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.

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 [13-15] 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 [16-18]. 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 [22-24].

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.

Table 1

M. purpureus CSU-M183 genome general features.

Job Metric	Value
Length of genome assembly (Mb)	23.75 (23,752,195 bp)
Contig number	69
G + C content (%)	49.4
Coverage	353X
Q30 (%)	87.1
SNP number	3
Number of protein-coding genes	7305
Average gene length (bp)	1693
Genes with function prediction	5182
Number of proteins with KEGG ortholog	3362
Number of proteins with COG	4157

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.

Table 2

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

COG classification	Percentage(%)
RNA processing and modification	0.77
Chromatin structure and dynamics	0.77
Energy production and conversion	5.08
Cell cycle control, cell division, chromosome partitior	2.98
Amino acid transport and metabolism	7.82
Nucleotide transport and metabolism	2.38
Carbohydrate transport and metabolism	8.52
Coenzyme transport and metabolism	4.04
Lipid transport and metabolism	4.38
Translation, ribosomal structure and biogenesis	6.90
Transcription	3.66
Replication, recombination and repair	4.40
Cell wall/membrane/envelope biogenesis	3.03
Cell motility	0.24
Posttranslational modification, protein turnover, chaperones	6.88
Inorganic ion transport and metabolism	3.80
Secondary metabolites biosynthesis, transport and catabolism	4.23
General function prediction only	19.08
Function unknown	4.81
Signal transduction mechanisms	2.36
Intracellular trafficking, secretion, and vesicular transport	2.02
Defense mechanisms	0.99
Nuclear structure	0.10
Cytoskeleton	0.77

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.

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.

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].

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.

Table 3

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

CDS	Length (bp)	Product	KO
gene = g3398	1762	Unnamed protein product
gene = g3399	1389	Hypothetical protein, orf7
gene = g3400	2796	ctnD	K00108
gene = g3401	403	Predicted protein	K00108
gene = g3402	3076	Dehydrogenase, orf1
gene = g3403	987	citrinin biosynthesis oxygenase, ctnA	K13821
gene = g3404	940	citrinin biosynthesis oxydoreductase, ctnB
gene = g3405	7780	citrinin polyketide synthase, pksCT
gene = g3406	1497	citrinin biosynthesis transporter, ctnC
gene = g3407	829	ctnF
gene = g3408	1498	Hypothetical protein, orf8
gene = g3409	2116	ctnR
gene = g3410	663	ctnG
gene = g3411	567	ctnG	K01673
gene = g3412	3163	ctnI

Table 4

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

CDS	Length (bp)	Product	Identity (%)	KO
gene = g1401	2388	Fungal specific transcription factor, MpigR	91.41
gene = g1402	1108	Alcohol dehydrogenase, MpigE	95.39
gene = g1403	819	Amino oxidase/esterase, MpigF	93.01
gene = g1404	1395	Amino oxidase/esterase, MpigF	94.03
gene = g1405	1027	Dehydrogenase, MpigH	97.66
gene = g1406	1449	Acyltransferase, MpigD	95.82	K22889
gene = g1407	892	Ketoreductase, MpigC	95.05	K11165
gene = g1408	1719	Citrinin biosynthesis transcriptional activator CtnR	95.48
gene = g1409	8071	Polyketide synthase, MpigA	96.51
gene = g1410	1032	Peptidyl-prolyl cis-trans isomerase Cpr7	98.68	K05864
gene = g1411	573	D-tyrosyl-tRNA(Tyr) deacylase, MpigO	95.58	K07560
gene = g1412	1849	TPA: CCR4-NOT transcription complex, subunit 3	84.23	K12580
gene = g1413	634	C2H2 finger domain protein	76.40
gene = g1414	1531	Dihydrolipoamide dehydrogenase	84.17	K00382
gene = g1415	1665	Fatty acid desaturase	73.58	K13076
gene = g1416	1792	Phosphoglucomutase	68.45	K01835

Table 5

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

CDS	Length (aa)	Homologue	Homologue Length (aa)	Identity
gene = g3061	3945	ABA02239.1_8 [gene = mkA]	3075	37.19%
gene = g2167	2593	ABA02240.1_1 [gene = mkB]	2547	38.62%
gene = g4491	499	ABA02241.1_9 [gene = mkC]	524	80.53%
gene = g1403	273	ABA02242.1_7 [gene = mkD]	263	25.00%
gene = g1402	369	ABA02243.1_6 [gene = mkE]	360	39.17%
gene = g4228	1133	ABA02245.1_4 [gene = mkG]	1052	52.61%
gene = g1429	426	ABA02246.1_3 [gene = mkH]	455	78.10%
gene = g1395	570	ABA02247.1_2 [gene = mkI]	543	54.21%

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.

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.

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.

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.

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.

The primers used in this study.

(DOCX)

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22 Feb 2022

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31 Mar 2022

ID: PONE-D-22-01622

Title: 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

Journal: Plos One

Dear Editor,

Thank you very much for your and reviewers' comments concerning our manuscript entitled “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（PONE-D-22-01622）”. We have studied the editor/reviewer comments and made corrections that we hope meet with your approval. The main corrections have been marked in red in the revised manuscript (PONE-D-22-01622) and our details responses to the comments are given below in bold type.

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Response：Thank you very much for your comment, we have changed the manuscript to comply with PLOS ONE's style requirements, including file naming requirements

2. Thank you for stating the following financial disclosure:

we would also like to thank Editage for English language editing. This work was supported by the National Natural Science Foundation of China (No. 32101906), Natural Science Foundation of Hunan Province (No. 2021JJ31146); Open Project Program of the Hunan Provincial Key Laboratory of Food Safety Monitoring and Early Waring (No. 2021KFJJ02), Education Department of Scientific Research Project of Hunan Province (No. 20B619) and Education Department of Postgraduate Research and Innovation Project of Hunan Province（No. CX20210863, CX20210898）

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Response：Thank you for your comment. Funders JL and SZ designed the study and annotated the genome, respectively. SZ and JL co-write the manuscript. All authors read and approved the final version of the manuscript.

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we would also like to thank Editage for English language editing. This work was supported by the National Natural Science Foundation of China (No. 32101906), Natural Science Foundation of Hunan Province (No. 2021JJ31146); Open Project Program of the Hunan Provincial Key Laboratory of Food Safety Monitoring and Early Waring (No. 2021KFJJ02), Education Department of Scientific Research Project of Hunan Province (No. 20B619) and Education Department of Postgraduate Research and Innovation Project of Hunan Province（No. CX20210863, CX20210898）

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Response：Thank you for your comment, we have deleted the funding information in the manuscript and submitted them in the Funding Statement.

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Response：Thank you for your comment, we have provided accession numbers for the relevant data in the Materials and Methods of the manuscript (page 9, line 9-13).

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Response：Thank you for your comment, we have moved the ethics statement to the Materials and Methods section of the manuscript (page 9, line 14-16).

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Response：Thank you for your comment, we have changed the supplemental information as requested (page 31, line 20-21).

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Response：Thank you for your comment, we have checked the reference list to make sure it is complete and correct.

Reviewers' comments:

Reviewer #1:

The Monascus purpureus CSU-M183 strain is of industrial importance and warrants a detailed genomic analysis. The authors here have done a good job describing its genomic contents, particularly in relation to pigment and citrinin metabolism. I have a couple of comments.

1. Annotation pipelines such as MAKER pipeline take into account various evidences to predict the genes in a genome. It might perform better than solely using augustus and SNAP.

Response：Thank you very much for your comment.

2. In methods section, the genome assembly process is not described in enough details that it can be reproduced. Parameters used in HGAP program are missing.

Response：Thank you very much for your comment, we have re-described the genome assembly process in detail in the Materials and methods section of WGS and assembling (page 7, line 4-7).

3. In introduction, it is not clearly rationalized why the genome sequencing of this particular strain is necessary while several other high pigment producing strains are already sequenced.

Response：Thank you very much for your comment. There are not many published whole genome sequences of Monascus so far, and there are certain differences between the Monascus species, such as some do not contain a complete monacolin K biosynthetic gene cluster, and some do not produce citrinin, etc. Besides, we illustrate that the cell wall is an ideal target for the regulation of fungal morphology, chitin is the main component of cell wall, but the number of genes encoding chitin synthase and chitin biosynthesis pathway in M. purpureus is still unclear (page 5, line 3-11). Therefore, we comprehensively predicted the gene encoding chitin synthase and the biosynthetic pathway of chitin in M. purpureus CSU-M183 through whole genome sequencing for future morphological studies.

4.With several M. purpureus genomes available, it might be helpful to conduct a comparative genomic analysis among them to elucidate likely mechanism that CSU-M183 strain is special in its metabolic capacity.

Response：Thank you very much for your suggestion. Based on the whole genome sequence of strain CSU-M183, we have performed genome resequencing of the mutant strains (morphologically different strain), focusing on the genes related to morphology, metabolism and growth, and to explore the relationship between the morphology, growth and metabolism of Monascus, for our future study.

Reviewer #2:

In this study, the authors employed a hybrid sequencing strategy (PacBio and Illumina) to get the whole-genome assembly of a Monascus purpureus mutant. The authors comprehensively addressed the predicted secondary metabolite biosynthetic clusters as well as the expression of chitin and Monascus pigments genes in mutant strain, Monascus purpureus CSU-M183. Although there are some concerns (given below), I find this study relevant for the research community.

Comments:

1. In the Materials and methods section of WGS and assembling, the information about the method of PaBioRS II library preparation is not described. The authors should add information about the PacBio sequencing library construction and parameters used for filtering the reads. The authors should also provide information on the Illumina NGS sequencing platform used, and the processing of Illumina reads.

Response：Thank you very much for you comment, we have added relavant information in the Materials and methods section of WGS and assembling (page 7 line 8 to page 8 line 6).

2. They used two sequencing platforms to generate the assembly of Monascus purpureus mutant. However, they did not describe how they validated the completeness of the genome assembly.

Response：Thank you very much for you comment. Indeed the genome assembly was performed using HGAP2 for PacBio RSII reads, and Illumina reads were mapped on assembled genome to correct possible SNPs and Indels. Though we finally obtained a high-quality genome with 69 contigs, it’s not assembled into chromosome level. So we did not estimate the completeness of the genome (page 7 line 8 to page 8 line 6).

3. I am confused about the description of how they obtained the M. purpureus mutant strain. If they created the mutant strain from parent M. purpureus LQ-6, the authors have to describe the mutagenesis and screening. Otherwise, they have to cite the previous study that reported the M. purpureus CSU-M183 mutant.

Response：Thank you very much for you comment, we have cited our previous research of the M. purpureus CSU-M183 mutant（page 6, line 2）.

4. Fig. 1 – I recommend giving the alphabets corresponding to each annotated function inside the pie chart. I find the pie chart shown in the manuscript not helpful because some colors are same for annotated functions. For example, the color for E, O, and Z are the same. The other option is providing the percentage of predicted genes with annotated function as a table instead of a pie chart.

Response：Thank you very much for you comment, the percentage of predicted genes with annotation functions have been presented in a table (Table 2).

5. In the methods section of RT-qPCR, the authors should add the statistical methods used for qPCR analysis. They did not mention how many replicates they used for qPCR experiments. Response：Thank you very much for you comment. We have supplemented Statistical analysis in the Materials and Methods to illustrate the statistical methods used in this study and the number of experimental replicates (page 9, line 17-22).

6. In the RT-qPCR results, the authors should give the p-values of significantly higher qPCR expression results instead of using terminologies such as “obviously higher”, “relatively significant”, “very significant,” or “extremely significant”. I find it hard to follow what the authors meant by “extremely significant” and “very significant”. They should also describe in the legend of Figure 4 on what each type of asterisks (**, ***) meant.

Response：Thank you very much for your comment, we gave the p-values of significantly higher qPCR expression results and supplemented in the legend of Fig 3 on what each type of asterisks meant (page 22, line 12-13).

7. Page #14 (Line #18) - Page # 15 (Line #3) – “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-N22 acetylglucosamine diphosphorylase (UAP1) [EC:2.7.7.23] (encoded by gene g6630) to yield UDP-N-acetyl-D-glucosamine (UDP-GlcNAc)”. The above description matches Fig 5, not Fig 4.

Response：Thank you very much for your comment，we have corrected it.

8. This manuscript requires language editing before accepting for publication. I have noticed the repetition of words, spelling, and grammatical errors. Although some paragraphs are fairly well written, I recommend revising the manuscript with the assistance of a native English speaker. Response：Thank you for your comment, the manuscript has been revised with the help of native English speakers.

9. Authors should correct the format of in-text citation throughout the manuscript. Also, keep consistency in the units' format (Ex: ml or mL)

Response：Thank you very much for your comment, we have changed the in-text citation format as requested and unified the unit of volume to ml.

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8 Apr 2022

22 Apr 2022

Editor' comments:

1. Please submit the PacBio sequencing reads (raw data) and Illumina data you generated to an appropriate database (e.g. NCBI SRA) and give the SRA accession number. Currently, it appears as if you only submitted the assembled genome, but not the raw sequence data, which are also important.

Response：Thank you very much for your comment. Firstly, due to the COVID-19 in Shanghai, the people were strongly asked to work at home, and the raw date is being stored on the server of sequencing company (Chinese National Human Genome Center at Shanghai). Secondly, we would submit the raw date to NCBI-SRA as soon as possible when the staff return to work. Thus, we just provided the raw date of SRA acceptance number with BioProject of PRJNA824977 in this revised manuscript (PONE-D-22-01622R2；Page 9，Line 14-15).

2. Some sections of the manuscript were apparently changed in an attempt to improve the English, but this is not actually better in many cases. Especially the abstract now contains a number of language problems in the changes sentences that were not present before. Please carefully check the manuscript and correct language problems.

Response：Thank you for your comment, the manuscript has been revised and marked in blue.

Reviewers' comments:

Reviewer #1:

1. Reviewer 1 asked about the reasons why this particular Monascus purpureus strain was sequenced. This is still not sufficiently explained in the introduction. Please explain what makes this strain interesting (not the species as such, but this particular strain).

Response：Thank you very much for your comment. We have explained what makes this strain interesting in the introduction（Page 5, Line 6-16）.

Reviewer #2:

Reviewer 2 suggested checking for genome completeness, which is something that can and should be done especially with genomes that are not assembled fully at chromosome level. You can use the presence of conserved genes to check for completeness (of the gene space at least), e.g. by using the BUSCO tool or other methods to check for the presence of expected conserved eukaryotic genes. Please do this and document the method used and the results in your manuscript.

Comments:

Response：Thank you very much for you comment. We have checked the completeness of the genome by using the BUSCO tool. The corresponding methods（Page 6, Line 20-22） and results（Page 10, Line 9-13）have been supplemented in the Manuscript.

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25 Apr 2022

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

PONE-D-22-01622R2

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Reviewers' comments:

19 May 2022

PONE-D-22-01622R2

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

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