Lichen-Derived Actinomycetota: Novel Taxa and Bioactive Metabolites

Abstract

Actinomycetes are essential sources of numerous bioactive secondary metabolites with diverse chemical and bioactive properties. Lichen ecosystems have piqued the interest of the research community due to their distinct characteristics. Lichen is a symbiont of fungi and algae or cyanobacteria. This review focuses on the novel taxa and diverse bioactive secondary metabolites identified between 1995 and 2022 from cultivable actinomycetota associated with lichens. A total of 25 novel actinomycetota species were reported following studies of lichens. The chemical structures and biological activities of 114 compounds derived from the lichen-associated actinomycetota are also summarized. These secondary metabolites were classified into aromatic amides and amines, diketopiperazines, furanones, indole, isoflavonoids, linear esters and macrolides, peptides, phenolic derivatives, pyridine derivatives, pyrrole derivatives, quinones, and sterols. Their biological activities included anti-inflammatory, antimicrobial, anticancer, cytotoxic, and enzyme-inhibitory actions. In addition, the biosynthetic pathways of several potent bioactive compounds are summarized. Thus, lichen actinomycetes demonstrate exceptional abilities in the discovery of new drug candidates.

1. Introduction

Lichens form important symbiotic communities in the ecosystem and are characterized by a symbiotic association between fungi and algae. They occupy 8% of the earth’s surface [1]. Some bioactive compounds, such as usnic acid, gyrophoric acid, diffractaic acid, polysaccharides, anthraquinones, and terpenes, have been isolated from lichens, and some of these compounds have been employed in clinical treatments [2]. Organisms with a slower growth rate reportedly exhibit strong resistance to external secondary metabolism [3]. Further, organisms that move slowly and thrive in low-resource environments produce large amounts of defensive metabolites for protection against their many predators. Lichens and their symbiotic organisms, especially actinomycetes, grow slowly. They are natural habitats for the production of beneficial bioactive compounds or metabolites.

Antibiotics produced by microorganisms contribute significantly to human health. Actinomycetes are an essential resource for the discovery of drug-lead compounds. Actinomycete drug resources have been utilized and developed for many years. Hence, identifying new active structural substances has become increasingly difficult [4]. Lichens are a unique group of organisms formed by symbioses between fungi and algae or cyanobacteria [5]. The wide variety of lichens can provide new sources of actinomycetes for use in the discovery of novel drugs [6]. Only a few national and international research groups have investigated actinomycete lichen resources. Moreover, few groups have reported active metabolites derived from actinomycetes.

Lichen-derived actinomycetota are potent producers of bioactive metabolites. This review summarizes the compounds isolated from lichen-derived actinomycetes. The compounds are classified into 11 types based on their different structures. Some secondary metabolites exhibit various biological activities, such as anti-inflammatory, antimicrobial, anticancer, cytotoxic, and enzyme-inhibitory activities. The chemical structures of 114 secondary metabolites isolated from lichen-associated actinomycetes and 25 novel actinomycetota species are listed in this review. Furthermore, the biological activities of some lichen actinomycetota have also been investigated, although the effective chemical components of these strains are still unknown. The biosynthetic pathways of some unique secondary metabolites from lichen-derived actinomycetes are also reported here.

2. Novel Actinomycetota Taxa

The phylum “Actinobacteria” was modified to “Actinomycetota” by Goodfellow in 2021 [7]. Therefore, we use “actinomycetota” in the present article. Actinomycetes are extensively dispersed. The 25 novel species isolated from lichens between 2007 and 2022 are described in

Table 1. Novel actinomycetota taxa isolated from lichens between 2007 and 2022.

Family	Genus	Species	Lichen Habitats	Media	Refs.
Aurantimonadaceae	Aureimonas	Aureimona leprariae	Yunnan Province, south-west PR China	Humic acid–vitamin agar and ISP2	[8]
Methylobacteriaceae	Methylobacterium	Methylobacterium planium	Yunnan Province, south-west PR China	Humic acid–vitamin agar	[9]
Microbacteriaceae	Glaciibacter	Glaciibacter flavus	The south bank forest of the Baltic Sea, Germany	Humic acid–vitamin agar	[10]
	Frondihabitans	Frondihabitans cladoniiphilus	The natural spruce forest at Koralpe, in the Austrian Alps	Tryptone–yeast extract medium and ISP2	[11]
	Leifsonia	Leifsonia lichenia	The Botanical Garden of the University of Tokyo	Modified Detmer medium and nutrient agar	[12]
	Naasia	Naasia lichenicola	The south bank of the Baltic Sea, Germany	Humic acid–vitamin agar and YIM 38 medium	[13]
	Schumannella	Schumannella luteola	Tokyo, Japan	Modified Detmer medium and nutrient agar	[14]
	Subtercola	Subtercola lobariae	Jiaozi Snow Mountain, Yunnan Province, China	Potato dextrose agar and ISP2	[15]
Micromonosporaceae	Actinoplanes	Actinoplanes lichenis	Maha Sarakham Province, Thailand	Humic acid–vitamin agar with nalidixic acid and cycloheximide and ISP2	[16]
		Actinoplanes lichenicola	Maha Sarakham Province, Thailand	Humic acid–vitamin agar with nalidixic acid and cycloheximide and ISP2	[17]
		Actinoplanes ovalisporus	Maha Sarakham Province, Thailand	Humic acid–vitamin agar with nalidixic acid and cycloheximide and ISP2	[17]
Nakamurellaceae	Nakamurella	Nakamurella albus	Yunnan Province, south-west PR China	Humic acid–vitamin agar and ISP2	[18]
	Nakamurella	Nakamurella leprariae	Yunnan Province, south-west PR China	Humic acid–vitamin agar and YIM 38 medium	[19]
Nocardioidaceae	Nocardioides	Nocardioides exalbidus	Izu-Oshima Island, Japan	IAM-A1 agar medium and trypticase soy agar	[20]
Promicromonosporaceae	Luteimicrobium	Luteimicrobium album	Rishiri Island, Japan	Humic acid–vitamin agar with nalidixic acid and cycloheximide and nutrient agar	[21]
Pseudonocardiaceae	Actinomycetospora	Actinomycetospora iriomotensis	Iriomote Island, Japan.	Humic acid–vitamin agar with nalidixic acid and cycloheximide and nutrient agar	[22]
		Actinomycetospora rishiriensis	Rishiri Island, Hokkaido, Japan.	Humic acid–vitamin agar with nalidixic acid and cycloheximide	[23]
	Pseudonocardia	Pseudonocardia	Nahuel Huapi National Park, Patagonia	Artificial soil agar and Emerson’s yeast extract–starch agar and KEHE agar	[24]
Rhodobacteraceae	Paracoccus	Paracoccus lichenicola	Yunnan Province, south-west PR China	Humic acid–vitamin agar and YIM 38 medium	[25]
	Rubellimicrobium	Rubellimicrobium rubrum	The south bank forest of the Baltic Sea, Germany	Humic acid–vitamin agar and YIM 38 medium	[26]
Streptomycetaceae	Streptomyces	Streptomyces lichenis	Chiang Rai Province, Thailand	Arginine–vitamin agar and ISP2	[27]
		Streptomyces	The tropical rainforest in Xishuangbanna, Yunnan, China	YIM 212 medium	[28]
		Streptomyces parmotrematis	Doi Suthep-Pui National Park, Chiang Mai Province, Thailand.	Humic acid–vitamin agar and starch casein nitrate agar with cycloheximide and nalidixic acid	[29]
Thermomonosporaceae	Actinomadura	Actinomadura violacea	Pong Phra Bat Waterfall, Chiang Rai Province, Thailand	Humic acid–vitamin agar with nalidixic acid and cycloheximide	[30]
		Actinomadura parmotrematis	Chiang Rai Province, Thailand	Starch–casein nitrate agar with nalidixic acid and cycloheximide	[31]

. In Figure 1, the red dots indicate the collection sites of the lichen samples from which new species of actinomycetes were identified. Most published literature suggests the distribution of new lichen-associated actinomycetota in Asia, especially in Yunnan Province, China. Among the 25 isolated species, 6 actinomycete species (24%) belonged to the family Microbacteriaceae. Three (12%) species belonged to each of the families of Micromonosporaceae, Pseudonocardiaceae, and Streptomycetaceae. The remaining actinomycetes came from the families Nakamurellaceae, Rhodobacteraceae, Streptomycetaceae, and others (Figure 2).

The humic acid–vitamin agar selective medium was mainly used to isolate most of the novel actinomycetota species (Table 1). A few novel species were isolated using standard growth media such as ISP2 medium and potato dextrose agar. Most species were incubated for 1–4 weeks at 25–30 °C.

3. Natural Products from Lichen-Associated Actinomycetota

A total of 114 compounds from lichen-associated actinomycetes were reported. Based on their structural characteristics, they were classified into diketopiperazines, peptides, indoles, furanones, quinones, isoflavonoids, etc. Some exhibit anti-inflammatory, antimicrobial, anticancer, cytotoxic, and enzyme-inhibitory activities. These compounds are promising small molecules for advancement as new drug and pesticide candidates.

3.1. N-Containing Compounds

3.1.1. Diketopiperazines

Diketopiperazines are the smallest cyclic dipeptides formed by the double condensation of two α-amino acids. They have a stable six-membered ring that serves as an important pharmacophore [32]. Diketopiperazines are structures with numerous biological functions of interest to natural product researchers.

Cyclo (Gly-L-Ala) (1) and 5-methyl-uracil (2) were isolated from the actinomycete Amycolatopsis sp. YIM 130687, collected from the Jinsha River region in Yunnan Province [33]. Compound 1 exhibited weak activity against Escherichia coli and Salmonella typhimurium, and 2 showed weak inhibitory activity against S. typhimurium. Cyclo (L-Pro-L-Val) (3) was isolated from Nocardia ignorata [34]. Cyclo-(Ala-Leu) (4), cyclo-(Gly-Phe) (5), cyclo-(Leu-Tyr) (6), and cyclo-(Phe-Tyr) (7) were obtained from Amycolatopsis sp. YIM 130932, associated with the lichen Punctelia rudecta found in Yunnan Province [35]. Cyclo (L-Pro-L-OMet) (8) was isolated from Nocardia ignorata [34]. Cyclo-(Phe-Pro) (9) and cyclo-(L-Leu-L-Pro) (10) were isolated from Streptomyces cyaneofuscatus MOLA 1488, associated with the marine lichen Lichina confinis [36]. The crude extract of this strain showed anticancer activity against murine melanoma cells (B16 cell line) and the HaCaT cell line (normal immortalized keratinocyte cell line). The half maximal inhibitory concentration (IC₅₀) values for B16 and HaCaT cell lines were 0.33 ± 0.2 μM and 0.25 ± 0.1 μM, respectively. Cyclo-(Pro-Tyr) (11) was isolated from the actinobacterium QHHL-09 from the Tibetan Plateau [37]. Two new brominated diketopiperazines, cyclo (D-Pro-L-Br-Tyr) (12) and cyclo (L-Pro-L-Br-Tyr) (13), were isolated from Nocardia ignorata [34]. All 13 diketopiperazines from the lichen derived actinomycetota described above are presented in Figure 3.

3.1.2. Peptides

Peptide natural products (PNPs) represent a unique class of compounds with fascinating structural motifs that impart important biological activities [38]. Cis-3-isobutyl-tetrahydroimidazo [1,2-a] pyridine-2,5-dione (14) was isolated from the lichen-associated actinobacterium QHHL-09, found on the Tibetan Plateau. It inhibited HIV-1 reverse transcriptase, glutamate receptor 2, and protein kinase ck2 [37]. Turnagainolide B (15) was obtained from the strain Streptomyces sp. YIM 130597 from the lichen Punctelia rudecta, found in Yunnan Province, China [35]. It displayed weak antibacterial activity toward S. typhimurium with a MIC value of 64 μg/mL.

Antipain (16), V2 (antipain dehydration product) (17), and lichostatinal (18) were isolated from a British Columbian lichen associated with Streptomycetes sp. L91-3 [39]. Compound 16 was a known potent cathepsin K (CatK) inhibitor, whereas 17 was a new dehydrated analog of antipain and a much weaker CatK inhibitor. Compound 18 was identified as a new potent CatK inhibitor using affinity crystallography. N-methyldactinomycin (19) was collected from Streptomyces cyaneofuscatus MOLA1488 from Erquy (France) [36]. Geninthiocin B (20) was isolated from Streptomyces sp. YIM 130001 obtained from the tropical rainforest in Xishuangbanna (Yunnan, China). It showed antibacterial activity against Bacillus subtilis [28]. Skyllamycins A–E (21–25) were isolated from a Streptomyces anulatus strain from the New Zealand lichen Pseudocyphellaria dissimilis. Antibacterial assays revealed that compound 24 possessed superior activity against B. subtilis E168 compared to previously reported congeners [40]. All 12 PNPs from the lichen derived actinomycetota described above are presented in Figure 4.

3.1.3. Indoles

Due to its diverse biological activities, the indole scaffold is a vital heterocyclic organic compound of medical and pharmaceutical interest [41]. Indole-carboxaldehyde (26) was isolated from the lichen Collema auriforme, found in the Austrian town of Kesselfallklamm [34]. Compound 26 showed weak cytotoxic activity against the HaCaT (IC₅₀ = 79 ± 6 μM) and B16 (IC₅₀ = 72 ± 6 μM) cell lines.

(3-Hydroxyacetyl) indole (27) and N-acetyl-β-oxotryptamine (28) were isolated from Lichina confinis, a marine lichen collected on the Brittany coast (Erquy, France) [36]. Acetotryptamide (29) was isolated from the fresh lichen Punctelia borreri from the Jinsha River region, Yunnan Province. It possessed antibacterial activity against S. typhimurium and E. coli [33]. 7-Prenylisatin (30), 5-isoprenylindole-3-carboxylate (31), JBIR-126 (32), and JBIR-149 (33) were isolated from culturable lichen actinomycetes found around Qinghai Lake on the Qinghai–Tibet Plateau [37]. Seven new compounds, cladoniamides A–G (34–40) were isolated from cultures of Streptomyces uncialis found on the surface of the lichen Cladonia uncialis collected near Pitt River, British Columbia. Cladoniamide G displayed significant cytotoxicity against human breast cancer MCF-7 cells in vitro at 10 μg/mL [42]. All 15 indoles from the lichen derived actinomycetota described above are presented in Figure 5.

3.1.4. Pyrrole Derivatives

Pyrrole derivatives are a distinct class of heterocycle compounds that contribute significantly to natural products [43]. Mminaline (41) and 1H-pyrrole-2-carboxamide (42) were isolated from Amycolatopsis sp. YIM 130687, isolated from the fresh lichen Punctelia borreri found in the Jinsha River region, Yunnan Province. Compound 41 displayed weak antibacterial activity against MRSA, and 42 showed weak antibacterial activity against Staphylococcus aureus and F. solani [33]. Metacycloprodigiosin (43) and undecylprodigiosin (44) were extracted from the QHHL-18 isolate associated with lichens found around the Qinghai Lake on the Qinghai–Tibet Plateau [37]. All 4 pyrrole derivatives from the lichen derived actinomycetota described above are presented in Figure 6.

3.1.5. Pyridine Derivatives

Pyridine is a crucial heterocyclic framework found in natural products. Methods have been developed for pyridine synthesis because of their importance and appeal in organic chemistry and natural product research [44]. Four new echinosporins, amycolasporins A–D (45–48), were derived from the lichen-associated actinomycete Amycolatopsis hippodrome. Compounds 46 and 47 demonstrated antibacterial activity against B. subtilis, S. aureus, and E. coli [45]. A novel compound, JBIR-120 (49), was isolated from Streptomyces sp. RI104-LiC104 from a lichen found on Rishiri Island, Hokkaido Prefecture, Japan [46]. It was weakly cytotoxic against 22Rv1 cells (human prostate carcinoma epithelial cell line) and effectively inhibited the growth of cells activated by dihydrotestosterone. Two known compounds, 4-methoxy-5-(methylthio)-[2,2′-bipyridine]-6-carbonitrile (50) and 7-methoxy-5-(pyridin-2-yl) isothiazolo [4,5-b] pyridine (51), were isolated from the Streptomyces strain YIM 130597 collected from the lichen Punctelia rudecta in Yunnan, China. Compound 51 exhibited strong antibacterial activity against S. aureus (MIC value of 64 μg/mL), E. coli (MIC value of 32 μg/mL), S. typhimurium (MIC value of 64 μg/mL), and MRSA (MIC value of 32 μg/mL) [35]. All 7 pyridine derivatives from the lichen derived actinomycetota described above are presented in Figure 7.

3.1.6. Aromatic Amides and Amines

One or both primary amino groups and the imino group in an aliphatic polyamine can interact with different acids, resulting in mono-, di-, or tri-substituted amide derivatives [47]. A new compound, (E)-3- hydroxy-2,4-dimethylhept-4-enamide (52), was derived from the marine actinomycete Streptomyces cavourensis YY01-17 [48]. 2-Acetamidophenol (53), phenacetamide (54), anthranilic acid (55), 4-(3-methylbut-2-enyloxy) benzamide (56), and 2-pyruvoylaminobenzamide (57) were isolated from the fresh lichen Punctelia borreri. Compound 53 effectively inhibited the growth of MCF-7 breast cancer cells. Compound 54 showed inhibitory activity against F. graminearum with a MIC value of 2 μg/mL and against S. aureus with a MIC value of 8 μg/mL [33]. Compounds 58 3-(4-hydroxyphenyl)-N-methylpropanamide and 59 N-(4-hydroxyphenethyl)-acetamide were isolated from actinomycetes from the Qinghai–Tibet Plateau near Qinghai Lake [37]. Amycophthalazinone A (60) was a new phthalazinone derivative isolated from Amycolatopsis sp. YIM 130642. It exhibited inhibitory activity against S. aureus, S. typhi, and Candida albicans with MIC values of 32, 32, and 64 μg/mL, respectively [49].

Compounds 61 2-carbamoyl-3-hydroxy-1,4-naphthoquinone and 62 (–)-chry-sogine were isolated from the fresh lichen Punctelia borreri found in the Jinsha River region of Yunnan. Compound 61 displayed antimicrobial activity against Botrytis cinerea, F. graminearum, S. aureus, and MRSA with MIC values of 1, 1, 2, and 2 μg/mL, respectively [33]. A new echinosporin derivative, amycolasporin E (63), and a known echinosporin (64) were obtained from the lichen-associated actinomycete Amycolatopsis sp. YIM 130415 [45]. All 13 aromatic amides and amines from the lichen derived actinomycetota described above are presented in Figure 8.

3.2. Furanones

Furanones are commonly utilized in synthesis. The products display important pharmacological properties such as antiviral, anticancer, and antimicrobial properties [50]. JBIR-89 (65) was a new butenolide from the lichen-derived Streptomyces sp. RI104-LiB101 collected from Rishiri Island, Hokkaido Prefecture, Japan [51]. (5S)-5-(6-Hydroxy-6-methyloctyl)-furan-2(5H)-one (66) and (5S)-5-(6-hydroxy-7- methyloctyl)-furan-2(5H)-one (67) were produced by lichen-associated actinomycetes collected on the Tibetan Plateau, China [37]. Six new compounds, actinofuranones D-I (68–73), and three known compounds, JBIR-108 (74), E-975 (75), and E-492 (76), were obtained from S. gramineus derived from the lichen Leptogium trichophorum collected from an evergreen broad-leaf forest in Benzilan, Diqing, Yunnan, China [52]. Compounds 71, 72, 75, and 76 inhibited nitric oxide synthase expression (in OS) in RAW 264.7 cells after LPS induction. In addition, 71, 72, 75, and 76 inhibited the LPS-induced proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). All 12 furanones from the lichen derived actinomycetota described above are presented in Figure 9.

3.3. Aromatic Compounds

3.3.1. Quinones

Quinones exhibit various biological activities, including antibacterial, antiplasmodial, antioxidant, trypanocidal, anticancer, and anti-HIV activities. All these activities are linked to the redox properties of their carbonyl groups [53]. Compound 77 (+)-4-hydroxy-1-teralone was obtained from the lichen-derived actinomycete Amycolatopsis sp. YIM 130687 collected from Yunnan Province, China [33]. Four novel nanomycin compounds, 4aβ,10aα-dihydroxynanaomycin βA (78), 4aβ,10aβ-dihydroxynanaomycin βA (79), 4aα,10aβ-dihy-droxynanaomycin βA (80), and 10β-hydroxynanaomycin αE (81), and two known compounds, nanaomycin αA (82) and nanaomycin βA (83), were produced by Streptomyces hebeiensis [54]. Compounds 82 and 83 displayed antibacterial activity against S. aureus and B. subtilis with MIC values ranging from 3.13 to 100 μg/mL, and modest antifungal activity against C. albicans.

Compound 84 4-deoxy-ε-pyrromycinone was isolated from lichen actinomycetes collected around Qinghai Lake [37]. JBIR-88 (85) was a new angucycline produced by the lichen-derived Streptomyces spp. RI104-LiC106, and it exhibited antibacterial activity against Micrococcus luteus. Furthermore, 85 showed cytotoxicity against HeLa cells with a MIC of 36 μM and ACC-MESO-1 cells with a MIC of 52 μM [51]. BE-24566B (86) was a new antibiotic produced by Streptomyces violaceusniger A24566, which was isolated from a lichen collected in Jyogasaki, Shizuoka Prefecture, Japan. This compound inhibited Gram-positive bacteria, including methicillin-resistant S. aureus [55].

The lichen-derived actinomycete Steptomyces sp. 0630c, collected from Zhaosu County of the Xinjiang Uygur Autonomous Region, China, yielded three compounds, steffimycin D (87), steffimycin E (88), and steffimycin F (89) [56]. Compound 89 was a new steffimycin-type antibiotic with weak cytotoxicity towards MCF-7 (human breast adenocarcinoma), HepG-2 (human liver hepatocellular carcinoma), and A2780 (human ovarian carcinoma) cell lines. Two known compounds, 88 and 89, exhibited potent antibacterial action against S. aureus with MIC values of 2 μg/mL. (7S*, 9R*, 10R*)-Pyrromycin (90) was isolated from a lichen actinomycete collected from Qinghai Lake [37]. Uncialamycin (91) was a novel enediyne antibiotic isolated from the British Columbian lichen Cladonia uncialis collected near the Pitt River [57]. It exhibited antibacterial activity against S. aureus, E coli, and Burkholderia cepacia with MIC values of 0.0000064, 0.002, and 0.001 μg/mL, respectively. 7-O-methylkoninginin D (92) and koninginin E (93) were obtained from Streptomyces sp. from the lichen Punctelia rudecta collected in Yunnan Province, China [35]. All 17 quinones from the lichen derived actinomycetota described above are presented in Figure 10.

3.3.2. Isoflavonoids

Isoflavonoids have a B-ring connected to their C-ring at the C-3 position (3-phenylchroman skeleton). They display a wide range of biological activities including antioxidant, anticarcinogenic, and antiproliferative properties. They also possess the ability to reduce osteoporosis and cardiovascular disease [58]. Seven isoflavonoid glycosides, namely genistein (94), formononetin (95), prunetin (96), kakkatin (97), isoformononetin (98), 7-O-methyl-5-O-α-L-rhamnopyranosylgenestein (99), and 7-O-α-D-arabinofur-anosyldaidzein (100), were isolated from Amycolatopsis sp. YIM 130642. Compounds 94 and 99 inhibited S. aureus and E. coli. Compound 96 inhibited E. coli with a MIC of 32 μg/mL, while 100 demonstrated bacteriostatic activity against S. typhi with a MIC value of 64 μg/mL [49]. All 7 isoflavonoids from the lichen derived actinomycetota described above are presented in Figure 11.

3.3.3. Phenolic Derivatives

Phenolic compounds occur mainly in soluble conjugate and insoluble forms and are covalently bound to sugar moieties or structural components of the cell wall. Phenolic compounds have been extensively studied due to their varied health benefits as antioxidants and their roles in preventing chronic inflammation, cardiovascular disease, cancer, and diabetes [59,60]. The novel compound (R)-3-methyl-1,3-dihydroisobenzofuran-4,6-diol (101) was obtained from Amycolatopsis hippodrome [45]. P-hydroxyphenethyl alcohol (102) and sorbicillin (103) were obtained from Amycolatopsis sp. YIM 130687. Compound 103 showed cytotoxicity against the hepatocellular carcinoma cell line QGY-7703 and inhibited the growth of C. albicans [33]. 2-(4-Hydroxybenzylacetone)-5-methoxyphenol (104), amycolabenzoyl (105), and amycolabenzosides A–B (106–107) were obtained from Amycolatopsis hippodrome. Compound 104 attenuated nitric oxide production by suppressing the expression of nitric oxide synthase (iNOS) in LPS-induced RAW 264.7 cells in a dose-dependent manner [44]. Usnic acid (108), a prevalent cytotoxic secondary metabolite in lichens, was isolated from Streptomyces cyaneofuscatus MOLA1488. It imparts a green to greenish yellow color to many lichens [36]. All 8 phenolic derivatives from the lichen derived actinomycetota described above are presented in Figure 12.

3.4. Linear Esters and Macrolides

Esters of linear long-chain unsaturated fatty acids with multiple alcohols, both linear and branched, are widely used in the lubricant industry [61]. Macrolides are a large and structurally diverse class of macrocyclic natural products. They are valuable targets in synthetic chemistry due to their biological and medicinal importance [62]. One new linear compound, 2(S)-3′-hydroxybutan-2′-yl 2-hydroxypropanoate (109), and a known compound, 2-hydroxy-3-methylbutanoic acid (110), were procured from the marine-derived actinomycete Streptomyces cavourensis YY01-17 [48]. Cyaneodimycin (111) and cyaneomycin (112) were isolated from marine-lichen-associated Streptomyces cyaneofuscatus. Compound 111 exhibited antiproliferative action against B16, HaCaT, and Jurkat cell lines with MIC values of 27 ± 4 μM, 47 ± 11 μM, and 18.5 ± 0.5 μM, respectively [35]. Macrolactin A (113) was isolated from a lichen actinomycete found on the Qinghai–Tibet Plateau [37]. All 5 linear esters and macrolides from the lichen derived actinomycetota described above are presented in Figure 13.

3.5. Sterols

Sterols are isoprenoid derivatives and structural components of biological membranes. They are currently being investigated for their structural, functional, and regulatory roles [63]. Campesterol (114) from the lichen-derived strain Amycolatopsis sp. YIM 130687 inhibited the growth of MRSA with a MIC of 128 μg/mL [33]. 1 sterol from the lichen derived actinomycetota described above are presented in Figure 14.

4. Bioactivity of Uncharacterized Compounds

The biological activities of many lichen actinomycetota have been investigated. However, these studies did not report any pure compounds or their structures. Several lichen-associated actinomycetota have been screened for their biological activities, such as antibacterial and antifungal activities, inhibition of β-glucosidase activity, etc. Such screenings without the structural elucidation of bioactive metabolites may not be useful for the discovery of new compounds. Nonetheless, these data highlight the possible utility of lichen-associated novel actinomycetota for the discovery of novel bioactive chemicals in the future [64].

Twelve actinomycete strains were isolated from lichens collected from the Maha Sarakham Province, Thailand. Among these, four Streptomyces isolates, LDG1-03, LDG1-15, LDG1-16, and LLG1-03, showed antimicrobial activity against B. subtilis ATCC 6633. LDG1-03 and LDG1-15 exhibited antimicrobial activity against S. aureus ATCC 25923, Kocuria rhizophila ATCC 9341, and C. albicans ATCC 10231. The Actinoplanes isolate LDG1-06 inhabited C. albicans ATCC 10231 [65]. Actinomycetes LC-23 was isolated from a lichen found growing on the bark of the Averrhoa carambola plant. Actinomycete pure strains were screened using agar diffusion on ISP2 agar medium to determine antimicrobial potency. The ethyl acetate extract of this strain displayed a positive inhibitory effect against S. aureus BTCC B-611 and M. luteus BTCC B-552 [66]. Lichen-associated Streptomyces olivaceus LEP7 was recovered from tree bark collected in the botanical garden of Nilgiris, Tamil Nadu, India. The extract of Streptomyces sp. LEP7 inhibited E. coli, S. aureus, and P. aeruginosa efficiently. The extract was found to contain cyclopentene upon GC-MS analysis. According to the report, the remarkable antimicrobial activity of Streptomyces olivaceus when tested against wound infections caused by microbial pathogens, and the derivation of cyclopentene from LEP7is a first step in this direction [67].

Extracts from the lichen Umbilicaria esculenta strongly inhibited mold and mammalian disaccharide hydrolytic enzymes (β-glucosidase). The inhibitory component of the extract was very stable, retaining more than 95% of its activity when treated with heat, acid, alkali, and some hydrolytic enzymes [68]. Streptomyces sp. DPUA 1542 and Nocardia sp. DPUA 1571, two actinomycetota strains isolated from Amazon River basin lichens, produced β-lactamase inhibitors which cured bovine mastitis [69]. Streptomyces sp. DPUA 1576, isolated from an Amazon basin lichen, yielded a fibrinolytic protease. This protease could potentially provide new and unexploited fibrinolytic enzymes for different therapeutic purposes [70,71].

5. Biosynthetic Pathways of Lichen Secondary Metabolites

Advances in synthetic biology and associated technologies such as DNA synthesis, sequencing, and analysis techniques have accelerated the DBT cycle for metabolic and protein engineering to the point where both can be deployed to engineer the biosynthesis of a particular molecule [72]. The genes encoding these natural products in actinomycetes tend to be clustered, which allows the transfer of entire biosynthetic pathways to an exogenous host for heterologous expression. This strategy also enables the genetic modifications of such pathways, allowing the generation of various natural product analogs as well as the optimization of production yield [73].

The production of the novel thiopetide antibiotic geninthiocin B (20) is due to the Gen B gene encoding a putative lantibiotic dehydratase in the biosynthetic gene cluster of the lichen-derived Streptomyces sp. YIM130001. As described in the literature, the production of associated genes includes precursor proteins, Yeao cyclodchydratase, lanthipeptide dehydratases, etc. (Figure 15A). The biosynthetic pathway of geninthiocin was proposed by Schneider et al. [28], and is exhibited in Figure 15B. The precursor peptide (GenA) unit, which possesses a 31 aa leader peptide (LP), is connected with a C-terminal 15 aa core peptide unit. The Yeao cyclodchydratase biosynthetic gene clusters GenG1 and GenG2 could catalyze the processing of azole rings formation. The proteins GenB and GenC show a high degree of similarity to lanthipeptide-like dehydratases and most likely catalyze the formation of the dehydroalanine (Dha) and dehydrobutyrine (Dhb) functional groups. The two Dha group residues from the serines Ser₁ and Ser₁₃ are then utilized by GenD for assembly of the central six-membered nitrogenous heterocycle. Finally, the cleavage of Ser₁₅ to afford the C-terminus amide and the hydroxylation of Val₇ is catalyzed by GenI and GenH, completing the biosynthesis of geninthiocin B.

The genome of Streptomyces uncialis includes halogenases and flavin reductase, indolocarbazole aglycone construction, new flavin-dependent oxygenases, and so on (Figure 16A). The biosynthetic pathway of cladoniamides were proposed by Ryan et al. [74] and is exhibited in Figure 16B. It shows that ClaH and ClaF are highly related to the characterized L-tryptophan chlorinases, and that chlorine is installed on the L-tryptophan in the first step of the related rebeccamycin pathway. Due to the action of ClaH and a partner flavin reductase ClaF, L-tryptophan is chlorinated at the C-5′ position. Then, 5-chloro-L-tryptophan reacts with ClaO, generating an indole-3-pyruvate imine. ClaD dimerizes two of these molecules to generate a chromo pyrrolic acid molecule. ClaY catalyzes the hydrolysis of an amide bond in the N-methylsuccinimide ring, which is followed by oxidative decarboxylation. Three enzymes unique to the indenotryptoline biosynthetic pathway include two putative flavin-dependent oxygenases (ClaX1 and ClaX2) and a putative α/β hydrolase (ClaY) shown in Figure 15A. The cladoniamide biosynthetic gene cluster is highly homologous to that of BE-54017. 1 (R₁ = Cl, R₂ = R₃ = H) and a methylated derivative of 2 (R₁ = R₂ = H) separately accumulate in the BE-54017 heterologous expression system when the genes abeX1 and abeX2 are mutated, respectively. The route to generate downstream metabolites, indenotryptoline-containing molecules such as 3, from substrate 2 via cleavage of the epoxide could be driven via ketone formation from one tertiary alcohol, causing sigma-bond rupture and epoxide hydrolysis, opening the indolocarbazole scaffold. This cleaved molecule could then close through attack on the ketone by the indolic nitrogen, restoring the tertiary alcohol and arriving at the indenotryptoline scaffold 3. Each of these enzymes is thought to catalyze the transfer of a methyl group to a phenolic oxygen, consistent with the likely role of ClaM3 in cladoniamide biosynthesis of installing a methyl group on the appended hydroxyl group to produce cladoniamides A–C.

6. Conclusions

The current review focused on lichen actinomycetota from four different perspectives. (1) Lichen-associated actinomycetes represent a promising but underutilized resource. A wide variety of novel actinomycetes have been isolated from lichens. (2) The potential of bioactive metabolites from lichen actinomycetes has been explored, and a total of 114 secondary metabolites from lichen-associated actinomycetes are summarized here. (3) Although the biological activities of many lichen actinomycetota have been investigated, their definite chemical components are still undetermined. Thus, the discovery of more novel bioactive compounds reveals new research prospects. (4) The biosynthetic pathways of some unique secondary metabolites isolated from lichen-derived actinomycetes are discussed.

It has become increasingly difficult to isolate new sources of actinomycetes from common environments such as the soil, sea, and plants. These resources no longer meet the increasingly urgent demand for new drug-leading compounds [6]. Therefore, researchers must explore potent microbial resources from unique environments [64]. Current research on lichen-associated actinomycetes has focused mainly on Asia, whereas lichens are globally distributed. This wide distribution range enables researchers to search for novel species. Lichen environments are understudied in terms of microbiology, but they should not be disregarded in the hunt for novel actinomycetota and their diversity of beneficial chemical compounds [71]. The novelty and variety of lichen actinomycetota are evident in this review. Furthermore, the study of biosynthetic pathways is a crucial process in the excavation of bioactive natural products [75]. Biosynthesis of other biologically active compounds is relatively less studied and requires more attention from researchers. Future work could attempt to reveal more silent biosynthetic gene clusters, so that to uncover more and more novel and interesting biologically active natural products from lichen-associated actinomycetota.