stability of azaphilone pigments from a new isolate of Talaromyces amestolkiae.

Red Monascus pigments, a series of natural azaphilone alkaloids, have been utilized in China as a traditional food colorant for over 1000 years. However, instability under an acidic condition is its drawback. A new strain of Talaromyces amestolkiae was isolated in the present work, which produced the azaphilone talaromycorubrin and the corresponding azaphilone alkaloid (N-MSG-talaromycorubramine) exhibiting good stability even at pH below 3. The azaphilone alkaloid with acidic stability, an alternative of Chinese traditional red Monascus pigments, is potential for application as natural food colorant in acidic foods. The acidic stability of azaphilone alkaloid also benefits for direct fermentation of N-MSG-talaromycorubramine under a low pH condition. More importantly, correlation relationship between the terminal carboxylation of branched carbon chain of azaphilone and the stability of azaphilone alkaloids under an acidic condition is set up for the first time, which makes designing other acidic stable azaphilone alkaloids via genetic engineering become possible.

Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering.

Monascus pigments have various food industry applications and are pharmacologically active. Genome sequencing-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been implemented to increase pigment production in Monascus. To increase pigment production in M. ruber KACC46666, the CRISPR/Cas9 system was used to introduce mutations in two negative regulator genes (MpigI and MpigI'), among other genes involved in the Monascus pigment biosynthesis pathway. Dual single-guide RNAs were constructed to inactivate MpigI and MpigI'. After CRISPR/Cas9 inactivation, yellow, orange, and red pigment expression in the resulting △MpigI16-7 strain (among several Cas9-mediated mutants studied) was 2.5-, 12.4-, and 18.5-fold, respectively, higher than that in the wild-type strain. This study provides valuable information regarding CRISPR-guided metabolic engineering for natural colorant production.

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.

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.

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.

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp.

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the Monascus azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein-protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

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.

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

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

Comparative transcriptome analysis of Monascus purpureus at different fermentation times revealed candidate genes involved in exopolysaccharide biosynthesis.

Exopolysaccharides (EPS), metabolites of the medicinal edible fungus Monascus purpureus, have antioxidant, immunomodulatory, and anti-inflammatory effects. However, the biosynthetic mechanism of EPS from M. purpureus is still unclear, which hinders its utilization. In this study, the fermentation conditions of M. purpureus were optimized and comparative transcriptomic analysis was performed to understand the mechanisms and effects of fermentation on EPS synthesis. The optimal medium composition was 40 g/L mannose, 4 g/L yeast powder, 1 g/L MgSO4·7H2O, 0.8 g/L KH2PO4, 1.6 g/L K2HPO4·3H2O, and 2 mL/L Tween 80, and the optimal cultivation conditions were an inoculum of 7 %, culture temperature 30 °C, initial pH 6.0, and 180 rpm for 4 d. A total of 8095 unigenes were obtained, and 17 key enzymes for EPS synthesis were identified. Interestingly, 12 carbohydrate metabolism subcategories were enriched in the group with 4 days of fermentation compared to 2 days, with most of the differentially expressed genes (DEGs) being upregulated, but only nine carbohydrate metabolism subcategories were enriched with longer fermentation time, with all DEGs being downregulated. This study provides a theoretical basis for enhancing the EPS content and reveals the dynamics of EPS synthesis in M. purpureus, providing important targets for future EPS molecular modifications and gene knockdown studies.

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.