Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing

  • Song Zhang,

    Roles Formal analysis, Writing – original draft

    Affiliation National Engineering Research Center of Rice and Byproduct Deep Processing, Central South University of Forestry and Technology, Changsha, Hunan, China

  • Xiaofang Zeng,

    Roles Methodology

    Affiliation College of Light Industry and Food Sciences, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, China

  • Qinlu Lin,

    Roles Resources

    Affiliation National Engineering Research Center of Rice and Byproduct Deep Processing, Central South University of Forestry and Technology, Changsha, Hunan, China

  • Jun Liu

    Roles Data curation, Writing – review & editing

    liujundandy@csuft.edu.cn

    Affiliations National Engineering Research Center of Rice and Byproduct Deep Processing, Central South University of Forestry and Technology, Changsha, Hunan, China, Hunan provincial Key Laboratory of Food Safety Monitoring and Early Waring, Changsha, Hunan, China

Abstract

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Introduction

Fermented products of Monascus spp. have been widely used in the food and pharmaceutical industry for more than 2000 years [1]. As the secondary metabolite produced by Monascus spp., Monascus pigments (MPs) are a mixture of azaphilones mainly composed of three colors (yellow, orange, and red) pigments, which possess various bioactivities, such as antimicrobial, anticancer, anti-inflammatory, and anti-obesity [2, 3]. Nowadays, due to the potential risks of allergies, carcinogenesis, and teratogenesis of synthetic pigments, natural MPs are widely used as food colorants and are well recognized by consumers [4, 5]. In addition, MPs have other applications in the pharmaceutical, textile, and cosmetics industries. Traditionally, MPs are mainly produced by solid-state fermentation (SSF) with rice as the substrate for high pigment concentration [6]. However, submerged fermentation (SF) was more widely applied in the industrial production at present due to high pigment production efficiency, an easy-to-control fermentation process, and avoidance of contamination [7, 8].

Different species of Monascus spp. have been isolated for the biosynthesis of various secondary metabolites. In general, M. fuliginosus [9, 10], M. ruber [11, 12] and M. pilosus [1315] have a strong capacity to produce monacolin K. Nevertheless, M. purpureus is the most predominant microorganisms for the efficient production of MPs because of its high efficiency to produce pigments [1618]. With the development of whole-genome sequencing (WGS) technology, the complete sequence analysis of Monascus has been used to reveal the chromosome evolution, regulatory mechanisms, and functional genes of M. purpureus, which lays the foundation for the production of secondary metabolites and biological researches. In 2015, Yang et al. published the first sequence information of M. purpureus YY-1, with a genome size of 24.1 Mb and a total of 7491 predicted genes. WGS analysis predicted the gene clusters related to pigment biosynthesis in M. purpureus YY-1 and explained the smaller size of the M. purpureus genome than that of related filamentous fungi, indicating a significant loss of genes [19]. Kumagai et al. reported the genome sequence information of the high pigment-producing M. purpureus GB-01 strain, with a genome size of 24.3 Mb and 121 chromosomal contigs [20]. Liu et al. identified the key genes (ERG4A and ERG4B) for ergosterol biosynthesis in M. purpureus LQ-6 (genome size: 26.8 Mb, 8596 protein-coding genes). Knocking out the ERG4 gene improved the permeability of the cell membrane and secretion of intracellular pigments; it also changed the morphology of M. purpureus LQ-6 in SF broth [21]. Although numerous studies on the morphological changes of Monascus in SF have been performed, the biological information on the metabolic engineering of morphology of Monascus remains unknown [2224].

Hyphae of filamentous fungi in SF mainly exist in three morphological forms, including free mycelia, mycelial pellets, and mycelial clumps [25], and the difference of metabolites is probably due to the different morphology of hyphae. The mycelium pellet is the optimal morphology for glucoamylase production by Aspergillus niger, while the fermentation production of citric acid is more biased to the mycelial morphology [26]. The veA gene globally regulates the propagation mode, mycelial growth, environmental tolerance, and secondary metabolites of fungi [27, 28]. Muller et al. disrupted the biosynthesis of chitin and changed the morphology of Aspergillus oryzae by regulating the transcription level of the chitin synthase gene chsB, and studied the relationship between morphology and α-amylase biosynthesis [29]. RNA interference technology has been applied to silence the expression of the chitin synthase gene chs4 in Penicillium chrysogenum, reducing the mutant growth rate, aggregation of dispersed hyphae into the mycelium, and increased penicillin production [30]. With the in-depth study of different phenotype mutants, it is found that the cell wall is an ideal target for morphological control. However, the differences were exsited in the encoding genes of chitin synthase and the regulation of chitin synthase on morphology in different fungi [31]. Furthermore, the specific encoding genes of chitin synthase and biosynthesis pathway of chitin in M. purpureus is still unclear.

M. purpureus CSU-M183 is a high pigment-producing industrial preparation strain obtained by atmospheric room temperature plasma (ARTP) mutation system. In this study, the whole genome of strain CSU-M183 was sequenced using the single-molecule real-time (SMRT, PacBioRS II) DNA sequencing and Illumina next-generation sequencing (NGS) platforms. We also investigated the molecular expression effects of ARTP mutagenesis on the secondary metabolic synthesis of Monascus by RT-qPCR. The results showed a comprehensive prediction of biosynthetic gene clusters (BGCs) for secondary metabolites and the biosynthetic pathway of chitin in M. purpureus CSU-M183. We expect this will provide a better strategy in morphological metabolic engineering of Monascus, for the industrial production of the secondary metabolites via submerged fermentation.

Materials and methods

Fungal strains, culture media, and growth conditions

M. purpureus CSU-M183 (CCTCC M 2018224, China Central for Type Culture Collection (CCTCC), Wuhan, China) was obtained using the ARTP mutation system from the parent strain M. purpureus LQ-6 (CCTCC M 2018600) [32]. Strains was cultivated on potato dextrose agar (PDA) and potato dextrose broth (PDB) medium at 30°C in the dark for 7 days.

To prepare the inoculum, spores were transferred from PDA slants to submerged culture medium and washed with sterile distilled water, and then diluted to approximately 3 × 107 spores/ml. The 10% (v/v) inoculum was transferred to the submerged culture medium and incubated 7 days. 10% (V/V) of the inoculum was transferred to 250 ml shark flasks containing 45 ml liquid medium and incubated for 7 days in a rotary shaker with parameters set at 30°C and 180 rpm, respectively.

DNA extraction

Mycelia were collected after centrifugation at 8228 ×g for 10 min and stored at—80°C. Genomic DNA was extracted from mycelia using the EasyPure® Genomic DNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. The quantity, quality, and purity of the genomic DNA were measured using Nanodrop2000 systems and 0.8% DNase-free agarose gel electrophoresis.

WGS and assembling

The whole genome of the M. purpureus CSU-M183 strain was sequenced using SMRT sequencing technology of PacBioRS II, and the sequencing quality was improved using Illumina NGS platform. The sequencing library was constructed using the TruSeqTM Nano DNA LT Sample Prep Kit–Set A (Illumina, USA) and amplified using the TruSeq PE Cluster Kit (Illumina, USA).

The quality of the assembled genome and annotated geneset were assessed first using the Benchmarking Universal Single-Copy Orthologs (version 3.1.0; BUSCO) with the fungi_odb9 dataset [33].

For raw data polymerase reads after PacBioRS II sequencing, subreads were obtained by removing the low-quality or unknown reads, adapters and duplications. The filtered reads were assembled de novo using the Hierarchical Genome Assembly Process (HGAP) algorithm version 2.0 [34].

For genome assembly, the default parameters of HGAP2 were used (Minimum Subread Length = 500, Minimum Polymerase Read Quality = 0.80, Minimum Polymerase Read Length = 100, Overlapper Error Rate = 0.06, Overlapper Min Length = 40) with input genome size as 30 Mb.

PacBio library construction.

High-quality DNA (10 μg in 200 μl 10 mM Tris–HCl pH8.5) was sheared using a Covaris g-tube (Covaris Inc.) with 6000RPM for 60seconds. Sheared DNA was purified by binding to 0.45X volume of pre-washed AMPure XP beads (Beckman Coulter Inc.), and eluted in EB to >140 ng/μl. The sheared DNA was quantified on an Agilent 2100 Bioanalyzer using the 12000 kit. 5 μg of sheared DNA was end-repaired using the PacBio DNA Template Prep Kit 2.0 (Part Number001-540-835) and incubated for 20 min at 37°C and then 5 min at 25°C prior to another 0.45X AMPure XP clean up, eluting in 30 μl EB. Blunt adapters were ligated before exonuclease incubation. Finally, two 0.45X AMPure bead clean ups are performed to remove enzymes and adapter dimers, and the final “SMRT bells” was eluted in 10 μl EB. Final quantification was carried out on an Agilent 2100 Bioanalyzer with 1 μl of library.

PacBio sequencing.

The diluted library was loaded onto the instrument, along with DNA Sequencing Kit 2.0 (Part Number 100-216-400) and a SMRT Cell 8Pac. In all sequencing runs, 90 min movies were captured for each SMRT Cell loaded with a single binding complex. Primary filtering analysis was performed with the RS instrument and the secondary analysis was used the SMRT analysis pipeline version 2.1.0.

Illumina sequencing.

Illumina library was sequenced on Hiseq X ten. Trimmomatic was used to trimm adaptor, low quality base(Q<20) and short reads(length <50bp).

Gene prediction and annotation

AUGUSTUS [35] and SNAP [36] were performed to predict coding genes. Genome functional annotation was performed using BLASTP, as well as NCBI non-redundant (NR), SwissProt, and Protein Information Resource (PIR) protein databases. All predicted genes were classified according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways and Cluster of Orthologous Groups of proteins (COG).

Prediction of secondary metabolites

To predict secondary metabolite biosynthesis of strain M. purpureus CSU-M183, the BGCs of secondary metabolites were annotated using antiSMASH fungi version 5.1.0 [37].

RT-qPCR analysis

RT-qPCR was performed according to the method described by Liu et al. [21], with β-Actin as the reference gene, the genes on the MPs and citrinin gene cluster were selected, and the expression of these genes was detected by qRT-PCR during the submerged fermentation of M. purpureus LQ-6 and M. purpureus CSU-M183, respectively. For removal of residual genomic DNA, RNA samples were treated with RNase-free DNaseI (Thermo Fisher Scientific, Massachusetts, USA) following the manufacturer’s protocol. The first-strand cDNA was synthesized using oligo-dT primers and EasyScript Reverse Transcriptase (TransGen Biotech, Beijing, China), according to the manufacturer’s protocol. qRT-PCR was performed using the TransStartGreen qPCR SuperMix UDG (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. 2−ΔΔCT was used to determine expression levels of the tested genes. The primers used in these analyses were listed in S1 Table.

Data availability

The assembled genome sequence of M. purpureus CSU-M183 has been deposited into the NCBI Genbank database with an accession number of JAACNI000000000. The BioProject and BioSample information are available at PRJNA599556 and SAMN13759458, respectively. The raw sequence data of M. purpureus CSU-M183 has been deposited into the NCBI Genbank database with an BioProject of PRJNA824977.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Statistical analysis

Each experiment was performed at least in triplicate and the results are shown as the mean ± standard deviation (SD). Statistical analyses were performed using the SPSS Statistics 23 (SPSS, Chicago, USA). Data were analyzed by one-way ANOVA, and tests of significant differences were determined by using Tukey multiple comparison or Student’s t-test at P < 0.05.

Results and discussion

Overview of WGS

In the previous study, we obtained a high pigment-producing M. purpureus CSU-M183 strain using the ARTP mutation system [32]. The morphological characteristics of Monascus are closely related to the production of secondary metabolites in SF. To further study the metabolic engineering of the morphology of M. purpureus CSU-M183, the WGS of strain M. purpureus CSU-M183 was carried out. BUSCO analysis indicated 92% completeness based on fungi reference genes. Among the 290 BUSCO groups searched, 280 BUSCO groups (including complete and fragmented BUSCOs) were identified, occupying 96.5% of the total BUSCO groups. Among them, 267 groups were complete BUSCO groups, occupying 92.0% of the total BUSCO groups. The M. purpureus CSU-M183 genome sequence of 23.75 Mb was generated by assembling approximately 9.25 Gb raw data (353× coverage), which had a GC content of 49.13% and 69 genomic contigs (Table 1). The genome functional prediction and annotation identified 7305 protein-coding genes, with an average gene length of 1693 bp.

To investigate the functions of the coding genes and metabolic pathways, all coding sequences (CDSs) were subjected to COG and KEGG analysis [38]. The COG database (http://www.ncbi.nlm.nih.gov/COG) classifies proteins by comparing all protein sequences in the genome [39]. In total, 4157 CDSs were allocated to COG categories (Table 2), with the maximum proportion of sequences related to “carbohydrate transport and metabolism” (8.52%), followed by “amino acid transport and metabolism” (7.82%), “translation, ribosomal structure and biogenesis” (6.90%), “posttranslational modification, protein turnover, and chaperones” (6.88%), and “energy production and conversion” (5.08%). Proteins that have not been fully identified in the genome of strain CSU-M183 were classified as “general function prediction only” (19.08%) and “function unknown” (4.81%) in COG categories.

thumbnail
Table 2. COG classification of predicted genes encoding proteins with annotated functions of M. purpureus CSU-M183 genome.

https://doi.org/10.1371/journal.pone.0263905.t002

KEGG enrichment analysis is essential for understanding the complex biological functions of genes in microorganisms, including metabolic pathways, genetic information transfer, and cytological processes [40]. Altogether, 3362 CDSs were allocated to five categories in the KEGG database, including “metabolism”, “cellular process” and “environmental information processing”, “genetic information processing”, and “organismal systems” (Fig 1). Annotation results showed that “metabolism” is the main category of KEGG annotations (1375, 40.90%), followed by “genetic information processing” (707, 21.03%) and “organismal systems” (519, 15.44%). Moreover, CDSs were significantly enriched in “translation” (282), “carbohydrate metabolism” (279), “amino acid metabolism” (266) and “transport and catabolism” (259) subcategories, indicating that M. purpureus CSU-M183 had the strong ability of protein translation, carbohydrate utilization and energy conversion.

thumbnail
Fig 1. Enrichment analysis of KEGG pathways for predicted genes of the M. purpureus CSU-M183 genome.

The y-axis represents the KEGG pathway and the x-axis denotes the number of genes.

https://doi.org/10.1371/journal.pone.0263905.g001

Identification of secondary metabolites BGCs

AntiSMASH is a widely used tool that can identify and annotate BGCs in bacterial and fungal genome sequences [41]. To further understand the biosynthesis of the secondary metabolites in strain M. purpureus CSU-M183, BGCs prediction of secondary metabolites were performed using antiSMASH fungi version 5.1.0. A total of 26 BGCs were detected, including terpene, non-ribosomal peptide synthetases (NRPS), type I polyketide synthases (T1PKS), β-lactone, and 18 putative gene clusters.

After the database search with antiSMASH, a BGC (contig 000002F, gene g3398-g3411) for citrinin within the genome sequence of strain CSU-M183 was predicted (Fig 2A), which was identical to the known citrinin BGC (GenBank accession number: AB243687.1, 21917 bp) [42], and the identity of homologous genes was 99%-100%, the predicted functions of the genes in citrinin BGC are listed in Table 3. Additionally, 81% of homologous genes were similar to those in citrinin BGC0001338, and 57% in citrinin BGC000894. A putative BGC responsible for the biosynthesis of MPs was identified in the genome of strain CSU-M183 with 41% of homologous genes showed similar to that in BGC0000027 (Fig 2B), including 16 genes (contig 000001F, gene g1401-g1416) listed in Table 4. As shown in Table 4, the identity of homologous genes was considerably high, such as gene g1409 was 96.51% similar to MpigA (Polyketide synthase), gene g1407 was 95.05% similar to MpigC (Ketoreductase), and gene g1406 was 95.82% similar to MpigD (Acyltransferase). Moreover, by using the known monacolin K BGC (GenBank accession number: DQ176595.1, 45000 bp) of M. pilosus as a reference [13], no complete monacolin K BGC was detected in the genome sequence of M. purpureus CSU-M183 (Fig 2C). All protein-coding genes in the genome sequence were analyzed by BLASTP, where gene g3061, g2167, g4491, g1403, g1402, g4228, g1429, g1395 were homologous to the genes mkA~mkI, respectively. However, these genes do not locat in a gene cluster in genome, and the homologous protein identities were low, especially gene g1403 (with mokD of M. pilosus with 25.00% identity) (Table 5). It has been reported that overexpression of mokD significantly enhanced the production of monacolin K by 200.8%, which illustrated this gene play a vital role in the synthesis of monacolin K [24]. These findings were similar to that of the parent strain M. purpureus LQ-6, which could not produce monacolin K [43].

thumbnail
Fig 2. Schematic representation of predominant secondary metabolites BGCs in the genome sequence of M. purpureus CSU-M183.

(a) Citrinin. (b) MPs. (c) Monacolin K.

https://doi.org/10.1371/journal.pone.0263905.g002

thumbnail
Table 3. Functional prediction of genes detected in the citrinin BGC of M. purpureus CSU-M183.

https://doi.org/10.1371/journal.pone.0263905.t003

thumbnail
Table 4. Functional prediction of genes detected in the MPs BGC of M. purpureus CSU-M183.

https://doi.org/10.1371/journal.pone.0263905.t004

thumbnail
Table 5. Functional prediction of genes detected in the monacolin K BGC of M. purpureus CSU-M183 by NCBI-BLASTP.

https://doi.org/10.1371/journal.pone.0263905.t005

Due to various species of Monascus and large gaps in available biological information, the development of basic theoretical research on Monascus has been relatively slow. With the continuous development of sequencing technology and bioinformatics, breakthrough progress has been made in biosynthetic pathways of the secondary metabolites of Monascus [19, 44, 45], among which MPs, citrinin and monacolin K are the most notable. However, studies on functional and comparative genomics—such as the annotation of unknown sequences, investigation of gene models, comparison of multiple sequence alignment analysis, and metabolic engineering of Monascus morphology, which can elaborate the relationship among secondary metabolite productivity, growth, and morphology in SF, are less.

The key gene PKS with 7838 bp responsible for citrinin biosynthesis was first identified from M. purpureus in 2005 [46] and then five genes encoding Zn(II)2Cys6 transcriptional activator, membrane transporter, dehydrogenase, oxygenase, and oxidoreductase for citrinin biosynthesis were cloned [47]. Microbial PKSs have been mainly classified into three types–type I PKSs (modular type I PKSs and iterative type I PKSs), type II PKSs, and type III PKSs. The PKS responsible for citrinin biosynthesis belongs to the iterative type I PKSs, which contains putative domains for ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), methyltransferase (MT), thioesterase (TE), and acyl carrier protein (ACP) [46]. The KS domain catalyzes the condensation of precursors to extend the polyketone chain, whereas the AT domain selects the precursors, and the ACP domain makes covalent bonds between the precursors and intermediates, which are necessary for the functioning of most PKSs [48]. In 2012, the citrinin BGC with the length of 43 kb from Monascus aurantiacus was first published [49], including 16 open reading frames (ORFs) for ctnD, ctnE, orf6, orf1, ctnA, orf3, orf4, pksCT, orf5, ctnF, orf7, ctnR, orf8, ctnG, ctnH, and ctnI, which are dramatically similar to those of the citrinin BGC of strain M. purpureus CSU-M183. These results revealed high homology of citrinin BGC in Monascus, especially the key gene PKS. In 2012, a putative 53 kb MP BGC of M. ruber was first reported, which consisted of genes encoding PKSs, fatty acid synthases, regulatory factors, and dehydrogenase [3]. Xie et al. reported that gene pigR (gene g1401 in Table 4) is a positive regulatory gene in MPs biosynthesis pathway [50], whereas gene MpigE (gene g1402 in Table 4) may be involved in the conversion of different MPs [51]. The genome size of M. purpureus was found to be smaller than that of related filamentous fungi, indicating a significant loss of genes [19]. A previous study reported that monacolin K cannot be produced due to the lack of monacolin K biosynthesis locus in some M. purpureus genomes [52]. After the prediction of monacolin K BGC in the genome of strain CSU-M183, we found that there was no complete monacolin K BGC in the strain CSU-M183, which was consistent with previous studies [43, 52]. Undoubtedly, the identification of BGCs has greatly facilitated the understanding of the biosynthetic pathways of secondary metabolites in Monascus, which can provide theoretical support for industrial production of Monascus secondary metabolites.

Expression level of Monascus pigments and citrinin clusters located genes

After 7 days of SF, the MPs and citrinin yields of M. purpureus LQ-6 and M. purpureus CSU-M183 were 43.97 U/ml, 1.27 mg/L and 83.77 U/ml, 5.34 mg/L, respectively (Fig 3A). To verify the effect of mutagenesis on the metabolism of MPs and citrinin, the relative expression levels of several key genes, MpigA, MpigR, MpigC, MpigD, MpigE, MpigF, MpigG, MpigH, MpigI, MpigJ, MpigK, MpigL, MpigM, MpigP, MpigQ, cit S, cit A, cit B, cit C, cit D and cit E were vestigated using RT-qPCR. As shown in Fig 3B and 3C, the relative expression levels of MpigM, MpigP, cit B, cit C, cit D and cit E in M. purpureus CSU-M183 were extremely significant (p<0.001) compared to that of M.purpureus LQ-6 at 4th day; while the relative expression levels of MpigA, MpigJ, MpigK, cit S, cit C in M. purpureus CSU-M183 were extremely significant (p<0.001) than that of M. purpureus LQ-6 at 7th day. During the fermentation process, in addition to the relative expression of extremely significant (p<0.001) genes, the expression levels of other genes in the MPs and citrinin biosynthesis gene clusters in CSU-M183 were very significant (p<0.01), such as MpigA, MpigC, MpigD, MpigF, MpigG and cit S at 4th day and MpigD, MpigG, MpigH and cit B at 7th day. The production of MPs and citrinin is directly or indirectly related to the function of genes in their biosynthetic gene clusters, and the relative expression of genes can directly reflect the contribution of genes in the fermentation process. A number of studies have performed functional analysis of MPs and citrinin gene clusters, such as: inactivating MpigA in M. ruber, Monascus lost its pigment production ability, which proved that PKS was involved in pigment synthesis [53]. MrpigJ(encoded by MrpigJ, a homolog of MpigJ) and MrpigK(encoded by MrpigK, a homolog of MpigK) form two subunits of the specialized fungal FAS, which produce the fatty acyl portion of the side chain of MPs [54]. Moreover, MrpigM, as an o-acetyltransferase, synthesized an O-11 acetyl intermediate in Chen et al’s Monascus model, and knocking out MrpigM(the homolog of MpigM) blocked the pathway of pigment synthesis intermediate [54]. The inactivation of genes in citrinin biosynthesis gene cluster led to a significant decline on citrinin production, even lower than the detection level, such as knocking out cit A, pksCT and cit B [42, 55]. It indicates that the increase of MPs and citrinin production may be caused by the increase of gene expression level in gene cluster caused by ARTP mutation, and these genes are very important for the synthesis of MPs and citrinin.

thumbnail
Fig 3.

(a) Production of MPs and citrinin in SF of M. purpureus LQ-6 and M. purpureus CSU-M183 for 7 days. (b) Expression levels of genes related to MPs and citrinin biosynthesis of M. purpureus LQ-6 (control) and M. purpureus CSU-M183 at 4th day. (c) Expression levels of genes related to MPs and citrinin biosynthesis of M. purpureus LQ-6 (control) and M. purpureus CSU-M183 at 7th day. * p<0.05, ** p<0.01, *** p<0.001.

https://doi.org/10.1371/journal.pone.0263905.g003

Analysis of the chitin biosynthesis pathway

As the main component of the fungal cell wall, chitin is important for the morphology of fungi. Based on the homology of amino acid sequence, chitin synthetases can be divided into three categories (class I-III) in Saccharomyces cerevisiae, four (class I-IV) in Candida albicans, and seven (class I-VII) in filamentous fungi. The numbers of gene encoding chitin synthetase in various filamentous fungi are different, generally containing 6–10 genes encoding chitin synthetase [56].

To date, information about chitin biosynthesis in M. purpureus has not been reported. To lay a foundation for the further study of morphological metabolism of M. purpureus, we analyzed the chitin biosynthesis of strain CSU-M183 and annotated the function of the relevant genes in the pathway. By matching the predicted chitin biosynthesis-related enzymes in CSU-M183 strain genome with the KEGG database, the biosynthetic pathway of chitin in M. purpureus was identified. As shown in Fig 4, phosphoacetylglucosamine mutase (PGM3) [EC:5.4.2.3] (encoded by gene g4907) converts N-acetyl-D-glucosamine 6-phosphate (GlcNAc-6P) to N-acetyl-alpha-D-glucosamine 1-phosphate (GlcNAc-1P), which is then dephosphorylated by UDP-N-acetylglucosamine diphosphorylase (UAP1) [EC:2.7.7.23] (encoded by gene g6630) to yield UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). Moreover, chitin synthase (chs1) [EC:2.4.1.16] (encoded by genes: g872, g920, g3078, and g5640) converts UDP-GlcNAc to chitin. Additionally, the other genes encoding the important enzymes in the biosynthetic pathway of chitin were annotated, such as N-acetylmuramic acid 6-phosphate etherase (murQ) [EC:4.2.1.126] encoded by gene g1905, chitinase [EC:3.2.1.14] encoded by genes g3222, g6372 and g1142, and glucosamine-phosphate N-acetyltransferase (GNPNAT1, GNA1) [EC:2.3.1.4] encoded by gene g2832.

thumbnail
Fig 4. Prediction of chitin biosynthetic pathway in M. purpureus CSU-M183 genome.

The corresponding enzymes involved in each bioconversion step are shown in green.

https://doi.org/10.1371/journal.pone.0263905.g004

Class III chitin synthases only exist in the cell wall of fungi with high chitin content and are essential for regulating the mycelial aggregation morphology of fungi. Gene chsB in Aspergillus fumigatus plays an important role in cell wall biosynthesis, hyphal growth, and asexual reproduction [57]. To provide genetic resources for further studies, we mainly identified the gene chsB (gene g4739, 3243 bp) encoding class III chitin synthase in M. purpureus CSU-M183.

Generally, the specific characteristic of their SF is the aggregation of mycelia that are affected by environmental conditions, leading to different rheological properties of the fermentation broth. Such changes affect the transfer of mass, heat, and momentum, as well as the biosynthesis and production efficiency of target products. Moreover, the morphology of hyphae is closely related to the biosynthesis of secondary metabolites, and changes in the mycelium morphology of Monascus can regulate the level of secondary metabolites [17, 24]. As the main component of the fungal cell wall, chitin affects the mycelial morphological changes such as apical extension, branch growth, and differentiation. Blocking the biosynthetic pathway of chitin inevitably changes the mycelial aggregation and regulates metabolic pathways of target products. With the rise of the SF technology of Monascus, the effects of mycelial morphology on the biosynthesis of secondary metabolites in the fermentation process have attracted much attention. However, research on the metabolic engineering of Monascus morphology is still in the blank stage. In this article, we commented the strategies for morphological regulation of filamentous fungi, and discussed the impact of calcium signal transduction and chitin biosynthesis on apical hyphal growth and mycelial branching. Furthermore, based on the WGS analysis of strain M. purpureus CSU-M183, we will use genetic engineering technology to disturb the chitin biosynthesis of M. purpureus CSU-M183, change the mycelial aggregation morphology in the process of SF, regulate the biosynthesis of secondary metabolites, and clarify the molecular mechanism of the regulation of morphological on secondary metabolism using genetic engineering technology and histochemical correlation analysis.

Conclusion

Genomic information of M. purpureus CSU-M183 reported here can serve as a reference genome for Monascus genomics research. It’s predicted that the secondary metabolites BGCs and the chitin biosynthetic pathway in the genome of M. purpureus CSU-M183. We verified that ARTP induced significantly the upregulated expression of most Monascus pigment and citrinin clusters located genes by RT-qPCR. In addition, we annotated and classified the chitin biosynthesis genes of M. purpureus CSU-M183, which offer a strategy of morphological metabolic engineering. In conclusion, we provided genomic resources for further biological studies on the metabolic engineering of the morphology of Monascus.

Supporting information

Acknowledgments

We would like to thank Editage for English language editing.

References

  1. 1. Patakova P. Monascus secondary metabolites: production and biological activity. Journal of Industrial Microbiology & Biotechnology. 2013;40(2):169–81. pmid:23179468
  2. 2. Kim D, S K. Beneficial effects of Monascus sp. KCCM 10093 pigments and derivatives: A mini review. Molecules. 2018;23(1):98. pmid:29301350
  3. 3. Feng YL, Shao YC, Cheng FS. Monascus pigments. Applied Microbiology & Biotechnology. 2012;96:1421–1440. pmid:23104643
  4. 4. Chen G, Yang S, Wang C, Shi K, Wu Z. Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation. Applied Microbiology and Biotechnology. 2020;104(6):2469–79. pmid:31993704
  5. 5. Downham A, Collins P. Colouring our foods in the last and next millennium. International Journal of Food ence & Technology. 2010;35(1):5–22.
  6. 6. Zhao L, Lu F, Zhang X, Wang Z. Isolation of ionizable red Monascus pigments after extractive fermentation in nonionic surfactant micelle aqueous solution. Process Biochemistry. 2017;61:156–62.
  7. 7. Liu J, Luo Y, Guo T, Tang C, Lin Q. Cost-effective pigment production by Monascus purpureus using rice straw hydrolysate as substrate in submerged fermentation. Journal of Bioscience and Bioengineering. 2019;129(2):229–236. pmid:31500988
  8. 8. Wang Y, Zhang B, Lu L, Huang Y, Xu G. Enhanced production of pigments by addition of surfactants in submerged fermentation of Monascus purpureus H1102. Journal of the Science of Food & Agriculture. 2013;93(13):3339–44. pmid:23595359
  9. 9. Lin L, Wang C, Li Z, Wu H, Chen M. Effect of Temperature-Shift and Temperature-Constant Cultivation on the Monacolin K Biosynthetic Gene Cluster Expression in Monascus sp. Food Technology and Biotechnology. 2017;55(1):40. pmid:28559732
  10. 10. Lin L, Wu S, Li Z, Ren Z, Wang C. High Expression Level of mok E Enhances the Production of Monacolin K in Monascus. Food Biotechnology. 2018;32(1):35–46.
  11. 11. Huang J, Liao NQ, Li HM. Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by activating mokH and mokA, and by accelerating cAMP-PkA pathway. International Journal of Biological Macromolecules: Structure, Function and Interactions. 2018;109:950–4.
  12. 12. Zhang , BB, Xing , Hong-Bo , Jiang , Bing-Jie , et al. Using millet as substrate for efficient production of monacolin K by solid-state fermentation of Monascus ruber. Journal of Bioscience and Bioengineering. 2017;125(3):333–338. pmid:29157871
  13. 13. Chen YP, Yuan GF, Hsieh SY, Lin YS, Wang WY, Liaw LL, et al. Identification of the mokH gene encoding transcription factor for the upregulation of Monacolin K biosynthesis in Monascus pilosus. Journal of Agricultural and Food Chemistry. 2010;58(1):287–93. pmid:19968298
  14. 14. Feng Y, Chen W, Chen F. A Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin. Food Science & Biotechnology. 2016;25(4):1115–22. pmid:30263383
  15. 15. Simu SY, Castro‐Aceituno V, Lee S, Ahn S, Lee HK, Hoang VA, et al. Fermentation of soybean hull by Monascus pilosus and elucidation of its related molecular mechanism involved in the inhibition of lipid accumulation. An in sílico and in vitro approach. Journal of Food Biochemistry. 2018;42(1): e12442.
  16. 16. Embaby AM, Hussein MN, Hussein A, Papp T. Monascus orange and red pigments production by Monascus purpureus ATCC16436 through co-solid state fermentation of corn cob and glycerol: An eco-friendly environmental low cost approach. Plos One. 2018;13(12):e0207755. pmid:30532218
  17. 17. Lv J, Qian GF, Chen L, Liu H, Xu HX, Xu GR, et al. Efficient Biosynthesis of Natural Yellow Pigments by Monascus purpureus in a Novel Integrated Fermentation System. Journal of Agricultural and Food Chemistry. 2018;66(4):918–925. pmid:29313328
  18. 18. Suraiya S, Siddique MP, Lee JM, Kim EY, Kim JM, Kong IS. Enhancement and characterization of natural pigments produced by Monascus spp. using Saccharina japonica as fermentation substrate. Journal of Applied Phycology. 2017; 30:729–742.
  19. 19. Yang Y, Liu B, Du XJ, Li P, Liang B, Cheng XZ, et al. Complete genome sequence and transcriptomics analyses reveal pigment biosynthesis and regulatory mechanisms in an industrial strain, Monascus purpureus YY-1. Sci Rep. 2015;5:8331. pmid:25660389
  20. 20. Kumagai T, Tsukahara M, Katayama N, Yaoi K, Fujimori KE. Whole-Genome Sequence of Monascus purpureus GB-01, an Industrial Strain for Food Colorant Production. Microbiology Resource Announcements. 2019;8(24): e00196–19. pmid:31196916
  21. 21. Liu J, Chai XY,Guo T, Wu JY, Yang PP, Luo YC, et al. Disruption of the Ergosterol Biosynthetic Pathway Results in Increased Membrane Permeability, Causing Overproduction and Secretion of Extracellular Monascus Pigments in Submerged Fermentation. Journal of Agricultural and Food Chemistry. 2019;67(49):13673–83. pmid:31617717
  22. 22. Chen G, Huang T, Bei Q, Tian XF, Wu ZQ. Correlation of pigment production with mycelium morphology in extractive fermentation of Monascus anka GIM 3.592. Process Biochemistry. 2017;58:42–50.
  23. 23. Lv J, Zhang BB, Liu XD, Zhang C, Chen L, Xu GR, et al. Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology. Journal of Bioscience and Bioengineering. 2017:452–8. pmid:28625612
  24. 24. Zhang C, Liang J, Zhang A, Hao S, Zhang H, Zhu Q, et al. Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus Azaphilone Polyketide Pathway. Journal of Agricultural and Food Chemistry. 2019;67(9):2563–69. pmid:30734557
  25. 25. Ibrahim D, Weloosamy H, Lim SH. Effect of agitation speed on the morphology of Aspergillus niger HFD5A-1 hyphae and its pectinase production in submerged fermentation. World Journal of Biological Chemistry. 2015; 6(3): 265–71. pmid:26322181
  26. 26. Sun XW, Wu HF, Zhao GH, Li ZM, Wu XH, Liu H, et al. Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing. Bioprocess and Biosystems Engineering. 2018;41(7): 1029–1038. pmid:29610994
  27. 27. Calvo AM. The VeA regulatory system and its role in morphological and chemical development in fungi. Fungal Genetics and Biology. 2008;45(7):1053–61. pmid:18457967
  28. 28. Tan YM, Wang H, Wang YP, Ge YY, Ren XX, Ren CG, et al. The role of the veA gene in adjusting developmental balance and environmental stress response in Aspergillus cristatus. Fungal Biology. 2018;122(10):952–64. pmid:30227931
  29. 29. Müller C, Mcintyre M, Hansen K, Nielsen J. Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis. Applied & Environmental Microbiology. 2002;68(4):1827–36. pmid:11916702
  30. 30. Liu H, Zheng Z, Wang P, Gong G, Wang L, Zhao G. Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum. Applied Microbiology and Biotechnology. 2013; 97(8):3363–72. pmid:23179625
  31. 31. Lenardon M, Munro CA, Gow N. Chitin synthesis and fungal pathogenesis. Current Opinion in Microbiology. 2010;13(4):416–23. pmid:20561815
  32. 32. Liu J, Guo T, Luo YC, Chai XY, Wu JY, Zhao W, et al. Enhancement of Monascus pigment productivity via a simultaneous fermentation process and separation system using immobilized-cell fermentation. Bioresource Technology. 2019;272:552–560. pmid:30396112
  33. 33. Simão SF, Waterhouse RM, Panagiotis I, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31(19):3210–2. pmid:26059717
  34. 34. Vasanthan J, Yasubumi S. Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data. Briefings in Bioinformatics. 2019; 20(3):866–876. pmid:29112696
  35. 35. Stanke M, Schffmann O, Morgenstern B, Waack S. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics. 2006;7:62. pmid:16469098
  36. 36. Ian K. Gene finding in novel genomes. BMC Bioinformatics. 2004;5(1):59.
  37. 37. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, et al. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Research. 2015;43:W237–W243. pmid:25948579
  38. 38. Minoru K, Susumu G, Shuichi K, Yasushi O, Masahiro H. The KEGG resource for deciphering the genome. Nucleic Acids Research. 2004;32:D277–D280. pmid:14681412
  39. 39. Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Research. 2000;(1):33–6. pmid:10592175
  40. 40. Xiang YP, Wang YM, Shen H, Wang DY. The Draft Genome Sequence of Pseudomonas putida Strain TGRB4, an Aerobic Bacterium Capable of Producing Methylmercury. Current Microbiology. 2020; 77(4): 522–527. pmid:31004191
  41. 41. Kai B, Simon S, Katharina S, Rasmus V, Nadine Z, Yup LS, et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nuclc Acids Research. 2019;(W1):W81–W87.
  42. 42. Xu MJ, Yang ZL, Liang ZZ, SN Z. Construction of a Monascus purpureus mutant showing lower citrinin and higher pigment production by replacement of ctn A with pks 1 without using vector and resistance gene. Journal of Agricultural and Food Chemistry. 2009;57(20):9764–8. pmid:20560630
  43. 43. Chai X, Ai Z, Liu J, Guo T, Bai J, Lin QL. Effects of pigment and citrinin biosynthesis on the metabolism and morphology of Monascus purpureus in submerged fermentation. Food Science and Biotechnology. 2020;29(7):927–937. pmid:32582455
  44. 44. Chen WP, He Y, Zhou Y, Shao YC, Chen FS. Edible filamentous fungi from the species Monascus: early traditional fermentations, modern molecular biology, and future genomics. Comprehensive Reviews in Food Science & Food Safety. 2015;14(5):555–67.
  45. 45. Li L, Shao YC, Li Q, Yang S, Chen FS. Identification of Mga1, a G-protein α-subunit gene involved in regulating citrinin and pigment production in Monascus ruber M7. Fems Microbiology Letters. 2010; 308(2):108–14. pmid:20500530
  46. 46. Shimizu T, Kinoshita H, Ishihara S, Sakai K, Nagai S, T N. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus. Applied and Environmental Microbiology. 2005. pmid:16000748
  47. 47. Shimizu T, Kinoshita H, Nihira T. Identification and in vivo functional analysis by gene disruption of ctnA, an activator gene Involved in citrinin biosynthesis in Monascus purpureus. Applied and Environmental Microbiology. 2007;73(16):5097. pmid:17586673
  48. 48. Chen A, Schnarr N, Kim C, Cane D, Khosla C. Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. Journal of the American Chemical Society. 2006;128(9):3067–74. pmid:16506788
  49. 49. Li YP, Yang X, Huang ZB. Isolation and characterization of the citrinin biosynthetic gene cluster from Monascus aurantiacus. Biotechnology Letters. 2012;34(1):131–6. pmid:21956130
  50. 50. Xie NN, Liu QP, Chen FS. Deletion of pigR gene in Monascus ruber leads to loss of pigment production. Biotechnology Letters. 2013; 35(9): 1425–32. pmid:23690031
  51. 51. Liu QP, Xie NN, He Y, Wang L, Shao YC, Zhao HZ, et al. MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7. Applied Microbiology and Biotechnology. 2014;98(1):285–96. pmid:24162083
  52. 52. Kwon HJ, Balakrishnan B, Kim YK. Some Monascus purpureus genomes lack the Monacolin K biosynthesis locus. J. Appl. Biol. Chem. 2016;59(1):45–47.
  53. 53. Shao YC, Lei M, Mao ZJ, Zhou YX, Chen FS. Insights into Monascus biology at the genetic level. Applied Microbiology and Biotechnology. 2014;98(9):3911–22. pmid:24633442
  54. 54. Chen WP, Chen RF, Liu QP, He Y, He K, Ding XL, et al. Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi. Chemical Science. 2017;8(7):4917–25. pmid:28959415
  55. 55. Li YP, Pan YF, Zou LH, Yang X, Huang ZB, He QH. Lower citrinin production by gene disruption of ctnB Involved in citrinin ciosynthesis in Monascus aurantiacus Li AS3.4384. Journal of Agricultural and Food Chemistry. 2013;61(30):7397–402. pmid:23841779
  56. 56. Zhang JJ, Jiang H, Du YR, Keyhani NO, Xia YX, K J. Members of chitin synthase family in Metarhizium acridum differentially affect fungal growth, stress tolerances, cell wall integrity and virulence. PLOS Pathog. 2019; 15(8): e1007964. pmid:31461507
  57. 57. Fukuda K, Yamada K, De Oka K, Yamashita S, Horiuchi H. Class III chitin synthase ChsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development. Eukaryotic Cell. 2009;8(7):945–56. pmid:19411617