Lichen-Derived Actinomycetota: Novel Taxa and Bioactive Metabolites

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.


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 Humic acid-vitamin agar [9] Microbacteriaceae Glaciibacter Glaciibacter flavus The south bank forest of the Baltic Sea, Germany Humic acid-vitamin agar [10] Frondihabitans
Humic acid-vitamin agar with nalidixic acid and cycloheximide and nutrient agar [22] Actinomycetospora rishiriensis 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.

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 JBIR-149 (33) were isolated from culturable lichen actinomycetes found around Qin Lake on the Qinghai-Tibet Plateau [37]. Seven new compounds, cladoniamides (34−40) were isolated from cultures of Streptomyces uncialis found on the surface o lichen Cladonia uncialis collected near Pitt River, British Columbia. Cladoniamid displayed significant cytotoxicity against human breast cancer MCF-7 cells in vitro g/mL [42]. All 15 indoles from the lichen derived actinomycetota described abov presented in Figure 5.  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.

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,

Isoflavonoids
Isoflavonoids have a B-ring connected to their C-ring at the C-3 position (3phenylchroman 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) [49]. All 7 isoflavonoids from the lichen derived actinomycetota described above are presented in Figure 11.

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.

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].

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.

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.

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].

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 lichenassociated 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].

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 Ser 1 and Ser 13 are then utilized by GenD for assembly of the central six-membered nitrogenous heterocycle. Finally, the cleavage of Ser 15 to afford the C-terminus amide and the hydroxylation of 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 Ltryptophan 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 sigmabond 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 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 (R 1 = Cl, R 2 = R 3 = H) and a methylated derivative of 2 (R 1 = R 2 = 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 sigmabond 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.

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

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 lichenassociated actinomycetota.