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

Background: Species under the genus Monascus are considered as economically important and have been widely used in the production of yellow and red food colorants. In particular, three Monascus species, namely, M. pilosus , M. purpureus , and M. 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 pigment-related biosynthetic pathways in three Monascus species ( M. pilosus , M. purpureus , and M. ruber ) at the metabolome and genome levels. Illumina MiSeq 300 bp paired-end sequencing generated 17 million high-quality short reads in each species, corresponding to 200 times the genome size. We measured the pigments and their related metabolites using potato dextrose liquid (PDL) media. The colors in the PDL media corresponding to the pigments and their related metabolites produced by the three species are very different from each other. The gene clusters for secondary metabolite biosynthesis of the three Monascus species also diverged, conrming that M. pilosus and M. purpureus are chemotaxonomically different. M. ruber has similar biosynthetic gene clusters for citrinin, monacolin K, and Monascus azaphilone pigments with M. pilosus and M. purpureus. The comparison of secondary metabolites produced also revealed divergence in the three species. Conclusions: Our ndings are important for improving the utilization of Monascus species in the food industry and industrial eld. 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.

The complete genome sequence of the industrial strain M. purpureus YY-1 is already available (Yang et al., 2015). However, the genome sequences of M. ruber and M. pilosus are still incomplete. Understanding the diversity of the pigments produced by these species at the genome level is remarkably important for their industrial applications. We analyzed M. pilosus, M. purpureus, and M. ruber to determine the diversity of the pigments based on metabolome data and pigment-related gene clusters. Several pigments are synthesized through the PKS and NPRS systems responsible for organizing gene clusters in the genome. Comparison of gene clusters between the three species will provide new insights to the potential production of novel pigments.
Monascus species produce a multitude of compounds, including polyketides, unsaturated fatty acids, phytosterols, pigments, and monacolins. Monacolins, especially monacolin K, inhibit 3-hydroxy-3methylglutaryl-coenzyme A reductase, which is the rate-limiting step in cholesterol biosynthesis. These compounds found in red yeast rice prevent the production of high cholesterol level that causes atherosclerosis (Gerards et al., 2015). Hence, it is expected that metabolites related with Monascus pigments can contribute to human healthcare. However, citrinin was found as an undesirable contaminant in red yeast rice (Fink-Gremmels et al., 1991).
Thus, it is required to clarify the diversity of pigment biosynthetic pathways in economically important Monascus species. In the present study, we determined the genome sequences of M. pilosus, M. purpureus, and M. ruber. The phylogenetic and chemotaxonomic differences between the three were characterized by analyzing the gene clusters of secondary metabolites. The pigment production in M.
pilosus was also further characterized.

Production of secondary metabolites
Monascus species can produce a several types of azaphilones, including 1H-isochromenes, nitrogenated azaphilones, citrinins, and monacolins (Gao et al., 2013). We measured the pigments and their related metabolites in three Monascus species using potato dextrose liquid (PDL) media, which is the frequently used and suitable culture media for Monascus growth and metabolite production (Carvalho et al., 2003).
As shown in Fig. 1a, the colors in the media cultured with the individual species are distinct. A gradient of orange to light yellow (from center toward edges) was observed in M. pilosus, gray to light yellow in M.
ruber, and pink in M. purpureus. We also observed the gradients from light orange in M. pilosus, from pale orange to black color in M. ruber, and from red to dark purple in M. purpureus. Thus, the colors were re ected by the different pigments produced by the three species cultured in identical conditions. We also analyzed the pigment-related metabolites using LC-MS and identi ed 14 metabolites (Fig. 1b). The reproducibility of metabolite quantities was con rmed by three iterative measurements.
The biosynthetic pathway from malonyl-CoA to1H-isochromenes was determined in Penicillium marneffei and M. ruber (Woo et al., 2014;Chen et al., 2017). Citrinin polyketide synthase (PKS) converts the PKS-bound product citrinin (He and Cox, 2016). The biosynthetic pathway from malonyl-CoA to monacolins was also determined in Aspergillus terreus, M. ruber, and M. purpureus (Cambell et al., 2010; Zhang et al., 2017). It should be noted that PKS-bound products are acted upon by two different types of PKS enzymes -one is an enzyme to produce Monascus azaphilone pigments which corresponds to the pathway from malonyl-CoA to 1H-isochromenes and nitrogenated azaphilones (Chen et al., 2017) and the other is citrinin polyketide synthase which corresponds to the pathway from PKS-bound product to citrinin (He and Cox et al., 2016). Figure 2 shows the metabolic pathways of ve major groups: (i) monacolins, (ii) citrinins, (iii) monaphilines, (iv) 1H-isochromenes, and (v) nitrogenated azaphilones. The color bars represent the levels of metabolites accumulated based on 2D clustering results in Fig. 1b. Among the three Monascus species, some of the metabolites related with 1H-isochromenes were accumulated through the biosynthetic pathways from malonyl-CoA to 1H-isochromenes. All eight metabolites related with 1H-isochromenes were only detected in M. pilosus. Pigments produced by M.
pilosus are more suitable for observation in PDL medium than the other two species. Citrinin was only observed in M. purpureus. Dehydromonacolin K, which is a precursor for monacolin K production, was detected in all three species.
Thus, the accumulation of pigment-related metabolites may be different among the three Monascus species, as observed using identical culture conditions in PDL medium. Metabolites related with 1Hisochromenes and nitrogenated azaphilones were observed among the three, but the metabolites were different between individual species. On the other hand, metabolites related with anka avin 1 and rubropunctatin were observed in M. pilosus, while monascorubramine was detected in the other two.

Discussion
The three Monascus species examined in the present study are commonly used for food fermentation in However, in view of food safety, we need to con rm whether the toxins produced by some Monascus strains exist in the genome or metabolome. Metabolites are generally classi ed into the primary metabolites that are essential for growth and reproduction and the secondary metabolites that are usually involved in mechanisms for ecological adaptation but are not essential to regular cellular processes. Metabolic pathways can be divided into two types: one is the general pathway shared by most fungi and the other are specialized pathways that have evolved in response to speci c ecologies of certain lineages and are consequently more narrowly distributed at the taxonomic level. Citrinin pathway belongs to the former as it is present in many Penicillium, Aspergillus, and Monascus species (Wong et al., 1977;Ma et al., 2000;Rasheva et al., 2003). However, the biosynthetic gene cluster of Monascus azaphilone pigments is limited in the Monascus genera. The biosynthetic process of secondary metabolites forms a cluster or non-clustered gene organization that is integral to the entire spectrum of fungal ecological strategies (e.g., saprotrophic, pathogenic, and symbiotic). Gene duplication (GD) is often implicated in the evolution of fungal metabolism (Floudas et al., 2012). A second source of metabolic gene innovation in fungi is horizontal gene transfer (HGT), which includes xenobiotic catabolism (Gardiner et al., 2012), toxin production (Friesen et al., 2006), and degradation of plant cell walls (Garcia-Vallve et al., 2000). GD and HGT were more frequently occurring in clustered genes than in their non-clustered counter parts (Wisecaver et al., 2014). 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. pilosus and M. purpureus clades, whereas M. ruber has either M. pilosus or M. purpureus trends. Monascus-speci c diverged pigments may have evolved because of GD and HGT events, resulting in the creation of clustered genes in their genomes; thus, a large number of gene clusters was observed (Table 1). Chemotaxonomy, including pigments, is the most useful role for the divergence in the Monascus genera.

Conclusions
In this study, the complete genome sequences of M. pilosus NBRC 4520, M. purpureus NBRC 4478, and M. ruber NBRC 4483 were obtained. Three biosynthetic gene clusters, speci cally monacolin K, citrinin, and azaphilon pigments that are involved in secondary metabolism, were analyzed and compared. The classi cation of strains according to the two clade groups, designated as (i) M. pilosus and (ii) M. purpureus, may play an important role in the food industry and industrial eld through the improved utilization of Monascus species. However, in view of food safety, further studies are needed to con rm whether the toxins produced by some Monascus strains originate from the genome and not from the metabolome.

Methods
Strains, culture condition, and metabolite detection Three Monascus species, speci cally, M. pilosus NBRC 4520, M. purpureus NBRC 4478, and M. ruber NBRC 4483, were obtained from the National Institute of Technology and Evaluation in Japan. The three species were cultured in potato dextrose liquid medium at 30°C for 7 days with 140 rpm shaking in TAITC BR-23FP. A solution of 10 mg freeze-dried PDL medium added with 1 mL methanol was sonicated for 30 min to extract secondary metabolites. The extracted metabolites were measured using Shimadzu LCMS-8040 (Shimadzu, Kyoto, Japan) with 300 mm ODS MonoBis columns (Kyoto Monotech Co., Ltd., Kyoto, Japan).

Genome sequencing and assembly
We isolated genomic DNA from the three species individually and sequenced them using Illumina MiSeq  (Chen et al., 2008). To identify the gene-coding regions, the nucleotide sequence of the assembled scaffolds was annotated using DIAMOND, a high throughput BLASTX compatible sequence alignment algorithm (Buch nk et al., 2015). The assembled sequences were also BLASTed against the UniProtKB/Swiss-Prot database (Pundir et al., 2017)

Authors' contributions
Conceptualization and design of the study were performed by YH, SK, and NO. Sample preparation and genomic DNA isolation were carried out by YH. Assembly and scaffolding of sequencing reads were performed by NO. Subsequent comparative genomic analysis were conducted by NO and YH. Statistical processing and gure creation were conducted by SK. Culture and LC-MS analysis were performed by YH.
Valuable comments and advice on writing papers were provided by AA, MK, and YSK. All authors have read and approved the nal manuscript. Tables   Due to technical limitations, tables are only available    Accumulation of three metabolite clusters corresponding to the pigments observed in Figure 1

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Tables20200121higa.pdf