It is believed that actinobacteria can adapt highly to harsh mangrove conditions by developing unique metabolic pathways, which can provide novel secondary metabolites (Tan et al. 2017). Of note, one of the less explored niches in the mangrove environments is the mangrove plants such as
Instead of traditional methods that have considerably slowed the chance of finding new compounds, whole-genome sequencing and genome mining have paved a new way to exploit the biosynthetic potential of bioactive
In addition, most BGCs remain poorly characterized and are silent under laboratory culture conditions. A novel anti-HIV compound streptoketides from soil
However, genes encoding functional proteins involved in the biosynthesis of these plant-derived compounds have not been exploited yet. More and more
In this study, we characterized biological activities and sequenced the genome of
Following the manufacturer’s protocol, the genomic DNA of strain SX6 was extracted using G-spin™ Total DNA Extraction Mini Kit (Intron Bio, Korea). PCR amplification for the 16S rRNA gene was performed as described previously (Quach et al. 2021). The identification of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities were carried out on the EzTaxon server (Chun et al. 2007). The phylogenetic tree was built by the maximum-likelihood method using Molecular Evolutionary Genetics Analysis (MEGA) software version 7 with Kimura-2-parameter distances.
Ethyl acetate was used to extract secondary metabolites from strain SX6 following the procedure described previously (Nguyen et al. 2019b). In brief, the mixture of cell-free supernatant:ethyl acetate (1:1 ratio) was vigorously shaken for 30 min and kept stationary 60 min until the separation of aqueous and organic phases. The organic phase was evaporated on the rotary evaporator (Scilogex RE100-Pro, USA) at 55°C and 80 ×
where A
where A
Heatmap presenting antibacterial activity against at least one tested pathogenic bacteria of endophytic actinobacteria isolated from
BC –
When grown on ISP1-7 media, the aerial mycelium of isolate SX6 formed monopodial branched hyphae and was well-developed with white color, while substrate mycelium was pale yellow (Table SII). The yellow pigment was observed in the ISP2 agar on which this isolate grew at the maximum level under cultivation temperature of 30°C, pH 7.0, and 1% NaCl. Spiral spore chain and warty spore surface were observed by SEM (Fig. 2A). In addition, the isolate SX6 assimilated various carbon sources such as glucosamine, fructose, sorbitol, trehalose, mannose but not myo-inositol, mannitol, and raffinose. Enzymatic tests revealed the production of cellulase, chitinase, protease, and xylanase (Table SII).
Identification of endophytic strain SX6.
A) Scanning electron microscopy of hyphae of strain SX6 grown on ISP2 medium; B) phylogenetic tree based on 16S rRNA gene sequences of strain SX6 and closely related
BLAST search of the 16S rRNA gene sequence of SX6 showed the highest similarity to
Biological activities determined in the
A) Selective antibacterial activity against MRSE ATCC® 35984™ and
DPPH and hydroxyl radical scavenging assays
Features of the SX6 and other
Species | Size (Mb) | GC (%) | Genes | CDSs | tRNAs | rRNAs |
---|---|---|---|---|---|---|
7.69 | 72.8 | 6,952 | 6,779 | 68 | 11 | |
7.69 | 71.5 | 6,881 | 6,866 | 66 | 3 | |
7.15 | 72.7 | 6,951 | 6,773 | 66 | 18 | |
7.74 | 72.5 | 7,030 | 6,832 | 66 | 3 |
For functional annotation, 6,779 CDSs (99.2%) were assigned to 21 functional categories. Almost all CDSs were associated with functions, including general function (1,090 genes), transcription (553 genes), amino acid transport and metabolism (427 genes), carbohydrate transport and metabolism (408 genes), and energy production and conversion (314 genes) (Fig. 4A).
Genome characterization of
A) Clusters of Orthologous Groups (COGs) of protein functions; B) whole genome-based phylogenetic classification of
The species status of strain SX6 was further confirmed by
Six gene clusters, including geosmin, albaflavenone, isorenieratene, hopene, sapB, and ectoine were identified comprising 100% of the genes from the know cluster. Clusters with 60–90% similarity included melanin (60%), citrulassin (60%), spore pigment (66%), desferrioxamin (83%), and coelichelin (90%) (Table SIII). Notably, cluster 2 with a predicted similarity of 67% to BGC of actinomycin D, a well-known antibiotic with high antibacterial and cytotoxic activities (Liu et al. 2016), was a hybrid cluster containing 11 genes homologous to
In addition, the largest cluster in the SX6 genome, cluster 19, showed moderate similarity at 48% to the BGC of streptovaricin from
Cluster 25 was predicted as a complex of NRPS, T1PKS, and other genes that also exhibited a similarity of 48% with the known polyoxypeptin BGC of
In supporting of the genomic finding, HPLC-DAD-MS analysis revealed the presence of daidzein in the extract of SX6 at the retention time of 9.103 min, which was relatively similar to the retention time of the standard daidzein compound (8.897 min) (Fig. 6C). Additionally, genistein was detected based on a 12.323-min retention time. Further confirmation by MS analysis showed that MS spectra of SX6 extract revealed two distinct MS peaks [M + H]+ = 255.44 m/z and [M + H] + = 271.40 m/z, corresponding to standard daidzein and genistein (Fig. SI and SII). The UV absorption spectra for the SX6 extract also displayed similar peaks with standard daidzein and genistein.
Cryptic secondary metabolite biosynthetic gene clusters identified in the genome of
Biosynthetic pathway of daidzein and genistein in
A) Using genome mining, the proposed biosynthetic pathways of daidzein and genistein present in
The strain SX6 belongs to the
Genomic analysis of
In addition, clusters 19 and 25 were identified as hybrid BGCs with low similarities with streptovaricin and polyoxypeptin BGCs, respectively. Compared to the reference streptovaricin cluster, a structurally-related macrolide antibiotic with a cluster size around 95 kb comprising 41 open reading frames (Liu et al. 2020; Luo et al. 2022), cluster 19 contained duplicates of five genes encoding type I modular PKSs responsible for the streptovaricin backbone. Likewise, cluster 25 only contained core genes involved in the production of polyoxypeptin a potent apoptosis inducer (Balachandran et al. 2014). The genome-wide comparison revealed that streptovaricin was only predicted in the
A highlight of this study was the presence of the biosynthetic pathways of plant-derived compounds, including daidzein and genistein. Daidzein and genistein are representative compounds of isoflavones found in plants, especially legumes (Sohn et al. 2021). Various
In this study, we reported for the first time a broad range of biological activities and the complete genome information for