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Article

Genome Characteristics of the Endophytic Fungus Talaromyces sp. DC2 Isolated from Catharanthus roseus (L.) G. Don

by
Nguyen Duc Quan
1,†,
Ngoc-Lan Nguyen
1,2,†,
Tran Thi Huong Giang
1,
Nguyen Thi Thanh Ngan
1,2,
Nguyen Thanh Hien
1,
Nguyen Van Tung
1,2,
Nguyen Hoang Thanh Trang
1,
Nguyen Thi Kim Lien
1,2 and
Huy Hoang Nguyen
1,2,*
1
Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Str., Cau Giay, Hanoi 100000, Vietnam
2
Department of Biotechnology, Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Str., Cau Giay, Hanoi 100000, Vietnam
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2024, 10(5), 352; https://doi.org/10.3390/jof10050352
Submission received: 7 March 2024 / Revised: 6 May 2024 / Accepted: 8 May 2024 / Published: 15 May 2024
(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)
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Abstract

:
Talaromyces sp. DC2 is an endophytic fungus that was isolated from the stem of Catharanthus roseus (L.) G. Don in Hanoi, Vietnam and is capable of producing vinca alkaloids. This study utilizes the PacBio Sequel technology to completely sequence the whole genome of Talaromyces sp. DC2The genome study revealed that DC2 contains a total of 34.58 Mb spanned by 156 contigs, with a GC content of 46.5%. The identification and prediction of functional protein-coding genes, tRNA, and rRNA were comprehensively predicted and highly annotated using various BLAST databases, including non-redundant (Nr) protein sequence, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Carbohydrate-Active Enzymes (CAZy) databases. The genome of DC2 has a total of 149, 227, 65, 153, 53, and 6 genes responsible for cellulose, hemicellulose, lignin, pectin, chitin, starch, and inulin degradation, respectively. The Antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH) analyses revealed that strain DC2 possesses 20 biosynthetic gene clusters responsible for producing secondary metabolites. The strain DC2 has also been found to harbor the DDC gene encoding aromatic L-amino acid decarboxylase enzyme. Conclusively, this study has provided a comprehensive understanding of the processes involved in secondary metabolites and the ability of the Talaromyces sp. DC2 strain to degrade plant cell walls.

1. Introduction

Catharanthus roseus (L.) G. Don is a flowering plant species in the family Apocynaceae [1]. C. roseus is widely distributed in the regions of America, Africa, Asia, southern Europe, Australia, and Vietnam [2]. The plant’s secondary metabolites exhibit a diverse range of beneficial effects in combating various diseases (leukemia, various types of cancer) and illnesses (sore throat, fever, indigestion, septic wounds, diabetes) [1]. Moreover, the plant is highly valued in the field of medicine due to the existence of numerous alkaloids with pharmaceutical properties, such as vindoline, vinblastine, catharanthine, vincristine, ajmalicine, reserpine, serpentine, horhammericine, tabersonine, leurosine, and lochnerine [1]. Among these alkaloids, vincristine, vindesine, and vinblastine have been recognized for their anticancer properties [3]. However, the plant only produces a limited quantity of these beneficial alkaloids. Many research efforts have been undertaken to enhance the production of vinca alkaloids.
Endophytic fungi have gained increased attention for their capacity to produce vinca alkaloids, such as Fusarium oxysporum [4], Talaromyces radius CrP20 [5], Curvularia verruculosa [6], Botryosphaeria laricina strain CRS1 [7], and Alternaria alternata AUMC14391 [8]. Several studies on C. roseus have discovered that the utilization of biotic elicitors, such as fungal concentrate, can effectively increase the synthesis of secondary metabolites under in vitro conditions. Several endophytic fungal strains elicit the accumulation of vinca alkaloids in the leaves of C. roseus. Inoculation of C. roseus with endophytes (Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6) was found to enhance vindoline content. This was achieved by upregulating genes associated with the terpenoid indole alkaloid biosynthesis in C. roseus [9]. Previous research also found that cell extracts of endophytic fungi, such as Fusarium solani RN1 and Chaetomium funicola RN3, greatly increased the accumulation of alkaloids in the cell suspension culture system [10].
The Talaromyces is a genus of approximately 80 fungal species within the Trichocomaceae family; each of them has unique functions. Many of them can cause fungal pathogens like Talaromyces marneffei, T. indigoticus, T. piceus, T. radicus, T. helicus, T. amestolkiae, and T. stollii [11]. Several species can produce bioactive compounds, like T. pinophilus, T. stipitatus, T. purpurogenus, and T. wortmannii [12]. Other members have a great impact on the food industry [13]. For instance, T. bacillisporus, T. flavus, T. helicus, T. macrosporus, T. stipitatus, T. trachyspermusi, and T. wortmannii cause spoilage of pasteurized juices. T. purpurogenus produces mycotoxins. T. islandicus causes rice yellowing. The ability to produce enzymes and soluble pigments makes Talaromyces an important genus for biotechnological purposes. For example, T. funiculosus generates cellulase and utilizes it to produce ethanol through the hydrolysis of sugar cane bagasse [14]. T. atroroseus [15], T. assiutensis [16], and T. albobiverticillius [17] can synthesize pigments that have application as cosmetic and food colorants. In addition, the Talaromyces species are also capable of producing a wide range of secondary metabolites, including esters, coumarins, isocoumarin, polyketones, anthraquinone, terpenoids, meroterpenoids, steroids, alkaloids, and others [18].
Many studies have also revealed that Talaromyces species possess gene clusters associated with cell wall-degrading enzymes and secondary metabolites. Whole genome se quencing of strain T. piceus 9-3 revealed that its genome had a diverse set of lignocellulolytic enzymes, including two cellobiohydrolases, one endo-β-1,4-glucanase, and ten β-glucosidase gene clusters [19]. The genome of strain T. pinophilus 1–95 contained two cellobiohydrolases, eight β-1,4-endoglucanases, 29 β-glucosidases, 97 hemicellulose-degrading enzymes, 24 α-amylases, and 52 secondary metabolism gene clusters [20]. The genome of T. albobiverticillius Tp-2 contained eight distinct gene clusters responsible for the biosynthesis of secondary metabolites [17]. The genome of T. albobiverticillius Tp-2 contained eight distinct gene clusters responsible for the biosynthesis of secondary metabolites. In this study, the whole genome sequencing of the Talaromyces DC2 strain was performed using PacBio Sequel and Illumina NovaSeq 6000 sequencing platforms. The Talaromyces DC2 strain has already been identified by our research as a prolific producer of vinca alkaloid with anticancer properties [21]. The acquired whole genome sequencing data enrich our understanding of the relationships between gene clusters and metabolic products in the DC2 strain. Our results demonstrated that the DC2 strain has the capability to degrade pectin and starch, synthesize xylooligosaccharides and short-chain fructooligosaccharides, and produce swainsonine, varicidin A, asperterpenoid A, squalestatin S1, ustethylin A, and ilicicolin H, as well as perform the decarboxylation of L-tryptophan. Furthermore, the obtained whole genome data can serve as a valuable resource for future bioengineering research.

2. Materials and Methods

2.1. Fungal Strain

Strain DC2 was isolated from the surface sterilized stem of the Catharanthus roseus (L.) G. Don plant cultivated in Hanoi, Vietnam, with a yellow-colored colony as previously described [21]. The protocol for obtaining endophytic fungi from plant materials has been elucidated in a previous study [21]. Strain DC2 has already been proven to have the ability to produce anticancer compounds, including vincristine and vinblastine. Identification and quantification of vincristine and vinblastine produced by the DC2 strain were conducted by ultra-high performance liquid chromatography/multiple reaction monitoring mass spectrometry analyses.

2.2. Extraction of Genomic DNA

For DNA extraction, isolated endophytic fungi were inoculated in 100 mL potato dextrose broth (PDB; Sigma, Saint Louis, MO, USA) medium and cultured in 250 mL Erlenmeyer flasks at 25 °C in the dark on a rotary shaker at 200 rpm. After 7 days, the fungal biomass was harvested by centrifugation at 10,000 rpm for 15 min on an Eppendorf 5810R centrifuge (Eppendorf, Hamburg, Germany) and used for DNA extraction by the cetyltrimethylammonium bromide (CTAB) method with minor adjustments for optimization [22]. Qualification and quantification of extracted DNA were measured using a Nanodrop®1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

2.3. Genome Sequencing

The genomic DNA of strain DC2 was sequenced using the PacBio Sequel system (Menlo Park, CA, USA) and the Illumina NovaSeq 6000 (San Diego, CA, USA). For the PacBio sequencing library, 5–10 µg of genomic DNA was sheared into 10–15 kb fragments using a g-TUBE device (PerkinElmer, Ho Chi Minh City, Vietnam). Then the library was constructed using the SMRTbell Express Template Preparation Kit 2.0 (Pacbio, Menlo Park, CA, USA), following the manufacturer’s protocol. In brief, the process involved amplifying the DNA fragments using barcoded DNA primers, resulting in a pooled collection of all the samples. For the Illumina sequencing library, the library was prepared using the VAHTS Universal Pro DNA Library Prep Kit (Vazyme, Nanjing, China) following the manufacturer’s protocol. The generated library was cleaned up, and the sequencing process was carried out using 2–150 paired-end (PE) and 10–15 kb read length configurations for Illumina and PacBio sequencing, respectively.

2.4. Assembly, Gene Prediction and Annotation

PacBio and Illumina reads were assembled using Hifiasm (v0.13-r308) and Canu (v1.7). The assembly result was corrected with Pilon (v1.22). The gene prediction was performed using Augustus (v3.3) with default parameters. The tRNAs, rRNAs, and non-coding RNAs were predicted using tRNA scan-SE (v1.3.1), barrnap (v0.9), and Rfam (v12.2) [23], respectively. The repeat sequences were detected using RepeatMasker (v4.0.6) using the Dfam database (v2.0) (http://www.repeatmasker.org, accessed on 6 December 2023).
BLAST searches of non-redundant (NR) protein sequences from the NCBI, Kyoto Encyclopedia of Genes and Genomes (KEGG) [24], Gene Ontology (GO) [25], Clusters of Orthologous Groups (COG/KOG) [26], Carbohydrate-Active Enzymes (CAZy), Pfam, Swiss-Prot, and Database of Fungal Virulence Factors (DFVF) databases were performed to annotate the gene products. Signal peptides were analyzed using the online software SignalP v.5.0 (http://www.cbs.dtu.dk/services/SignalP/, accessed on 6 December 2023). The polypeptide chain of a transmembrane protein was analyzed using the online software TMHMM v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 6 December 2023).
Secondary metabolite biosynthetic clusters were identified using the antiSMASH web server (fungal version 7.0.1) with the default settings [27].

3. Results and Discussion

3.1. Genome Sequencing, Assembly, and Genomic Features

The Illumina sequencing data yielded a total of 39,360,260 clean reads, which corresponds to 5,900,310,550 bases. These readings had 91.83% of their bases with a quality score of Q30. On the other hand, the PacBio sequencing data produced 107,913 raw reads, totaling 340,232,173 bases. The N50 value was 3423 bp. After assembly, a total of 156 scaffolds were obtained, with a total size of 34,575,287 bp (Table 1). The final assembly revealed a GC content of 45.94%. The genome size of strain DC2 was compared to the recently available 75 genome sizes in NCBI, which range from 26.6 Mb of Talaromyces piceae strain 9-3 (GCA_001657655.1) to 42.5 Mb of Talaromyces nanjingensis strain JP-NJ4 (GCA_031010415.1) (Supplementary Table S1).

3.2. The Gene Functions of Talaromyces sp. DC2

A total of 11,131 genes were annotated in the genome of strain DC2 (Table 1). The highest number of functional genes in DC2 was determined by the NR database (10,780 genes, 96.85%), followed by Pfam (8735 genes, 78.47%), Swiss-Prot (8165 genes, 73.35%), GO (6682 genes, 60.03%), KEGG (6509 genes, 58.48%), KOG (6353 genes, 57.07%), DFVF (2658 genes, 23.88%), and CAZy (1230 genes, 11.05%) (Table 1). The number of protein-coding genes in DC2 is lower than that of T. pinophilus 1-95, which consisted of 13,472 protein-coding genes [20], but higher than that of T. albobiverticillius Tp-2, which consisted of 10,380 protein-coding genes [17]. Variations in the number of protein-coding genes may result from variations in the quality of the input DNA quality, the sequencing technique employed, and/or the size and native sequence of the genome.
The 6509 genes were mapped to known enzyme pathways in six KEGG types: cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems (Figure 1). The most abundant pathways in DC2 include carbohydrate metabolism (978), amino acid metabolism (908), signal transduction (824), and xenobiotics biodegradation and metabolism (796). The abundance of genes in the xenobiotics biodegradation and metabolism, as well as signal transduction pathways, suggests that strain DC2 is capable of metabolizing xenobiotics in its environments.

3.3. Carbohydrate-Active Enzymes in Talaromyces sp. DC2

Carbohydrate-active enzymes (CAZymes) play important roles in carbohydrate degradation. We detected a total of 1230 genes classified as CAZymes in strain DC2. The total number of CAZymes in DC2 is greater than those of other Talaromyces strains, for example, T. pinophilus strain 1–95 (803 CAZymes) [20] and T. albobiverticillius strain Tp-2 (750 CAZymes) [17]. DC2 possesses a total of 527 glycoside hydrolases (GHs), 340 glycosyltransferases (GTs), 165 carbohydrate-binding modules (CBMs), 107 auxiliary activities (AAs), 83 carbohydrate esterases (CEs), and 8 polysaccharide lyases (PLs) (Figure 2A). The number of GH family genes in Talaromyces sp. DC2 (527 genes) is higher than those of T. albobiverticillius strain Tp-2 (427 genes) [17] and T. cellulolyticus (249 genes) [28]. GHs accounted for 42.85% of the CAZymes and were found in 74 families. The most prevalent families were GH18 (chitinase) with 56 out of 527 members and GH43 (β-xylosidase) with 36 out of 527 members. GTs comprised 43 families, consisting of 74 cellulose synthases from the GT2 family and 52 sucrose synthases from the GT4 family. CBMs included 25 families, with the highest prevalence found in family XIII of the cellulose-binding domain, accounting for 37 out of 165 members. The primary families of AAs were predominantly AA3 (34 out of 107), AA7 (28 out of 107), and AA1 (15 out of 107) families. The CEs were categorized into families of CE0-CE6, CE8-9, CE11-12, and CE14-CE16. PLs were distributed in PL0-PL1, PL4, PL7, and PL10.
In DC2, 507 out of 1230 CAZymes (41.22%) contained a signal peptide and/or transmembrane (Figure 2B and Supplementary Table S1). There are 54 CAZymes that have both signal peptides and transmembrane domains. Additionally, there are 301 CAZymes that have signal peptides but do not have transmembrane domains, and 202 CAZymes that have transmembrane domains but lack signal peptides. The 301 CAZymes possessing signal peptides but lacking transmembrane domains were classified as secreted CAZymes. It was found that the DC2 strain secrets a similar number of CAZymes to T. pinophilus strain 1–95, specifically, 323 CAZymes [20].
Strain DC2 was isolated from the stem of C. roseus [21]; therefore, we focused on plant cell wall-degrading CAZymes as described in previous studies [29,30]. Strain DC2 possess 653 CAZymes responsible for plant cell wall degradation. They are involved in degradation of cellulose (149 CAZymes), hemicellulose (227 CAZymes), lignin (65 CAZymes), pectin (153 CAZymes), starch (53 CAZymes), and inulin (6 CAZymes) (Table 2). Of these, β-glucosidases (27 genes) were involved in both cellulose and hemicellulose degradations; β-galactosidases (23 genes) and α-L-arabinofuranosidases (6 genes) were involved in both hemicellulose and pectin degradations (Table 2). The plant cell wall-degrading CAZymes of strain DC2 have been found to exhibit greater diversity compared to those of T. pinophilus strain 1–95 [20].
Cellulose is one of the three most abundant polysaccharides in plant cell walls and has a basic structure of β-1,4-linked D-glucose molecules [34]. Cellulose degradation involves the activities of endocellulases, exoglucanases, cellobiohydrolases, and β-glucosidases [35]. Strain DC2 consisted of 6 genes encoding exoglucanases (also known as cellobiohydrolases) (GH55), 43 genes encoding endoglucanases, 27 genes encoding β-glucosidases, and 3 cellobiose dehydrogenases. However, it did not have a gene encoding lytic polysaccharide monoxygenase (LPMO) (Table 2). Other fungal strains, in contrast, have a different number of genes encoding cellulose-degrading enzymes. Specifically, Trichoderma reesei QM6a is the only strain that harbors six genes encoding exoglucanases (GH23: two genes and GH55: four genes) [32], while information regarding the exoglucanases of the remaining five fungal strains is not available. In endoglucanases, there are 18 genes in the T. reesei QM6a, 9 genes in the Aspergillus niger CBS 513.88, 14 genes in the T. amestolkiae CIB, 10 genes in the T. pinophilus 1–95, 9 genes in the T. cellulolyticus Y-94, and 12 genes in the Penicillium oxalicum HP7-1 (Table 2). In β-glucosidase, the T. reesei QM6a, A. niger CBS 513.88, T. pinophilus 1–95, T. cellulolyticus Y-94, and P. oxalicum HP7-1 strains consist of 13, 18, 24, 19, and 8 genes, respectively (Table 2), while LPMO has only been found in the T. amestolkiae CIB (one gene), T. pinophilus 1–95 (one gene), T. cellulolyticus Y-94 (one gene), and P. oxalicum HP7-1 strains (three genes). Polysaccharide monooxygenase has only been found in the T. reesei QM6a (three genes) and A. niger CBS 513.88 (seven genes). The data suggest that the Talaromyces DC2 strain and T. reesei QM6a are the only two strains that have a nearly complete set of enzymes for effectively degrading cellulose.
Hemicellulose is composed of β-1,4-xylan and β-1,4-D-xylopyranoside [36]. Xylan degradation depends primarily on two enzymes, endo-β-1,4-xylanase and β-1,4-D-xylosidase [37]. In strain DC2, one endo-β-1,4-xylanase family (GH10) and one β-xylosidase family (GH43) were detected (Table 2). There are 36 genes encoding β-xylosidases. Furthermore, strain DC2 also contained various side-chain cleaving hydrolases, such as α-L-arabinofuranosidases (6 genes), α-glucuronidases (1 gene), and acetyl xylan esterases (42 genes) (Table 2). Similarly, the six comparative fungal strains also harbor a range of genes encoding hemicellulose-degrading enzymes. Specifically, the T. reesei QM6a, A. niger CBS 513.88, T. amestolkiae CIB, T. pinophilus 1–95, T. cellulolyticus Y-94, and P. oxalicum HP7-1 strains consist of 6, 4, 16, 9, 7, and 10 endo-1,4-β-xylanase-encoding genes, respectively, as well as 3, 10, 16, 10, 7, and 12 genes encoding β-xylosidase, respectively. The results support the high xylanase activity of strain DC2, which is comparable to that of T. pinophilus 1–95 [20]. The byproducts of xylan breakdown include xylo-oligosaccharides and xylose, which have potential applications in several industries such as health care, food, pharmaceuticals, and cosmetics [38]. Thus, it is evident that strain DC2 shows potential as a valuable source for the synthesis of xylo-oligosaccharides, a potential material that has many applications in different industries.
Lignin is a complex polymer that embeds in cellulose and hemicellulose to strengthen the structure of the plant cell wall. The primary enzymes involved in lignin degradation are the laccase and peroxidase families [39]. Our data analysis showed that the DC2 strain contains 15 genes that encode laccase and 1 gene that encodes peroxidase. This suggested that that the DC2 strain has the potential to break down the lignin matrix (Table 2). The six fungal strains, interestingly, do not possess any genes that encode liginiolytic enzymes.
Pectin is a heteropolysaccharide abundant component of the plant’s primary cell wall [40]. Pectin consists of α-1,4-linked D-galacturonic acid and several sugars such as rhamnose, arabinose, galactose, and other sugars [41,42]. Strain DC2 consisted of a complete set of CAZymes for pectin degradation, including glycoside hydrolases (GH2, GH28, GH35, GH51, GH53, GH54, GH78, GH88, GH93, and GH105), polysaccharide lyases (PL1, PL4, PL7, and PL20), carbohydrate esterases (CE8, CE12, and CE16), and carbohydrate-binding modules (CBM13, CBM32, and CMB51) (Table 2). Strain DC2 exhibits a pectin degradation profile that is comparable to that of T. pinophilus 1–95, T. cellulolyticus Y-94 and P. oxalicum HP7-1 strains but more diverse than that of the T. reesei QM6a, A. niger CBS 513.88, and T. amestolkiae CIB (Table 2). As suggested by Benoit et al. [43], the increase in pectin-related genes resulted in improved growth in pectin. This suggests that strain DC2 has the ability to thrive on different pectin sources, such as citrus pectin and apple pectin. Thus, strain DC2 exhibits significant promise for the breakdown of pectin.
Starch is an α-1,4-linked D-glucose polymer that is synthesized by plants as a means of energy storage [44]. Strain DC2 consists of 13 GH13 genes encoding α-amylases, 2 GH15 genes encoding glucoamylases, 15 GH31 genes encoding α-glucosidase, 1 GT35 gene encoding starch phosphorylase (1 gene), and 21 genes encoding starch-binding (18 CBM20 and 3 CBM21) (Table 2). Comparative analysis revealed that only T. reesei QM6a and A. niger CBS 513.88 contain genes encoding starch-degrading enzymes, while these genes were not found in the remaining four fungal strains. Furthermore, our study revealed that strain DC2 possesses a higher number of genes that encode α-amylases and α-glucosidase than T. pinophilus 1–95 [20] and 85 fungal strains belonging to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota [45].
Inulin is a fructan polysaccharide found in plants that serves as a storage carbohydrate. It consists of glucose molecules at the terminal end [46]. The process of inulin conversion involves the utilization of the glycosyl hydrolase families GH32 and GH91, which include enzymes such as inulinase, invertase, levanase, 1-exohydrolases, fructan-fructosyltransferases, and sucrose fructosyltransferases [47]. The genome of strain DC2 contained five genes encoding endo-inulinase from the GH32 family, which is consistent with the discovery made in Apgergillus niger [48]. Endo-inulinase breaks out the glycosidic bond β(2→1) to produce short-chain fructooligosaccharides [49]. Furthermore, strain DC2 was found to contain a gene encoding the inulin binding domain, CBM38. The data suggest that strain DC2 is capable of degrading inulin as an endobiont. In addition, the short-chain fructooligosaccharides serve as prebiotics [49]. Therefore, strain DC2 has the potential to be a great source for producing fructooligosaccharides that are similar to those produced by different Aspergillus strains [33,50,51].

3.4. The Secondary Metabolism in the DC2

AntiSMASH analysis suggested that strain DC2 possessed 37 biosynthetic gene clusters (BGCs) related to secondary metabolism. Among them, 20 out of 37 genes exhibited gene homologies with known clusters in the MIBiG database. These clusters included 12 Type I polyketide synthases (T1PKSs), five nonribosomal peptide synthetases (NRPSs and NRPS-like), and three terpene synthases (Terpenes) (Table 3). Strain DC2 has a lesser number of secondary metabolism BCGs compared to T. pinophilus strain 1-95 (68 clusters) [20] and T. albobiverticillius strain Tp-2 (62 clusters) [17].
There are six secondary metabolism clusters that have a gene similarity of 100% with six known biosynthetic clusters. These known clusters produce substances such as monascorubrin, YWA1, alternariol, ochratoxin, choline, and cyclic depsipeptide (Table 3). In region 9.1, one T1PKS was responsible for the biosynthesis of monascorubrin. This compound has been used as a natural red colorant for a wide range of foods in Asian countries [52]. Monascrorubin has been identified in Talaromyces species such as T. marneffei [52] and T. atroroseus [53]. In region 29.1, one T1PKS has been found to be responsible for the biosynthesis of naphthopyrone YWA1. This compound is considered a precursor of dihydroxynaphthalene (DHN)-melanin in Aspergillus nidulans [54] and aurofusarin in Fusarium graminearum [55]. In region 30.2, one T1PKS was found to be accountable for production of alternariol, a toxic metabolite in Alternaria that showed multiple potential pharmacological effects [30]. The alternariol has also been identified in T. pinophilus AF-02 [56]. Region 34.1 was responsible for the biosynthesis of ochratoxin A, a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species. This toxin can be detected in fungal contaminated food, beverages, and feed [57]. However, ochratoxin A was not included in the list of 238 secondary metabolite substances produced by Talaromyces species [18]. In region 43.2, one NRPS-like was responsible for the biosynthesis of choline, which is an essential metabolite for the growth of filamentous fungi and the regulation of mycelial morphology [58]. In region 69.1, one NRPS was identified as the catalyst for the production of cyclic depsipeptide. This occurs when the amide groups in the peptide structure are substituted with lactone bonds, which is facilitated by the presence of a hydroxylated carboxylic acid [59]. Cyclic peptides were discovered in T. wortmannii [60].
Seven BGC clusters exhibit the similarities ranging from 60% to 75%, including swainsonine (66%), varicidin A (71%), asperterpenoid A (66%), squalestatin S1 (60%), ustethylin A (70%), trichobrasilenol/xylarenic acid B/brasilane A/F/E/D (60%), and ilicicolin H (75%) (Figure 3). In region 7.1, one T1PKS was responsible for the biosynthesis of swainsonine, an indolizidine alkaloid that is produced by endophytic fungi and has the potential to be used as a drug for cancer therapy [61,62]. In region 9.2, a single NRPS was found to be linked to the production of varicidin A. Varicidin A is an naturally occurring antifungal compound that is produced by a Diels–Alderase reaction [63]. In region 24.1, a single terpene was responsible for the production of asperterpenoid A, a compound which exhibits strong inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B [64,65,66]. In region 30.1, a specific terpene was found to be accountable for the biosynthesis of squalestatin S1, which acts as a highly potent picomolar inhibitor of squalene synthase [67]. Additionally, squalestatin S1 exhibits a wide range of antifungal properties and serves as a lead structure for the development of cholesterol–lowering drugs [68]. In region 40.2, one T1PKS was responsible for the biosynthesis of ustethylin A, a compound synthesized by Aspergillus ustus [69]. In region 78.1, a specific terpene was identified to be responsible for the biosynthesis of trichobrasilenol/xylarenic acid B/brasilane A/F/E/D. This is an unusual sesquiterpene alcohol synthesized by a sesquiterpene cyclase from Trichoderma sp. [70]. In region 95.1, one NRPS was found to be accountable for the biosynthesis of ilicicolin H. This compound is a broad-spectrum antifungal agent that acts on mitochondrial cytochrome bc1 reductase [71,72,73,74,75]. This study, to our knowledge, is one of the first to report the presence of BGC clusters that encode swainsonine, varicidin A, asperterpenoid A, squalestatin S1, ustethylin A, trichobrasilenol/xylarenic acid B/brasilane A/F/E/D, and ilicicolin H in the Talaromyces genus.

3.5. The Indole Alkaloid Biosynthesis in the DC2

Decarboxylation of L-tryptophan leads to the formation of tryptamine, which serves as a common backbone for many secondary metabolites. One such metabolite is the pathway of terpenoid indole alkaloids in plants [76]. In strain DC2, a DDC gene (Gene ID: g533) that encodes an aromatic L-amino acid decarboxylase (AADC) was identified (Figure 4).
The open reading frame (ORF) of g533 had a length of 1536 nucleotides and corresponded to the coding sequence for 512 amino acids (Supplementary Figure S1). The g533 shared the closest genetic similarities to those of Talaromyces islandicus (CRG88687.1; 93.58%) and Talaromyces rugulosus7 (XP_035346356.1; 90.91%). The three sequences also formed a clade in the phylogenetic gene (Figure 5).
In contrast to AADCs from animals and plants, fungal AADCs have not been extensively studied. The first description of a fungal AADC was reported by Niedens et al. [77]. The authors demonstrated its broad substrate specificity, including L-tryptophan, L-tyrosine, L-phenylalanine, o-fluorophenylalanine, and p-fluorophenylalanine. Later, Kalb et al. [78] reported on the Ceriporiopsis subvermispora aromatic L-amino acid decarboxylases (CsTDCs) that were heterologously produced in a laboratory setting. The study identified that CsTDC exhibited strict specificity towards L-tryptophan and 5-hydroxy-L-tryptophan. Interestingly, AADC of strain DC2 in our study also contains the same sequence, 368LGRRFR373, as CsTDC’s sequence, 350LGRRFR355, where G351 is the active site. However, CsTDC has a phenylalanine at residue 329, whereas that of g533 has tyrosine. This is similar to the amino acid sequence of PcDHPAAS, which is capable of converting L-3,4-dihydroxyphenylalanin to 3,4-dihydroxylphenylacetaldehyde [79]. However, further investigation is required to assess the decarboxylation capacity of g533 towards aromatic amino acids.

4. Conclusions

In summary, whole genome sequencing has provided a comprehensive understanding of Talaromyces sp. DC2, encompassing its overall functions of CAZymes and secondary metabolites. Genome analysis showed that strain DC2 might serve as a potential source for the degradation of pectin and starch, the synthesis of xylo-oligosaccharides and short-chain fructooligosaccharides, and the production of swainsonine, varicidin A, asperterpenoid A, squalestatin S1, ustethylin A, and ilicicolin H. Additionally, it has the ability to carry out the fungal decarboxylation of L-tryptophan. Furthermore, the obtained genome sequencing data can serve as a valuable resource for future bioengineering research. However, further investigations are required to confirm the distinct characteristics and feasibility of the Talaromyces DC2 strain.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10050352/s1, Figure S1: The ORF and amino acid translation of DDC gene in the genome of strain DC2; Table S1: Comparison analysis of genome features of strain DC2 and 75 available Talaromyces strain in NCBI.

Author Contributions

Conceptualization, H.H.N.; methodology, H.H.N. and N.-L.N.; software, N.V.T.; investigation, N.-L.N., N.D.Q., T.T.H.G., N.T.T.N., N.T.H., N.V.T., N.H.T.T. and N.T.K.L., writing—original draft preparation, N.D.Q. and N.-L.N.; writing—review and editing, N.D.Q., N.-L.N. and H.H.N.; supervision, H.H.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Vietnam Academy of Science and Technology, grant number TĐCNSH.02/20-22.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw sequencing data were deposited in the NCBI Sequence Read Archive (SRX21294582—Illumina sequencing data and SRX21294583–PacBio sequencing data). The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBCLNQ000000000. The version described in this paper is version JBCLNQ010000000.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification of Talaromyces sp. DC2.
Figure 1. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification of Talaromyces sp. DC2.
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Figure 2. Distribution of carbohydrate-active enzyme (CAZyme) familes (A), and signal and transmembrane CAZymes (B) in Talaromyces sp. DC2.
Figure 2. Distribution of carbohydrate-active enzyme (CAZyme) familes (A), and signal and transmembrane CAZymes (B) in Talaromyces sp. DC2.
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Figure 3. Comparison of biosynthetic gene cluster constituents in strain DC2 with identified biosynthetic gene clusters for biosynthesis of swainsonine (66%), varicidin A, asperterpenoid A, squalestatin S1, ustethylin A, trichobrasilenol/xylarenic acid B/brasilane A/F/E/D, and ilicicolin H.
Figure 3. Comparison of biosynthetic gene cluster constituents in strain DC2 with identified biosynthetic gene clusters for biosynthesis of swainsonine (66%), varicidin A, asperterpenoid A, squalestatin S1, ustethylin A, trichobrasilenol/xylarenic acid B/brasilane A/F/E/D, and ilicicolin H.
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Figure 4. The indole alkaloid biosynthetic pathway gene found in Talaromyces sp. DC2 is depicted by the red-colored box. AADC, aromatic L-amino acid decarboxylase.
Figure 4. The indole alkaloid biosynthetic pathway gene found in Talaromyces sp. DC2 is depicted by the red-colored box. AADC, aromatic L-amino acid decarboxylase.
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Figure 5. Phylogenetic tree includes the amino acid sequences of putative aromatic L-amino acid decarboxylases. MUSCLE v.5.0 was used for sequence alignment and a neighbor-joining algorithm was used to construct the tree in the Mega-X v.10.2.6.
Figure 5. Phylogenetic tree includes the amino acid sequences of putative aromatic L-amino acid decarboxylases. MUSCLE v.5.0 was used for sequence alignment and a neighbor-joining algorithm was used to construct the tree in the Mega-X v.10.2.6.
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Table 1. Genome summary statistics for Talaromyces sp. DC2 and related strains.
Table 1. Genome summary statistics for Talaromyces sp. DC2 and related strains.
CharacteristicsTalaromyces sp. DC2Talaromyces pinophilus
1–95 [20]		Talaromyces albobiverticillius Tp-2 [17]
Value% of Total
Genome assembly (bp)34,575,287100%36,480,44338,354,882
Contigs156-114
N50 length (bp)346,458-4,804,1684,594,200
Minimum length (bp)3491-2,941,929-
Maximum length (bp)1,007,571-7,684,6676,575,826
G+C content15,883,165 bp45.94%46.25%45.78%
Coding region17,667,222 bp51.10--
Total genes11,13110013,57910,584
RNA genes1421.28107204
Protein-coding genes10,98998.7213,47210,380
NR10,78096.8512,9469782
KEGG650958.4868178844
GO668260.0381627412
KOG635357.07-2160
CAZy123011.05803750
Pfam873578.47-7412
Swiss-Prot816573.35-3657
DFVF265823.88-2058
NR, non-redundant; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; KOG, Clusters of Orthologous Groups; CAZy, carbohydrate-active enzymes; DFVF, Database of Fungal Virulence Factors.
Table 2. Plant cell wall-degrading carbohydrate-active enzymes (CAZymes) in Talaromyces sp. DC2 and other fungal strains.
Table 2. Plant cell wall-degrading carbohydrate-active enzymes (CAZymes) in Talaromyces sp. DC2 and other fungal strains.
SubstrateEnzymatic
Activity		CAZy FamilyTalaromyces sp. DC2T. pinophilus 1-95 [20]T. cellulolyticus Y-94 [20]T. amestolkiae CIB [31]Trichoderma reesei QM6a [32]Aspergillus niger
CBS 513.88 [33]		Penicillium oxalicum HP7-1 [20]
CelluloseEndoglucanaseGH5174414435
GH6110n/a121
GH7211n/a222
GH12322n/a223
GH45122n/a001
GH645n/an/an/a30n/a
GH7111n/an/an/a40n/a
GH812n/an/an/a20n/a
GH1311n/an/an/a00n/a
ExoglucanaseGH230n/an/an/a20n/a
GH556n/an/an/a40n/a
β-glucosidaseGH1355n/a234
GH3242419n/a11158
Polysaccharide monooxygenaseGH610n/an/an/a37n/a
Cellulose-bindingCBM111n/an/an/an/an/an/a
CBM218n/an/an/an/an/an/a
CBM31n/an/an/an/an/an/a
CBM65n/an/an/an/an/an/a
CBM101n/an/an/an/an/an/a
Lytic polysaccharide monoxygenase (LPMO)AA160n/an/an/an/an/an/a
AA90111n/an/a3
Cellobiose dehydrogenaseAA83n/an/an/an/an/an/a
FAD-dependent (GMC) oxidoreductaseAA334n/an/an/an/an/an/a
HemicelluloseEndo-1,4-β-xylanaseGH101111113
GH113869335
GH300n/an/a6202
XyloglucanaseGH740n/an/a311n/a
β-xylosidaseGH43361071631012
β-glucosidaseGH1355n/a234
GH3242419n/a11158
α-mannosidaseGH4718n/an/an/a80n/a
GH920n/an/an/a60n/a
α-L-arabinofuranosidaseGH51233n/a033
GH54455n/a211
GH620333112
α -1,6-mannanaseGH7621n/an/an/a70n/a
Β-mannanaseGH260n/an/an/an/a11
Endo-α-1,5-arabinanaseGH931443n/a003
β-glucuronidaseGH797n/an/an/a40n/a
α-glucuronidaseGH671n/an/a1111
α-galactosidaseGH27944n/a852
GH361243n/a22n/a
β-galactosidaseGH21366n/a642
GH3510n/an/an/a15n/a
α-L-fucosidaseGH29143n/a01n/a
GH95153n/a321
Acetyl xylan esteraseCE17221n/an/a1
CE2122n/an/an/a1
CE3375n/an/an/an/a
CE47n/an/an/an/an/an/a
CE55213n/an/a1
CE619n/an/an/an/an/an/a
Arabinoxylan-bindingCBM425n/an/an/an/an/an/a
LigninLaccaseAA115n/an/an/an/an/an/a
PeroxidaseAA21n/an/an/an/an/an/a
FAD-dependent (GMC) oxidoreductaseAA334n/an/an/an/an/an/a
Vanillin alcohol oxidaseAA46n/an/an/an/an/an/a
Copper radical oxidaseAA53n/an/an/an/an/an/a
Benzoquinon reductaseAA62n/an/an/an/an/an/a
Cellobiose dehydrogenaseAA83n/an/an/an/an/an/a
4-O-methyl-glucuronoyl methylesteraseCE151n/an/an/an/a0n/a
PectinPolygalacturonaseGH282232n/a4218
α-L-arabinofuranosidaseGH51233n/a003
GH54455n/a201
Exo-α-L-1,5-arabinanaseGH931443n/a003
β-galactosidaseGH21366n/a642
GH3510n/an/an/a15n/a
d-4,5 unsaturated β-glucuronyl hydrolaseGH883n/an/an/a001
unsaturated rhamnogalacturonyl hydrolaseGH105244n/a101
α-L-rhamnosidaseGH7817n/an/an/a18n/a
β-1,4-galactanaseGH53111n/a001
Pectate lyasePL1122n/an/a61
Rhamnogalacturonan lyasePL41n/an/an/an/a21
α-L-guluronate lyasePL71n/an/an/an/a0n/a
Endo-β-1,4-glucuronan lyasePL201n/an/an/an/a0n/a
Pectin methylesteraseCE8433n/an/a33
Pectin acetylesteraseCE12211n/an/a22
AcetylesteraseCE167n/an/an/an/a0n/a
Galactose-binding
(48 genes)		CBM1337n/an/an/an/an/an/a
CBM329n/an/an/an/an/an/a
CMB512n/an/an/an/an/an/a
Starchα-amylaseGH1313n/an/an/a17n/a
GlucoamylaseGH152n/an/an/a21n/a
α-glucosidaseGH3115n/an/an/a47n/a
GH631n/an/an/a20n/a
Starch phosphorylaseGT351n/an/an/an/an/an/a
Starch-bindingCBM2018n/an/an/an/an/an/a
CBM213n/an/an/an/an/an/a
InulinEndo-inulinaseGH325n/an/an/an/a1n/a
Inulin-bindingCBM381n/an/an/an/an/an/a
n/a: not applicable.
Table 3. Putative biosynthetic gene clusters of Talaromyces sp. DC2 showed similarity to known gene clusters in the minimum information about a biosynthetic gene cluster database.
Table 3. Putative biosynthetic gene clusters of Talaromyces sp. DC2 showed similarity to known gene clusters in the minimum information about a biosynthetic gene cluster database.
No.RegionTypeLocationMost Similar Known ClusterSimilarity
17.1T1PKS309,280–374,892Swainsonine66%
29.1T1PKS4143–41,865Monascorubrin100%
39.2NRPS142,422–212,589Varicidin A71%
422.1T1PKS296,509–349,694Etanone C/probetaenone42%
524.1Terpene236,960–270,368Asperterpenoid A66%
629.1T1PKS69,255–135,872YWA1100%
730.1Terpene76,815–108,403Squalestatin S160%
830.2T1PKS219,673–284,128Alternariol100%
934.1T1PKS81,492–145,061Ochratoxin100%
1040.1T1PKS526,164–588,670Waikikiamide A/B/C18%
1140.2T1PKS601,377–668,240Ustethylin A70%
1243.2NRPS-like181,715–301,302Choline100%
1352.1T1PKS183,397–251,045Waikikiamide A/B/C36%
1461.1T1PKS194,350–261,0943’-methoxy-1,2-dehydropenicillide/pestalotiollide B/C10%
1563.1T1PKS32,753–101,221Gregatin33%
1665.1T1PKS71,173–135,718Crytosporioptide B/A/C15%
1769.1NRPS40,492–110,190Cyclic depsipeptide100%
1878.1Terpene200,100–231,945Trichobrasilenol/xylarenic acid B/brasilane A/F/E/D60%
1993.1NRPS1–41,092Dihydroisoflavipucine/isoflavipucine31%
2095.1NRPS246,099–307,834Ilicicolin H75%
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Quan, N.D.; Nguyen, N.-L.; Giang, T.T.H.; Ngan, N.T.T.; Hien, N.T.; Tung, N.V.; Trang, N.H.T.; Lien, N.T.K.; Nguyen, H.H. Genome Characteristics of the Endophytic Fungus Talaromyces sp. DC2 Isolated from Catharanthus roseus (L.) G. Don. J. Fungi 2024, 10, 352. https://doi.org/10.3390/jof10050352

AMA Style

Quan ND, Nguyen N-L, Giang TTH, Ngan NTT, Hien NT, Tung NV, Trang NHT, Lien NTK, Nguyen HH. Genome Characteristics of the Endophytic Fungus Talaromyces sp. DC2 Isolated from Catharanthus roseus (L.) G. Don. Journal of Fungi. 2024; 10(5):352. https://doi.org/10.3390/jof10050352

Chicago/Turabian Style

Quan, Nguyen Duc, Ngoc-Lan Nguyen, Tran Thi Huong Giang, Nguyen Thi Thanh Ngan, Nguyen Thanh Hien, Nguyen Van Tung, Nguyen Hoang Thanh Trang, Nguyen Thi Kim Lien, and Huy Hoang Nguyen. 2024. "Genome Characteristics of the Endophytic Fungus Talaromyces sp. DC2 Isolated from Catharanthus roseus (L.) G. Don" Journal of Fungi 10, no. 5: 352. https://doi.org/10.3390/jof10050352

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