Journal of Applied Biology & Biotechnology Vol. 12(3), pp.

229-235, May-Jun, 2024

Available online at http://www.jabonline.in
DOI: 10.7324/JABB.2024.154874

Genome mining and AntiSMASH analysis of an Endophytic

Talaromyces sp. reveal biosynthetic pathway gene clusters for novel
bioactive compounds

Priyanka N. Shenoy1, Sneha Bhaskar1, M. Manu1, M. P. Likitha1, N. Geetha1, Shailasree Sekhar2*, K Ramachandra Kini1
1
Department of Studies in Biotechnology, University of Mysore, Mysuru, Karnataka, India.
2
Division of Biochemistry, School of Life Sciences, JSS Academy of Higher Education and Research, Sri Shivarathreeshwara Nagara, Mysuru, Karnataka, India.

ARTICLE INFO ABSTRACT

Article history:
Medicinal plants and their endophytes are one of the efficient producers of diverse secondary metabolites with
Received on: November 29, 2023
therapeutic importance. In the present study, an endophytic fungus Talaromyces spp. isolated from Syzygium
Accepted on: February 27, 2024
samarangense was subjected to whole-genome sequencing and Antibiotics and Secondary Metabolite Analysis
Available online: April 20, 2024
Shell (AntiSMASH) annotation to identify biosynthetic gene clusters (BGCs) of secondary metabolites and their
biosynthetic pathways. The Funannotate results revealed that the Talaromyces isolate has a total of 30.5Mb genome
Key words:
consisting of 372 contigs, 372 scaffolds and 47.64% GC content. In addition, 114 tRNA, 12722 functional mRNA,
Endophytic fungi,
Secondary metabolites, 12721 CDS transcripts, and 12721 protein coding sequences were predicted and annotated using various BLAST
Antibiotics and secondary metabolite databases. AntiSMASH revealed the presence of 76 BGCs, including 28 T1 Polyketide synthase (T1 PKS), 10
analysis shell, Nonribosomal peptide synthetases (NRPSs), 9 terpene, 1 Indole, 12 NRPs like, 4 T1 PKS and NRPs like, 2 T1
Talaromyces, PKS Indole, 1 NRPs like terpene, 3 NRPs T1 PKS, 1 Indole NRPs, 2 betalactone, 1 phosphonate, 1 fungal-RiPP T1
Biosynthetic gene clusters, PKS and 1 Other type. The analysis also predicted the occurrence of enzymes involved in the biosynthesis of some
Whole genome Sequence. of the important secondary metabolites such as Pyranonigrin, Squalestatin -S, Azanigerone -A, Asperterpenoid -A,
Napthopyrone, Clavaric acid, and Fusarin.

1. INTRODUCTION Whole genome sequencing (WGS) of fungi followed by genome

annotation offers an attractive option for the identitification of novel
Fungal endosymbionts are rich reservoirs for a wide variety of
and unexplored secondary metabolites and their biosynthetic genes.
phytochemicals and phytohormones [1]. Endophytes residing
Bioinformatic tools such as Funannotate and SMuRF assist researchers in
inside plant systems contribute to the production of numerous novel
genome pipeline assemblies, annotations and natural product discovery
compounds which have significant bioactive potential such as anti-
cancerous, anti-inflammatory, anti-leishmanial, anti-microbial, and through in silico strategies with precise, non-laborious, and rapid
anti-malarial properties [2-4]. Several of these compounds synthesized analysis. An important tool to identify Biosynthetic gene clusters (BGCs)
under biotic and abiotic stress conditions during interdependent in fungal genomes is Antibiotics and Secondary Metabolite Analysis
interaction between plants and their endophytes increases the survival Shell (AntiSMASH), which has been widely used to analyze several
value of both organisms. The fungus Talaromyces was first reported by fungal genomes for secondary metabolites. Several compounds from
mycologist Chester Ray Benjamin in 1955 under Trichocomaceae. The Talaromyces spp. have been extensively studied for their bioactivities.
genus exhibits tightly interwoven hyphae with soft, cottony, yellowish However, genome mining of the species has not been carried out so
fruit bodies (ascocarp) encompassing granules [5]. Talaromyces has far. In the present work, endophytic Talaromyces spp. isolated from
been reported to synthesize several important secondary metabolites, S. samarangense was subjected to WGS and genome mining using
including Huperzine A, a potent acetylcholine esterase inhibitor [6], AntiSMASH to identify any novel putative bioactive compounds.
and amestolkaloids, known for anti-inflammatory properties [7].
2. MATERIALS AND METHODS

2.1. Isolation and Sub-culturing of Endophytic Fungal Strain-YS-1

*Corresponding Author:
The explant material (S. samarangense) leaves and stems were
Shailasree Sekhar,
Division of Biochemistry, School of Life Sciences, JSS Academy of Higher collected from Vajamangala village, Mysore district. The explant
Education and Research, Sri Shivarathreeshwara Nagara, Mysuru, was thoroughly washed under running tap water. The leaf and stem
Karnataka, India. E-mail: shailasreesekhar@jssuni.edu.in bits were surface sterilized using 70% ethanol for 4 min, followed by

© 2024 Priyanka N. Shenoy, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
230 Shenoy, et al.: Journal of Applied Biology & Biotechnology 2024;12(3):229-235

sterile distilled water wash and treated with 1% sodium hypochlorite 2.5. Genome Mining for Natural Product Discovery
solution for 4 min. They were again washed with sterile distilled AntiSMASH tool: Fungal version 6.1.1 pipeline was used for the
water for 2–3 times, and the leaf bits were placed on solidified potato identification of potent secondary metabolite BGCs [16].
dextrose agar (PDA) media supplemented with ampicillin 100 mg/mL.
The Petri dishes were sealed with parafilmTM and incubated at 25 3. RESULTS AND DISCUSSION
± 2°C for 15 days with alternate light and dark cycles. Endophytic
fungal mycelia which were observed on leaf and stem explants, 3.1. YS-1 Strain Identification
were subcultured by point inoculation on PDA and potato dextrose The cultured fungal organism showed yellow-colored cottony mycelial
broth media. Samples were monitored for further mycelial growth mat with white dots covering the entire plate after being incubated
and were subjected to staining with lactophenol cotton blue in their for 15 days at 25 ± 2°C. Annotation and phylogenetic analysis results
sporulation state. Among the four endophytic fungal strains isolated showed that the organism belongs to Talaromyces spp. and the
from S. samarangense, strain YS-1 was selected for the present study. stereomicroscopic view of strain YS-1 was examined [Figure 1a and
and b].

2.2. Genomic DNA isolation and WGS of YS1 strain 3.2. Genome Assembly and Sequencing
DNA was isolated from the fungal mycelia by CTAB method [8]. The genome sequence of strain YS-1 was deposited in the NCBI GenBank
Isolated DNA was eluted with 50 µL of TE buffer and stored at −20°C. database (Accession No. PRJNA953512), and the Genome assembly
The purity of isolated DNA was determined using NanoDrop™ 2000C of Talaromyces spp. was accessed by assemblers such as Biosynthetic
spectrophotometer (Thermo Fisher Scientific). Genomic DNA samples spades, Spades, and Megahit. These assemblers were compared and
were electrophoresed on 1% agarose gel to check purity and integrity. analyzed using Quast (Quality assessment tool for genome assemblies).
The high-quality DNA of strain YS1 was sent for whole genome Megahit was found to be an effective assembler that provided data on an
sequence analysis to Hi-Gx360® Himedia Laboratories Pvt. Ltd., organism’s complex metagenomic assemblies. The whole genome size
Mumbai, India. The sample analysis was as follows: 250 ng of total of Talaromyces spp. was found to be 30.5Mb, consisting of 605 contigs
DNA was used as input for library preparation using QIASeq FX DNA (≥0 bp), 372 contigs (≥1000 bp), 208 contigs (≥5000 bp), 168 contigs
kit to fragment and obtain adapter ligated and indexed library as per (≥10000 bp), 149 (≥25000 bp), 125 contigs (≥50,000 bp) and the largest
manufacturer’s instructions. The indexed library was sequenced on an contig was found to be 1112360. The GC content was 46.74%, N50
Illumina Miseq by paired-end chemistry of 300 cycles. The parameters was 3.33Mb, N75 was 1.86Mb. L50 value was 30 and the L75 value
used to check the raw data quality are included in a MultiQC report was 65. The Funannotate analysis indicated the presence of 114tRNA,
and are used to understand the quantity of data obtained for each of the 12722 functional mRNA, 12721 CDS transcripts, and 12721 protein-
paired read files for an individual sample [9]. The fastp tool (v0.12.4) coding sequences in Talaromyces spp. YS-1 strain. The completeness
was used to remove adapter contamination [10]. of genome assembly, as measured by BUSCO, gave a score of 92.9%.

2.3. Genome Assembly and Annotations 3.3. Phylogenetic Analysis and Phylogenomic Tree
A de novo de-brujin graph-based assembly was carried out to assemble The phylogenetic analysis of the Talaromyces spp. sequenced in the
the short reads into larger stretches of DNA called contigs. These present study (YS-1 Strain) was compared with the isolates available
contigs are the starting material used for executing a genome annotation in the database. Three groups could be identified in the phylogenetic
that assigns functions to various regions of the genome of the organism
in question. Quality assessment for genome assemblies generated by
Spades, bioSpades, and Megahit assemblers was performed using
the Quast tool [11-13]. It was noticed that the assembly generated by
the megahit assembler had a comparatively higher number of contigs
with sizes >10kb. Hence, assembly generated in bioSpades mode used
for phylogenomics analysis and assembly generated using megahit
assembler was masked and further used for annotations and natural
product discovery.

A new pipeline carried out fungal gene structural and functional

a b
annotation, Funannotate, which uses several gene predictors such as
glimerHMM, Augustus, SNAP, and Coding-Quarry to improve its Figure 1: (a) Mycelial growth on potato dextrose agar plate.
prediction [14]. Proteins, transcripts, and nucleic acid annotation were (b) Stereomicroscopic view of YS-1 strain.
executed by using software such as MEROPS (version 12), UniProt
(version 2022_03), dbCAN (version 11), Pfam (version 35), RepeatsDB
(version 1), GO (version 2022-07-01), MIBiG (version 1.4), InterPro
(version 90), gene2product (version 1.8).

2.4. Phylogenomic Analysis

The Phylogenomic analysis was carried out by using the Benchmarking
Universal Single-Copy Orthologs (BUSCO) quantitative genome
analysis tool which basically compares different genomic data sets
containing whole, identical and disintegrated genes based on gene
prediction strategies [15]. Figure 2: Phylogenomic tree of fungal Talaromyces strain YS-1.
 Shenoy, et al.: Genome mining of Talaromyces sp. for BGCs 2024;12(3):229-235 231

tree, with the Talaromyces YS-1 strain forming a close relationship applications in pharmaceuticals and drug discovery can be analyzed
with Talaromyces amestolkiae, Talaromyces marneffei, and using this comprehensive analysis tool for the fungal genome.
Talaromyces stipitatus in the first group. The second group consisted
AntiSMASH analysis showed the presence of 76 secondary metabolite
of Talaromyces proteolyticus and Talaromyces rugulosus. The other
BGCs, which include 28 T1Polyketide synthases, 10 Nonribosomal
species Talaromyces atroroseus, formed a separate group [Figure 2].
peptide synthetases (NRPSs), 9 terpene, 1 Indole, 12 NRPs like 4 T1
Polyketide synthase (T1 PKS) and NRPs like, 2 T1 PKS Indole, 1
3.4. Genome Mining for Natural Product Discovery NRPs like terpene, 3 NRPs T1 PKS, 1 Indole NRPs, 2betalactone 1
AntiSMASH provides information about the secondary metabolite phosphonate, 1 fungal-RiPP T1 PKS and 1 Other type [Table 1]. Over
genes by comparing the query gene cluster with the BGC library. 40% of these BGCs indicated gene homologies with known clusters in
Potential gene clusters of certain secondary metabolites such as the MIBiG database. Several clusters responsible for coding bioactive
terpenes, alkaloids, and phenolic compounds having industrial compounds such as Pyranonigrin-A, Alternariol, Asperterenoid-A,

h
Figure 3: Comparison of BGCs in Talaromyces spp. YS-1 (a-h) with identified BGCs for biosynthesis of various bioactive compounds (ⅰ-ⅷ).
232 Shenoy, et al.: Journal of Applied Biology & Biotechnology 2024;12(3):229-235

Table 1: Predicted secondary metabolite biosynthetic gene clusters of strain YS-1.

Region From To Type Most similar known cluster
Region 1.1 27,682 357,538 T1PKS Pyranonigrin E (100%)
Region 1.2 710,748 273,103 T1PKS Unknown
Region 2.1 126,776 425,900 T1PKS Terretonin (50%)
Region 2.2 498,524 46,806 NRPS-like Unknown
Region 2.3 681,748 149,220 T1PKS, NRPS-like Zearalenone (18%)
Region 3.1 291,287 267,110 T1PKS, indole Trypacidin (35%)
Region 3.2 454,298 424,526 T1PKS Viriditoxin (22%)
Region 3.3 736,454 271,795 T1PKS Tricholignan A (50%)
Region 4.1 5,132 141,371 T1PKS Unknown
Region 5.1 169,403 349,214 T1PKS Alternariol (100%)
Region 5.2 358,147 224,543 NRPS-like, terpene Squalestatin S1 (60%)
Region 5.3 689,574 171,848 NRPS-like Unknown
Region 6.1 641 74,424 NRPS Unknown
Region 6.2 316,319 122,148 T1PKS Unknown
Region 6.3 605,092 179,953 NRPS, T1PKS Emericellamide A/emericellamide B (40%)
Region 7.1 86,321 155,050 T1PKS, indole Unknown
Region 8.1 108,656 127,159 T1PKS Unknown
Region 8.2 429,656 35,955 indole Fumigaclavine C (45%)
Region 9.1 117,817 133,974 NRPS-like Unknown
Region 10.1 114,007 51,896 NRPS-like Unknown
Region 10.2 208,461 48,783 NRPS-like Unknown
Region 10.3 531,819 174,717 NRPS-like Unknown
Region 10.4 598,279 61,197 other Unknown
Region 11.1 257,617 139,510 NRPS-like Unknown
Region 11.2 491,748 72,951 T1PKS Azanigerone A (26%)
Region 13.1 268,179 210,071 Terpene Asperterpenoid A (100%)
Region 14.1 64,395 71,552 NRPS Unknown
Region 14.2 515,735 185,810 Betalactone Unknown
Region 15.1 350,690 147,843 Terpene Unknown
Region 16.1 411,349 176,667 NRPS Unknown
Region 17.1 277,101 48,170 NRPS Unknown
Region 18.1 1 154,701 NRPS Unknown
Region 18.2 113,116 43,527 T1PKS Unknown
Region 20.1 152,443 119,563 NRPS, T1PKS curvupallide-B (11%)
Region 20.2 298,332 19,116 T1PKS Griseofulvin/epidechlorogriseofulvin/norlichexanthone/
dehydrogriseofulvin/4-desmethylgriseofulvin/griseoxanthone B (14%)
Region 21.1 225,214 49,633 T1PKS Citrinin (31%)
Region 21.2 382,522 82,811 Indole, NRPS Clapurines (27%)
Region 22.1 1613 76,023 T1PKS Unknown
Region 22.2 87,957 45,887 T1PKS Azanigerone A (26%)
Region 22.3 219,037 48,863 T1PKS Ankaflavin/monascin/rubropunctatine/monascorubrin (29%)
Region 22.4 390,717 46,696 NRPS Unknown
Region 24.1 206,270 31,408 T1PKS Unknown
Region 28.1 99,615 357,538 T1PKS Fujikurin A/fujikurin B/fujikurin C/fujikurin D (66%)
Region 29.1 302,469 273,103 T1PKS Unknown
Region 31.1 179,642 425,900 NRPS Pyranonigrin E (100%)
Region 33.1 123,646 46,806 T1PKS Unknown

(Contd...)
 Shenoy, et al.: Genome mining of Talaromyces sp. for BGCs 2024;12(3):229-235 233

Table 1: (Continued).
Region From To Type Most similar known cluster
Region 34.1 52,652 149,220 Terpene Unknown
Region 37.1 78,840 267,110 NRPS-like Unknown
Region 38.1 128,355 424,526 NRPS, T1PKS Unknown
Region 39.1 111,254 271,795 NRPS-like Unknown
Region 40.1 105,708 141,371 Terpene Unknown
Region 41.1 1 349,214 Phosphonate Unknown
Region 41.2 90,852 224,543 NRPS-like Unknown
Region 47.1 27,647 171,848 Betalactone Unknown
Region 48.1 2189 74,424 T1PKS Naphthopyrone (100%)
Region 49.1 67,645 122,148 fungal-RiPP, T1PKS Ankaflavin/monascin/rubropunctatine/monascorubrin (20%)
Region 54.1 13,165 179,953 T1PKS Pyranonigrin E (100%)
Region 54.2 116,565 155,050 Terpene Unknown
Region 57.1 19,253 127,159 T1PKS, NRPS-like Unknown
Region 59.1 196,661 35,955 Terpene Unknown
Region 60.1 49,877 133,974 Terpene Sordarin (11%)
Region 63.1 163,422 51,896 Terpene Clavaric acid (100%)
Region 67.1 92,953 48,783 T1PKS Chrysoxanthone A/chrysoxanthone B/chrysoxanthone C (8%)
Region 68.1 132,828 174,717 NRPS-like Unknown
Region 69.1 4601 61,197 T1PKS Melanin (100%)
Region 69.2 105,785 139,510 T1PKS Fusarin (100%)
Region 71.1 502 72,951 NRPS Unknown
Region 71.2 73,873 210,071 NRPS Unknown
Region 73.1 1 71,552 Terpene Unknown
Region 97.1 1 185,810 T1PKS Sorbicillin (71%)
Region 98.1 21,324 147,843 T1PKS, NRPS-like Shearinine D (9%)
Region 107.1 27,684 176,667 T1PKS Chrodrimanin B (100%)
Region 127.1 3370 48,170 NRPS-like Unknown
Region 129.1 3888 154,701 T1PKS Depudecin (33%)
Region 130.1 1 43,527 NRPS Unknown
Region 139.1 1 119,563 T1PKS, NRPS-like Fusarin (100%)

Naphthopyrone, Clavaric acid, Melanin, and Fusarin showed 100% from Hypholomas ublateritium (Gen Bank EU665687.1) for synthesis
similarity. of Clavaric acid, a potential anti-tumor compound [21] [Figure 3e].
Biosynthetic cluster region 69.1 showed homology with Melanin
AntiSMASH analysis of Talaromyces spp. strain YS-1indicated that the BGC [Figure 3f]. Melanin is found predominantly in PKS clusters of
gene clusters in the regions 1.1, 31.1, and 54.1 has significant similarity Fungi and is useful for adjusting to different environmental conditions
with the BGCs of Pyranonigrin E from Aspergillus niger ATCC 1015 [22]. The fungal YS-1 strain in regions 69.2 and 139.1 encodes BGC
(GenBank: ACJE01000019.1.) [Figure 3a]. Pyranonigrin- E, isolated with BLAST hits for Fusarin BGC from Fusarium fujikuroi (Gen
from Aspergillus niger, is a pyranopyrrole having effective anti-oxidant Bank: JX308619.1) [Figure 3g]. Fusarin BGCs were been identified
and radical scavenging activities [17]. BGC region 5.1 noted significant in Fusarium spp., along with different polyketide gene clusters [23].
BLAST hits with Alternariol from Parastagono sporanodorum SN15 BGC region 107.1 of fungal YS-1 strain presented 100% similarity with
(GenBank: KP941080.1) [Figure 3b]. Alternariol is reported to be BGC from Talaromyces verruculosus (Gen Bank: LC422696.1) meant
an effective anti-cancerous and antibacterial metabolite [18]. BGC for biosynthesis of secondary metabolite chrodrimanin B [Figure 3h].
13.1 exhibited high similarity with known clusters implicated in Different classes of secondary metabolites and a new monoterpenoid
the synthesis of Asperterpenoid-A from Talaromyces wortmannii Chrodrimanin-T have been reported from T. amestolkiae [24].
(GenBank: MK140602.1) [Figure 3c]. Asperterpenoid-A has effective Chrodrimanin-B- B isolated from Talaromyces funiculosus showed a
anti-inflammatory and antibacterial activities [19]. BGCs found in broad range of anti-bactericidal activity against Staphylococcus aureus,
region 48.1 of the YS-1 strain showed similarity with Naphthopyrone Mycobacterium smegmatis, Mycobacterium phlei, Micrococcus
BGC from Aspergillus nidulans FGSC A4 (Gen Bank: BN001302.1) tetragenus and Escherichia coli [25]. In addition, several other
[Figure 3d]. Li et al. [20] have reported that naphthopyrone is bioactives and Antibiotics such as Terretonin, Zearalenone, Trypacidin,
a potential antiproliferative compound produced from A. niger. Viriditoxin, Tricholignan -A, Squalestatin -S, Emericellamide -A,B,
A notable BLAST hit for the BGC region 63.1 was similar to BGC Fumigaclavine -C, Azanigerone -A, Curvupallide -B, Griseofulvin,
234 Shenoy, et al.: Journal of Applied Biology & Biotechnology 2024;12(3):229-235

Citrinin, Clapurines, Fujikurin A,B,C,D, Ankaflavin, Sordarin, experiments and collected the data. PSN, SB, SS and KRK analysed
Chrysoxanthone -A,B,C, Sorbicillin, Shearinine -D, and Depudecin the data and finalized the manuscript. All authors have read and
have been identified in the present study. approved the final manuscript.

Earlier reports have indicated that Talaromyces species produce

7. CONFLICT OF INTEREST
several important bioactive compounds. Quinoid compounds such
as erythroskirin, islandicin, skyrin, luteoskyrin, regulosin and The authors report no financial or any other conflicts of interest in this
work.
anthroquinone exhibiting antitumor and antioxidant activities were
identified in Talaromyces islandicus [26]. This species also produces 8. ETHICAL APPROVALS
mycotoxins and cyclochlorotine, with hepatotoxic and mutagenic
activities. Several isocoumarin derivatives and benzofurones identified This study does not involve experiments on animals or human
in the endophytic fungus T. amestolkiae exhibited antibacterial and subjects.
α-glucosidase inhibitory activities [27,28].
9. DATA AVAILABILITY
Genome fungiSMASH analysis of T. islandicus detected 19 different
types 1 PKS, anthrol reductase BGCs involved in the synthesis of The data generated and analyzed are included within this research article.
potent alkaloid anthroquinone [29]. Talaropeptins A&B synthesizing
NRPs gene cluster was identified in a marine isolate of Talaromyces 10. PUBLISHER’S NOTE
purpureogenus CX11 [30]. These compounds had antifungal activity.
This journal remains neutral with regard to jurisdictional claims in
Li et al. [31] have reported the presence of Talaromycolides in
published institutional affiliation.
Talaromyces pinophilus, which showed antibacterial activity toward
S. aureus resistant to methicillin.
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