Divergence of metabolites in three phylogenetically close Monascus species (M. pilosus, M. ruber, and M. purpureus) based on secondary metabolite biosynthetic gene clusters

- phylogenetically close Monascus species ( M..
- purpureus ) based on secondary metabolite biosynthetic gene clusters.
- Background: Species of the genus Monascus are considered to be economically important and have been widely used in the production of yellow and red food colorants.
- ruber, are used for food fermentation in the cuisine of East Asian countries such as China, Japan, and Korea.
- These species have also been utilized in the production of various kinds of natural pigments..
- Results: We examined the diversity of secondary metabolite production in three Monascus species (M.
- The colors in the liquid media corresponding to the pigments and their related metabolites produced by the three species were very different from each other.
- The gene clusters for secondary metabolite biosynthesis of the three Monascus species also diverged, confirming that M.
- ruber has similar biosynthetic and secondary metabolite gene clusters to M.
- The comparison of secondary metabolites produced also revealed divergence in the three species..
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- Conclusions: Our findings are important for improving the utilization of Monascus species in the food industry and industrial field.
- However, in view of food safety, we need to determine if the toxins produced by some Monascus strains exist in the genome or in the metabolome..
- Species of the genus Monascus are economically import- ant because they have been widely used in the produc- tion of yellow and red food colorants.
- ruber are commonly used for food fermentation in the cuisine of East Asian coun- tries including China, Japan, and Korea [1–3].
- It has been reported that these compounds are effective in lowering cholesterol, as well as in the treatment of diabetes, cardiovascular diseases, and some cancers [10]..
- ruber NBRC4483 to determine the diversity of the pig- ments based on metabolome data and secondary metabolite-related gene clusters such as monacolins, ci- trinin and azaphilone pigment.
- Several pigments are syn- thesized by PKS (poly ketide synthase) enzyme and NRPS (nonribosomal peptide) enzyme systems, which are encoded by large gene clusters in the genome.
- Com- parison of such gene clusters between the three species will provide new insights into the potential production of novel pigments..
- Monascus species produce a multitude of compounds, including polyketides, unsaturated fatty acids, and phy- tosterols.
- Therefore, further studies of genes involved in the synthesis of secondary metabolites by Monascus need to be carried out.
- In the present study, we determined the genome sequences of M.
- The phylogenetic and chemotaxonomic differ- ences between these three species were characterized by analyzing the gene clusters associated with secondary metabolites..
- Monascus species can produce several types of azaphi- lones, including nitrogenated azaphilones, N-glucosyl azaphilones, amino acid derivative azaphilones, and citri- nins [4].
- We cultivated the three Monascus species to compare the production of the pigments and their.
- Figure 1b showed the concentration of 14 metabolites quantified in three biological replicates in the three species as a heatmap with two-dimensional hierarchical clustering to display their similarity.
- This re- sult confirmed that not all these metabolites are synthe- sized in the three species.
- Dehydromonacolin K, rubro- punctatin, monascin, and ankaflavin 2 were commonly produced by all three Monascus species.
- Of the three species, M.
- 1 Pigments for three Monascus species for PD agar and PDL cultures.
- a Monascus species are cultured in PD agar and PDL medium at 30 °C..
- These resutls showed that the production of secondary metabolites are distinct among these three Monascus species.
- pilosus, while monascorubramine was detected in the other two species.
- pilosus, while monascorubramine was detected in the other two species..
- Diversity and classification of secondary metabolites produced by the three Monascus species.
- Azaphilones are pigments with pyrone- quinone structures containing a highly oxygenated bi- cyclic core and a chiral quaternary center and produced mainly by Monascus species [21].
- To examine the me- tabolite divergence in the three Monascus species, we examined the diversity of azaphilones in the abovemen- tioned five major metabolic groups.
- 3a) and identified the ubiquitous and species- specific metabolites in the five groups using Venn dia- grams.
- Results showed that the three Monascus species can produce unique 1H-isochromene compounds.
- Comparison of secondary metabolite gene clusters in the three Monascus species.
- In order to measure genetic and evolutional similarity of the enzymes related in these three species, we have ana- lyzed their whole genome sequences and annotated the sequences of the enzymes related in the biosysthesis pathways of these metabolites.
- We can expect that the genome size for each species is around 24 Mb, since in the literature, the genome sizes reported for M..
- Furthermore, to evaluate the clusters of the enzymes related with the secondary metabolic pathways we ana- lyzed their genome sequences using antiSMASH 4.0 [31], and we obtained 24 gene clusters for M.
- scores of the 92 gene clusters (Fig.
- 4), obtaining 22 groups with multiple gene clusters and 32 singletons..
- Gene clusters with identical gene organization were merged into a single group.
- We also obtained 54 groups of gene clusters (Table 1) that are displayed as Venn diagrams (Fig.
- ABC transporters are associated with PKS and NRPS gene clusters in several fungi and are respon- sible for the export of the corresponding secondary me- tabolites [33, 34]..
- Taking gene organization in gene clusters into consid- eration, only two gene clusters were common in the three species.
- In all, 85% of the identified secondary metabolic biosynthetic gene clusters were assigned to each Monascus species.
- Thus, the organization of genes in the genome of each Monascus species is highly di- verse (Fig.
- Otherwise, if gene-clusters are classified based on DNA sequence similarity, 16 (30%) were found to be common in the three species.
- Each Venn diagram classifies metabolies into reported species using a total of 74 previously reported secondary metabolites (a),citrinins (b1),monaphilones (b2),1H-isochromenes (b3),nitrogenated azaphilones (b4),and monacolins (b5), specific to Monascus-species .
- The results of the present study strongly suggest that the three Monascus species have greatly diverged gene clusters.
- We further analyzed the differences in the Monascus species at the DNA level by comparing the 8144-bp re- gion where a Monascus azaphilone pigment biosynthetic gene cluster was localized [35].
- ruber in the concatenated phylogeny based on the ITS, BenA, CaM LSU, and RPB2 gene re- gions of 46 relative strains [36].
- ruber has similar bio- synthetic gene clusters for citrinin, monacolin K, and Monascus azaphilone pigments with M.
- Comparison of citrinin biosynthetic gene clusters in the three Monascus species.
- To address this issue, we compared the peptide sequences of citrinin biosynthetic genes in the three Monascus species with those in M.
- 4 Dendrogram of secondary metabolite biosynthetic gene clusters observed in three Monascus species.
- The secondary metabolism gene clusters of M.
- Table 1 Classification of gene clusters (ID1 – 54) corresponding to the dendrogram in Fig.
- 5 Venn diagrams of secondary metabolite biosynthetic gene clusters observed in three Monascus species.
- (a1):Venn diagram classifying 82 secondary metabolic synthesis gene clusters of three Monascus species.
- (a2):Venn diagram classifying 82 secondary metabolic synthesis gene clusters of three Monascus species based on DNA sequence homology.
- The total number of gene clusters was 54.
- Venn diagram classifying 54 secondary metabolic synthesis gene clusters of three Monascus species based on DNA sequence homology: T1PKS (b1), NRPS (b2), T1-PKS-NRPS (b3), Terpenes (b4), Others (b5), Cf-Putative or Cf-fatty acid (b6).
- Comparison of monacolin biosynthetic gene clusters in the three Monascus species.
- In the present study, we also examined the monacolin gene clusters in three Monascus species..
- The three Monascus species examined in the present study are commonly used for food fermentation in the cuisine of East Asian countries [1–3].
- The three Monascus species can produce ubiquitous and species-specific pigment-related com- pounds (Figs.
- Analysis of gene-organization re- vealed 54 greatly diverged gene clusters in the three Monascus species studied (Fig.
- On the other hand, taking the pigment biosynthetic gene clusters into consideration, M.
- Thus, based on the findings of the present study, the Monascus species studied here can be classified into two groups: (i) the M..
- The mycotoxin citrinin is produced by various Penicil- lium, Aspergillus, and Monascus species .
- In the analysis of the monacolin K gene cluster, four M.
- ruber NBRC 4483 have a complete set of monacolin K gene clusters.
- ruber NBRC 4483 can produce monacolin K but lack a complete set of citrinin biosynthetic gene clusters..
- purpureus may play an important role in the food in- dustry and industrial field through the improved utilization of Monascus species.
- However, in view of food safety, we need to confirm whether the toxins produced by some Monascus strains exist in the gen- ome or metabolome.
- The citrinin pathway be- longs to the former as it is present in many Penicillium, Aspergillus, and Monascus species .
- However, the biosynthetic gene cluster of Monas- cus azaphilone pigments is limited in the Monascus genera.
- Gene duplication (GD) is often implicated in the evolution of fungal metabolism (Floudas et al., 2012).
- In the biosynthetic gene clusters of Monascus azaphilone pigments and citrinin, the.
- common trends in the strains of the three Monascus species are explained by the suggested M.
- Monascus-spe- cific diverged pigments may have evolved because of GD and HGT events, resulting in the creation of clustered genes in their genomes.
- thus, a large num- ber of gene clusters was observed (Table 1).
- More genome sequences of Monascus species will need to be determined to better under- stand the production of secondary metabolites in these organisms..
- Three biosynthetic gene clusters, specifically monacolin K, citrinin, and Table 3 Percent identity of the amino acid sequence of citrinin biosynthetic genes in three Monascus species.
- Reference peptide sequence is Monascus purpureus citrinin biosynthesis gene clusters (https://www.ncbi.nlm.nih.gov/nuccore/AB243687.1.
- Table 4 Percent identity of the amino acid sequence of monacolin biosynthetic genes in three Monascus species.
- Reference peptide sequence is Monascus pilosus monacolin biosynthesis gene clusters (https://www.ncbi.nlm.nih.gov/nuccore/DQ176595.1).
- purpureus, may play an import- ant role in the food industry and industrial field through the improved utilization of Monascus species.
- We further analyzed the genomes using antiSMASH pipeline [55] to extract the functional gene clusters such as PKS, in each Monascus species..
- Secondary metabolites of the fungus Monascus: a review.
- Exploring the distribution of citrinin biosynthesis related genes among Monascus species..
- A new hypocholestrerolemic agent produced by a Monascus species.
- Phylogenetic relationships of Monascus species inferred from the ITS and the partial β - tubulin gene.
- antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences

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