1. Introduction
Bioactive metabolites are mostly produced from various genera of actinomycetes, including members of
Streptomyces,
Actinomadura,
Nonomuraea,
Micromonospora, and
Verrucosispora [
1].
Streptomyces is the most important and dominant genus within the actinomycete groups. This genus belongs to the family
Streptomycetaceae, which have a high G+C content in their DNA and form extensively branched substrate and aerial mycelia that later differentiate into spore chains [
2].
Streptomyces can be differentiated from closely related genera by the presence of
LL-diaminopimelic acid (
LL-DAP) in the cell-wall peptidoglycan, but without diagnostic sugars in a whole-cell hydrolysate [
3]. Streptomycetes are widely distributed in nature and can be found everywhere, including in terrestrial soils and marine samples [
4,
5]. In addition, some
Streptomyces strains are associated with plants, lichens, and insects [
6,
7,
8]. Streptomycetes are a major source of bioactive metabolites, with more than 70% of known antibiotics being derived from this genus [
1]. Streptomycetes have become very useful in the search for bioactive metabolites because they can produce different chemical core-structures, such as macrolides, polyketides, terpenes, and non-ribosomal peptides [
9].
Cancer is one of the leading causes of death. According to the Global Cancer Statistic 2020, it was estimated that in 2020 there were 19.3 million new cancer cases and 10.0 million deaths from cancer [
10].
Streptomyces species are a source of small-molecule anticancer compounds. According to a genome mining study, the genomes of streptomycetes harbor a variable distribution of antitumor biosynthetic gene clusters (BGCs) and so these bacteria are a promising source of molecules for antitumor drug discovery [
9]. Examples of anticancer drugs derived from streptomycetes are actinomycin, bleomycin, and doxorubicin [
11].
Peat swamp forests are found mainly in Southeast Asia. These ecosystems represent a unique character, distinct from other soil habitats, in terms of their acidic and waterlogged conditions [
12]. Peat swap forest soils harbor a large amount soil microbes and have been recognized as a promising source of new actinobacteria. In the past few decades, several novel actinobacteria, including
Dactylosporangium sucinum,
Streptomyces actinomycinicus, and
Nocardia rayongensis, have been isolated from these ecosystems [
13,
14,
15]. In this present study, we describe a taxonomic study of the new
Streptomyces strain RCU-064
T based on a polyphasic approach. The bioactive metabolites produced by the strain were elucidated using column chromatography, including Sephadex LH-20 column and high-performance liquid chromatography (HPLC) for purification of the compound, and nuclear magnetic resonance (NMR) and mass spectral analyses for structure determination. The isolated compounds were analyzed for their (i) cytotoxicity against cell lines derived from oral human epidermoid carcinoma (KB), human breast cancer (MCF-7), and human small cell lung cancer (NCI-H187), (ii) anti-malarial activity against the multidrug-resistant
Plasmodium falciparum K-1, and (iii) anti-bacterial and anti-fungal activities.
3. Discussion
Based on the results of this polyphasic approach and genomic evidence, it is clear that strain RCU-064
T is a novel species in the genus
Streptomyces, for which the name
Streptomyces rugosispiralis sp. nov. is herein proposed. Previous studies have highlighted the presence of novel
Streptomyces in various ecological niches, including soil environments. For instance,
Streptomyces have been isolated from diverse soil types worldwide, including agricultural, forest, and grassland soils [
19,
20,
21]. Although actinomycetes have been isolated from soil for a century, novel species are still being reported from this type of habitat [
4], including peat swamp forest soils. In the past decade, several novel species have been isolated from peat swamp forest soils in Thailand. For example,
Actinomadura rayongensis, Amycolatopsis acidicola, Dactylosporangium sucinum, Nocardia rayongensis, Nonomuraea rhodomycinica, Streptoyces actinomycinicus, Streptomyces acididurans, and
Streptomyces humicola [
13,
14,
15,
22,
23,
24,
25]. These studies underline the adaptability of actinobacteria to different soil conditions and their significant contribution to the microbial diversity in terrestrial ecosystems. In addition, this indicates the unique characteristic of peat land harbors a high diversity of novel actinomycetes.
It is important to note that the physicochemical characteristics of peat swamp forest soil are unique due to its specific habitat. Peat swamp soils are typically characterized by a high organic matter content, acidic pH, waterlogged conditions, and low nutrient availability [
26]. These distinct conditions pose challenges for actinomycetes isolation, as certain species may have specific growth requirements or preferential adaptation to different environmental parameters.
In terms of the isolation of actinomycetes, there are a number of essential factors to consider. The selection of suitable isolation techniques is essential for capturing a diverse population. Common techniques include serial dilution, plating on selective media supplemented with particular carbon or nitrogen sources, and altering the pH conditions [
27]. These selective methods promote the development and isolation of actinomycetes, such as
Streptomyces, while inhibiting the growth of competing microorganisms.
Actinomycetes are renowned for their remarkable ability to produce a diverse array of secondary metabolites, many of which possess significant bioactive properties. These bioactive compounds have attracted considerable attention in various fields, including pharmaceutical, agricultural, and industrial applications. Among the numerous secondary metabolites produced by actinomycetes, a key precursor in the biosynthesis of several important classes of compounds is 3-amino-5-hydroxybenzoic acid (AHBA).
Importantly, AHBA serves as a starter unit or building block in the assembly of polyketide or nonribosomal peptide backbones, leading to the formation of diverse bioactive compounds [
28]. It can be classified into three distinct structural classes. The predominant class comprises ansamycins, wherein AHBA acts as a starter unit for the synthesis of a polyketide chain, leading to the formation of a macrocyclic lactam. Another class includes the mitomycins, which involve the combination of AHBA with an aminosugar component, resulting in the formation of unique tricyclic structures. Lastly, the saliniketals constitute a third class, characterized by being “degraded ansamycins,” although this class has only been observed so far in saliniketal compounds [
28].
The ansamycins are a family of polyketides that contain naphthalene/benzene or napthaquinone/benzoquinone rings that are connected at nonadjacent positions by an aliphatic chain. These natural compounds exhibit a variety of biological activities and have clinical applications [
29]. Many reports have revealed that they have a wide range of biological activities, including anticancer, antiviral, and antibacterial effects. These compounds are distinguished by the presence of a macrocyclic system that is comprised of an aromatic moiety embedded in an alicycle, which has drawn considerable interest from chemical synthesis and biosynthetic researchers [
30].
In this study, two classes of bioactive compounds—ansamycin and cyclic peptides—were isolated from strain RCU-064
T. Geldanamycin (
1) is the benzoquinone ansamycin, first isolated from the culture broth of
Sreptomyces hygroscopicus [
31]. It exhibited moderate antimicrobial activity against bacteria and fungi with minimum inhibition concentration (MIC) values ranging from 4 to >100 μg/mL. Geldanamycin has been reported to have in vivo anti-parasitic activity against
Syphacia oblevata but not against
Plasmodium berghei [
31]. However, in this study, the antimicrobial activity against
Bacillus cereus and
Mycobacterium tuberculosis of all compounds was negative (MIC > 50 μg/mL). This difference may be attributed to the variation in the tested microorganisms between these studies.
Geldanamycins (
1) are potent anticancer and antifungal agents, exhibiting their anticancer activity through the inhibition of heat shock protein 90 (Hsp90), a key chaperone protein involved in the stabilization of cancer cell growth and survival [
32,
33,
34]. By inhibiting the function of Hsp90, geldanamycin (
1) disrupts multiple signaling pathways involved in cancer progression, leading to cell cycle arrest and apoptosis [
35]. Previous work showed that geldanamycin (
1) inhibited the growth of HPV-18-positive HeLa cells, which are a type of cervical cancer cell line, and also showed antifungal activity against
Setosphaeria turcica plant pathogens [
36].
Geldanamycin (
1) and its derivatives have been reported to be produced as secondary metabolites in several
Streptomyces species. In Thailand,
Streptomyces sp. PC4-3 was isolated from a soil sample collected at Samed Island, Rayong province, Thailand, using starch-casein nitrate agar. The culture broth of
Streptomyces sp. PC4-3 was extracted with ethyl acetate, concentrated under low pressure to obtain the crude extract, and then subjected to silica gel flash column chromatography and NMR spectroscopic analysis, revealing the active component to be geldanamycin (
1) [
37]. Likewise, the crude extracts of
Streptomyces sp. BCC71188, which was isolated from a soil sample at Nakhon Si Thammarat Province, Thailand, were subjected to Sephadex LH-20 column chromatography followed by HPLC to yield 19 compounds, including 17-O-demethylgeldanamycin (
2) [
38].
Since geldanamycin (
1) has demonstrated cytotoxic effects, numerous attempts have been made to identify alternative effective anticancer drugs derived from its molecular structure. These efforts have involved the preparation and biological evaluation of many semi-synthetic derivatives of geldanamycin, often involving modifications at the C-17 position [
39,
40]. Despite these attempts, an anticancer agent based on the geldanamycin pharmacophore has yet to be approved for clinical use [
41]. However, previous research has reported the production of 17-O-demethylgeldanamycin (
2) by
Streptomyces DEM20745. This is of particular interest due to its potential as a starting material for the development of new semi-synthetic geldanamycin derivatives for clinical evaluation (41). For example, 17-arylgeldanamycins, synthesized via a triflation/Suzuki coupling approach using synthetic 17-O-demethylgeldanamycin, have been shown to have potent inhibition of Hsp90 [
42]. Nevertheless, the limited availability of 17-O-demethygeldanamycin (
2) has restricted further synthetic work in this area [
43]. Therefore, it is of interest that our results revealed the production of 17-O-demethyl-geldanamycin (
2) as a natural product by
Streptomyces RCU-064
T, which provides future development of a production strain for this potentially valuable compound.
Reblastatin (
3) and 17-Demethoxyreblastatin (
4) are phenolic analogues of geldanamycin. Screening for novel compounds with the ability to inhibit phosphorylation of the retinoblastoma protein revealed reblastatin (
3) [
44], isolated as a minor constituent from the cultivation of
Streptomyces hygroscopicus, which is also known for producing the potent Hsp90 disruptor geldanamycin (
1) [
31,
45]. In the original study, reblastatin (
3) demonstrated significant inhibition of the proliferation of the human histiocytic lymphoma U-937 cell line, with an IC
50 value of 0.43 μg/mL [
44]. Furthermore, it was found to exhibit a potent inhibitory activity in a cell-based oncostatin M signaling assay, with an IC
50 value of 0.16 μM [
46].
17-Demethoxyreblastatin (
4) is a derivative of reblastatin (
3), a natural product originally isolated from
Streptomyces species. It is structurally related to reblastatin (
3), differing in the presence of a demethoxy group at the C-17 position [
45]. This modification alters the chemical properties and potentially affects the biological activities of the compound. Evaluations of its cytotoxic potential have revealed inhibitory effects on cancer cell growth and proliferation, similar to those observed with reblastatin (
3). However, some research findings indicate that 17-demethoxyreblastatin (
4) displayed unique biological characteristics when compared to reblastatin (
3). The specific potency, selectivity, and mechanisms of action may vary between these two compounds due to the structural alteration [
47].
Some
Streptomyces strains have been reported to produce reblastatin (
3) and 17-demethoxyreblastatin (
4). The extraction of crude compounds from
Streptomyces hygroscopicus JCM4427 using ethyl acetate, followed by purifying using octadecyl silica column chromatography and HPLC, revealed the presence of reblastin (
3) and 17-demethoxyreblastatin (
4) that exhibited Hsp90 ATPase inhibition activity with IC
50 values of 0.32 μM and 1.82 μM, respectively, [
48]. Despite 17-demethoxyreblastatin (
4) being a derivative of reblastatin (
3), there is limited literature available regarding its production from
Streptomyces species. This scarcity of reports can be attributed to variations in the expression of the BGCs among different
Streptomyces species [
32].
Nocardamine (
5), also called desferrioxamine, is a cyclic peptide siderophore found in several species of bacteria, including
Nocardia, Pseudomonas, and
Streptomyces species [
49]. Siderophores are compounds synthesized by microorganisms to scavenge and bind iron from the surrounding environment. They act as an iron chelator, facilitating the uptake and utilization of this crucial nutrient by bacteria that require it for their growth and viability. Siderophores exhibit a strong affinity for iron and form stable complexes with the metal, effectively preventing its precipitation or interaction with other molecules present in the environment. Clinically, the nocardamine-type siderophore, Desferol, is used for the treatment of iron intoxication [
50,
51,
52]. In addition, dehydroxynocardamine (
6) is a derivative that is structurally related to nocardamine (
5), with the key difference being the absence of hydroxyl groups in its chemical structure [
53].
Nocardamine (
5) was originally isolated as an antibacterial metabolite from a
Nocardia strain [
52], while nocardamine (
5) and dehydroxynocardamine (
6) were isolated from the culture broth of a marine-derived
Streptomyces isolated from a sponge. Later, both compounds were also reported from soil
Streptomyces sp. strain TS-2-2 and
Streptomyces sp. BCC71188 [
37,
54]. This indicates that the ability to produce nocardamine (
5) could be found in both terrestrial and marine
Streptomyces species. In this study, nocardamine (
5) and dehydroxynocardamine (
6) were both inactive in the antimicrobial activity test at a concentration of 50 μg/mL, in accord with a previous report [
49] that found no antimicrobial activity of both compounds at a final concentration of up to 200 μg/mL. However, in another study, nocardamine (
5) showed a weak antimicrobial activity against
E. faecium and
B. subtilis but no activity against
V. alginolyticus and
C. albicans [
55]. Moreover, in the same study, nocardamine (
5) did not inhibit cell proliferation of tumor cell lines, including T-47D, SK-Mel-5, SK-Mel-28, and PRMI-7951, but did inhibit their colony formation [
55]. Furthermore, the genetic engineering of
Streptomyces atratus SCSIO ZH16, a deep-sea-derived
Streptomyces, by in-frame deletion to activate putative orphan gene clusters, led to the production of new compounds, including nocardamine (
5) [
52].
In the analysis of BGCs, the detection of NRPS-like and T1PKS similar to the previous known BGCs of geldanamycins and the detection of desferrioxamine in the genome of strain RCU-064T supported that strain RCU-064T is the producer of geldanamycins (1) and nocardamines (5). This highlights the remarkable capacity of this genus to function as a potential source for drug production and a rich source of bioactive metabolites. The discovery and production of actinobacterial metabolites has been facilitated by advances in genome sequencing, bioinformatics, and genetic engineering techniques. These approaches have enabled the identification and manipulation of the BGCs responsible for compound synthesis, allowing researchers to optimize system production, enhance yields, and generate novel derivatives with improved properties. Moreover, the exploration of diverse environments, such as soil, marine sediments, and plant-associated microbiomes, has led to the discovery of novel Actinobacterial strains and expanded the diversity of known actinobacterial metabolites.