Actinomycetes as Producers of Biologically Active Terpenoids: Current Trends and Patents

Terpenes and their derivatives (terpenoids and meroterpenoids, in particular) constitute the largest class of natural compounds, which have valuable biological activities and are promising therapeutic agents. The present review assesses the biosynthetic capabilities of actinomycetes to produce various terpene derivatives; reports the main methodological approaches to searching for new terpenes and their derivatives; identifies the most active terpene producers among actinomycetes; and describes the chemical diversity and biological properties of the obtained compounds. Among terpene derivatives isolated from actinomycetes, compounds with pronounced antifungal, antiviral, antitumor, anti-inflammatory, and other effects were determined. Actinomycete-produced terpenoids and meroterpenoids with high antimicrobial activity are of interest as a source of novel antibiotics effective against drug-resistant pathogenic bacteria. Most of the discovered terpene derivatives are produced by the genus Streptomyces; however, recent publications have reported terpene biosynthesis by members of the genera Actinomadura, Allokutzneria, Amycolatopsis, Kitasatosporia, Micromonospora, Nocardiopsis, Salinispora, Verrucosispora, etc. It should be noted that the use of genetically modified actinomycetes is an effective tool for studying and regulating terpenes, as well as increasing productivity of terpene biosynthesis in comparison with native producers. The review includes research articles on terpene biosynthesis by Actinomycetes between 2000 and 2022, and a patent analysis in this area shows current trends and actual research directions in this field.

In the last 15-20 years, it has become obvious that bacteria also produce terpenes and terpenoids and that most of the produced metabolites are represented by new compounds. Currently, the search for microorganisms synthesizing terpene derivatives is underway and microbial biosynthetic platforms are developed using such microorganisms [3]. Microbial biosynthesis has advantages over traditional methods of obtaining terpenoids: a short life cycle of microorganisms, which reduces the production time of Table 1. Potential applications of secondary metabolites produced by actinomycetes in various fields of human activities.
Terpene biosynthesis by actinomycetes is an actual research area discussed in research and review publications. However, the specialized reviews are focused on certain genera of actinomycetes and/or groups of terpene derivatives [41,42], bacterial terpenome [43], and evolution and ecology of microbial terpenoids [44]. The present review aims at assessing the biosynthetic potential (via the patent analysis in particular) of various representatives of Actinomycetes as producers of a wide range of biologically active terpenoids, including hybrid metabolites (meroterpenoids). The data can be used to create technologies for the biocatalytic production of practically valuable terpene derivatives using actinomycetes.
In writing this review, various databases were used: scientific articles and reviews were searched through platforms such as Web of Science, Scopus, and NCBI, and WIPO (World Intellectual Property Organization, https://patentscope.wipo.int/, accessed on 25 March 2022) was used to search for patents. To fully cover the topic, the review includes patents and articles (from 2000 to 2022) dedicated to terpene biosynthesis by representatives of Actinomycetes (according to the modern classification).

Terpene Biosynthesis by Actinomycetes
Terpene biosynthesis is one of the secondary metabolic pathways in actinomycetes, regulated by biosynthetic gene clusters (BGCs). BGCs include promoters, genes encoding carbon skeleton formation enzymes and post-modification enzymes, and regulatory genes. All terpenes are synthesized from the C5 isoprenoid precursors, namely isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are converted to isoprenyl diphosphates of varying lengths by isoprenyl transferases (Figure 2). Further formation of terpenes is catalyzed by a group of enzymes, namely terpene synthases (cyclases) (TSs) catalyzing the cyclization of geranyl (GPP), farnesyl (FPP), geranylgeranyl (GGPP), and geranylfarnesyl (GFPP) diphosphates to yield mono-, sesqui-, di-, sester-, and triterpenes. Unlike the basic biosynthetic enzymes, bacterial TSs have low homology of conserved sequences, providing an extremely diverse group. The main feature of TSs is that one enzyme can produce dozens of hydrocarbon skeletons significantly different from each other. A number of remarkable reviews have been devoted to bacterial and plant terpene synthases [5,6,45,46]. Modification of the terpene skeleton is achieved through the addition of various functional groups mediated by specialized enzymes, mainly those from the cytochrome (P450s) family.
A variety of methods (bioinformatics, genetic, analytical, biochemical, molecular) are employed to study terpene biosynthesis by actinomycetes. Direct screening of compounds from the microbial cultivation medium and their subsequent identification is a basic method of searching for new terpene derivatives; however, it is labor-and time-consuming. Currently, recently developed "genome mining" methods, namely a bioinformatics search for TS genes using the BLAST program and web-based tools such as ClustSCAN, NP.searcher, GNP/PRISM, and antiSMASH, are used to search for actinomycetes capable of producing terpene derivatives. Simultaneous discovery of new compounds and biosynthetic genes and enzymes is one of the most important advantages of the coordinated use of genome analysis and direct analysis of the metabolites. Using this approach, a few dozen terpenes (many of which are unique), several new cyclization mechanisms, and more than 120 putative genes of bacterial terpene synthases have been discovered [47].
Methods of genetic modification (e.g., gene knockout, presumably responsible for the terpene synthesis; editing of individual sections of BGCs, in particular, by introducing additional native or engineered promoters; influence on the regulatory gene expression) and heterologous expression (e.g., cloning of the interest gene in bacteria that are not capable of synthesizing the target product) are used to confirm the functional activity of the studied genes. E. coli or mutant strains Streptomyces avermitilis SUKA 2-22 with deletion of all endogenous BGCs [48], Streptomyces lividans [49], Streptomyces coelicolor, Streptomyces albus, etc. [50,51], can serve as host bacteria. The transformants are used either for the direct terpene synthesis or for the production of recombinant proteins subsequently incubated with acyclic allyl diphosphate substrates. Molecular and biochemical methods allow studying the crystal structure, kinetic and mechanistic parameters of isolated and purified TSs and mechanisms of terpene cyclization [47]. In addition, omics technologies have been actively developed to search for secondary metabolites, terpenoids in particular, to study the diversity, distribution, and evolution of BGCs [52]. MEP

Terpene Derivatives Produced by Streptomycetes and Their Enzymes
The analysis of published data indicates that most of the identified actinomycete terpene derivatives are synthesized by streptomycetes. The spectrum of produced compounds varies from mono-to tetraterpenes and their derivatives.
Epi-isozizaene (103), tricyclic sesquiterpene, was generated by several Streptomyces species and initially sparked interest as a candidate jet fuel on account of having a specific energy similar to that of jet fuel A-1 [110,111]. Heterologous epi-isozizaene synthase from S. coelicolor A3(2) and pentalenene synthase from Streptomyces sp. UC5319 produced 103, pentalenene (107) and α-isocomene (108) [111]. Using the genetic engineering techniques increased the yields of 108, 103, and 107 in E. coli to 77.5 mg/L, 727.9 mg/L, and 780.3 mg/L, respectively, while the yield of 107 was improved to 344 mg/L in Saccharomyces cerevisiae (US20200239796).

197
Genome mining of S. venezuelae ATCC 15439 revealed ven, a silent biosynthetic cluster responsible for the synthesis of diterpenoids venezuelaenes A (198) and B (5-oxo-venezuelaene A) (199) with a unique 5-5-6-7 tetracyclic skeleton [166].  performed a mechanistic study of two diterpene cyclases, spiroviolene synthase from S. violens NRRL ISP-5597 and tsukubadiene synthase from S. tsukubaensis NRRL 18488, which catalyze the formation of 200 and 201. Although the structures of 200 and 201 are significantly different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, which proved their phylogenetic similarity [167,168]. The generation of a new tetracyclic diterpene cattleyene (202) was observed using the recombinant TS CyS from S. cattleya NRRL 8057 [169].  The heterologous expression of hopA and hopB (encoding squalene/phytoene synthases) and hopD (encoding farnesyl diphosphate synthase) from S. peucetius ATCC 27952 in E. coli provided an acyclic triterpene squalene (230) with a yield of 11.8 mg/L [173]. Another acyclic triterpene, botryococcene (231), was produced by activating the Fur22 regulator and simultaneous expression of the biosynthetic genes of S. reveromyceticus SN-593. The yield of the target product was 0.3 g/L, which is comparable to the levels of other microbial producers [174].
Hopanoids are unusual pentacyclic triterpenes present in bacterial species. Hop-22(29)-ene (290) was isolated from wild-type [175,176] and genetically modified strains of streptomycetes [72,177]. A genome-wide analysis of S. scabies 87-22 detected a hopanoid biosynthetic cluster responsible for the synthesis of 232 [178]. The squalene-hopene cyclase (spterp25) catalyzing the complex cyclization of 230 to the pentacyclic triterpene 232 was described for S. peucetius ATCC 27952 [179]. Meroterpenoids are products of mixed biosynthetic origin that consist of terpenoid scaffold combined with polyketide, alkaloid, phenol, or amino acid. According to their different biosynthetic origins, meroterpenoids can be divided into two groups, polyketide and non-polyketide terpenoids. Meroterpenoids have attracted researchers' attention due to their unusual chemical structures and a wide range of biological properties [180].
Naphthoquinone-based meroterpenoids are large chemically diverse group including napyradiomycins, merochlorins, marinones, furaquinocins, etc., some of which have a high therapeutic potential. Naphthoquinone-based meroterpenoids derived from streptomycetes are described in the review published in 2020 [181], so our review highlights the most active producers and the derivatives with promising biological activity, as well as compounds isolated after 2020.
Biosynthesis of naphthoquinone-based meroterpenoids includes regioselective addition of aromatic polyketide (1,3,6,8-tetrahydroxynaphthalene) to a terpene diphosphate catalyzed by ABBA prenyltransferase (PTase). After the initial prenylation, oxidation, halogenation and cyclisation steps occur. Genome mining of streptomycetes as producers of naphthoquinone-based meroterpenoids led to the discovery of unique prenyltransferase and vanadium-dependent haloperoxidase (VHPO) enzymes, which differ significantly from those previously described for algae and fungi [182,183]. For instance, the high-resolution crystal structures of two homologous members of the VHPO family associated with napiradiomycin biosynthesis, NapH1 and NapH3, were characterized [184].

Terpene Derivatives Produced by Others Actinomycetes and Their Enzymes
Although most of the found actinomycete terpene derivatives are synthesized by streptomycetes, there is an increasing number of publications on terpene biosynthesis by representatives of the genera Nocardiopsis, Amycolatopsis, Isoptericola, Saccharopolyspora, Salinispora, Kitasatosporia, Verrucosispora, etc. The compounds produced are represented mainly by sesqui-and diterpenes and their derivatives.
Hopanoid lipids (465-482) were found in the genus Frankia [289] with the highest level among all known organisms. Short stretches of DNA have been identified that are thought to contain squalene-hopene cyclase genes (shc) [290]. A new sesquarterpenoid identified as heptaprenylcycline B (483) was isolated from the cell walls of nonpathogenic mycobacteria [291,292]

Discussion
The present review demonstrates that actinomycetes synthesize a wide variety of terpene derivatives ranging from monocyclic monoterpenes to polycyclic tri-and tetraterpenes and their various derivatives. Most actinomycete terpene derivatives are produced by Streptomyces, however, terpene biosynthesis by Allokutzneria, Amycolatopsis, Frankia, Kitasatosporia, Nocardia, Salinispora, Verrucosispora, etc., have been recently reported (Figure 3). The total number of identified terpenes and their derivatives exceeds 500. Among terpenes and terpenoids, sesqui-and diterpenoids predominate. The ability of streptomycetes to synthesize a wide range of hybrid metabolites (meroterpenoids), the total number of which exceeds 190, was shown. More than 350 actinomycete-derived terpenoids and meroterpenoids are novel compounds and frequently with unique carbon skeletons (Figure 4).  An extensive development of genome-sequencing technologies and bioinformatics tools have allowed the discovery of BCGs (including silent ones) in the genome of actinomycetes. That terpenoids and meroterpenoids are predominantly found among Streptomyces strains is presumably due to plenty of available genetic information about this group of actinomycetes. As of 26 June 2022, 1784 scaffold-level and 745 complete-level genome sequences of Streptomyces strains were available in the NCBI database. Recent genetic studies have shown that the biosynthetic potential of these actinomycetes is enormous. A genome-wide analysis of 22 Streptomyces species revealed more than 900 biosynthetic clusters; for most of these, the products are still unidentified [309]. In addition, Streptomyces are preferred hosts for the heterologous expression of terpene biosynthetic clusters from other microorganisms [48,50,310]. Since 2015, high biosynthetic potential of actinomycete genera such as Saccharopolyspora [311], Nocardiopsis [312], Rhodococcus [313,314], Salinispora [315], Verrucosispora [316], and Actinomadura [317] have been demonstrated. For instance, a genome-wide analysis of terpentecin-or brasilicardin-producing strains K. griseola MF730-N6 [318] and N. terpenica IFM0406 [319] revealed 15 and 47 BGCs yielding unidentified natural products, respectively. One of the main problems in terpene biosynthesis is that most biosynthetic clusters are silent; therefore, searching for methods of their activation is an urgent research direction. Currently, great success has been achieved in this field due to methods of heterologous expression and/or genome editing of the native producer [320]. Genomic data of the described actinomycete species demonstrated that 90% of the biosynthetic potential of these microorganisms is untapped yet and the possibility of discovering novel terpenoids with potential therapeutic effects remains [15,52,310,321]. Microbial collections can serve as a "springboard" for the discovery and patenting of new producers of bioactive terpene derivatives, as they include identified and well-characterized pure microbial cultures. For instance, the Regional Specialized Collection of Alkanotrophic Microorganisms (acronym IEGM, Perm, Russia; World Federation for Culture Collections # 285; USU 73559; http://www.iegmcol.ru/strains, accessed on 25 March 2022) contains more than 3000 strains of actinomycetes with a wide range of metabolic capabilities, which are promising for biocatalytic production of terpene derivatives [322][323][324][325][326] (RU0002529365).
Unlike the biosynthesis of well-studied secondary metabolites, such as polyketides and nonribosomal peptides, the prediction of terpene structures requires detailed understanding of the cyclization mechanisms and the structural characteristics of bacterial TSs [321,327]. In this regard, a separate research area is isolation of individual actinomycete terpene synthases, and description of their structural and mechanistic characteristics, as well as the study of terpene cyclization mechanisms. The crystal structures of linalool/nerolidol, 2-methylisoborneol, germacradienol/germacrene D, selina-4(15),7(11)diene, epi-zizaene, pentalenene, cucumene, (E)-biformene synthases, and other TSs isolated from streptomycetes were characterized. In turn, genome mining of streptomycetes as producers of naphthoquinone-based meroterpenoids led to the discovery of unique prenyltransferase (PTase) and vanadium-dependent haloperoxidase enzymes (VHPO) [182,183]. For instance, the high-resolution crystal structures of two homologous members of the VHPO family associated with napiradiomycin biosynthesis, NapH1 and NapH3, were characterized [184]. It has been found that bacterial TSs, PTases, and VHPOs differ significantly from the plant or fungi ones as well as from each other. Moreover, they are capable of producing dozens of different compounds, which distinguishes them from most bacterial biosynthetic enzymes [46]. By the example of an epi-zizaene synthase, the successful application of site-directed mutagenesis of the enzyme to control the range of the compounds produced was proved [110,122] (WO2015120431).
Actinomycetes produce terpenoids with various biological and pharmacological activities such as antimicrobial, anticancer, antioxidant, antiviral, anti-inflammatory, immunosuppressive, etc. (Table 2). However, the bioactivity for most of the new actinomycetederived terpenoids has not yet been determined but may be discovered in the future. For instance, napyridymycins A1 and A80915 A, B, C, D were originally known as antimicrobial agents, but after 2010, their high antiviral and cytotoxic activity have been determined. Among the biologically active actinomycete terpenoids, compounds with pronounced antimicrobial activity predominate ( Figure 5A). They seem to inhibit the growth of extraneous microflora and render actinomycetes competitive in the microbial community. This statement is confirmed by the fact that some actinomycetes begin to produce terpenoids in the presence of other microorganisms. Thus, S. cinnabarinus PK209 and S. hygroscopicus HOK021 (NITE P-02560) synthesize the diterpene lobocompactol and the antibiotic platensimycin in the presence of the Gram-negative Alteromonas sp. KNS-16 [140] and the Gram-positive Tsukamurella pulmonis TP-B0596 (JP2019149945), respectively. The effectiveness of actinomycete terpenoids and meroterpenoids, namely pentalenolactone, albaflavenone, platensimycin, platencin, terpentecin, lavanducyanin, marinocyanins A-C, furaquinocin L, 3-dechloro-3-bromonapyradiomycin A1, napyradiomycin A1, and merochlorin A, as promising antibiotics has been proven. This is true for cyslabdan, which enhances the action (1000-fold) of the antibiotic imipenem against MRSA. In addition to high antibacterial activity, many meroterpenoids, such as napyradiomycins B1, B3, B4, A80915A, B, C, furaquinocins A and B, murayaquinone, marinocyanin A-C, and saccharoquinoline, exhibit a high cytotoxic activity against different cancer cell lines ( Figure 5B). (The x-axis indicates the number of compounds with a certain type of activity (for meroterpenoids isomers also were counted)).
The high biological activity of meroterpenoids is probably associated with the addition of an isoprene fragment to the pharmacophore polyketide part that increases the affinity for biological membranes. The unique biological and structural properties of meroterpenoids contribute to the search for methods of their total and semi-synthetic synthesis [328][329][330].
Actinomycete-derived terpenoids participate in specific interactions with macroorganisms (plants and animals), regulate the bacterial life cycle, perform protective functions, or serve as taxonomic markers. Bacterial terpenoids are often optical isomers of plant terpenoids and may represent two chemical communication channels that do not overlap even if the same habitat is occupied by prokaryotic and eukaryotic organisms producing terpenes [103]. Soil-smelling terpenoids geosmin and 2-methylisoborneol were shown to play the role of signaling molecules for springtails (Collembola), which spread Streptomyces spores in the soil [331]. According to other reports, these terpenoids are aposematic signals used to indicate the unpleasant taste qualities of toxin-producing microbes, preventing predation by eukaryotes [332]. Čihák et al. (2017) pointed out that during germination of S. coelicolor M145 spores, they synthesize albaflavenone, which may coordinate the development of the producer (quorum sensing) and/or play a role in the competitive repression of microflora (quorum suppression) in the natural environment [117]. In the liquid culture, S. coelicolor A3(2) does not produce aminobacteriohopanetriol or produces this compound in negligible amounts. However, the triterpene generation increased sharply during the formation of an aerial mycelium and sporulation, which may be associated with structural changes in the membrane and protection against water loss [176]. In addition, some TSs and terpene derivatives are so unique that they can become a taxonomic trait and be used to identify different groups of actinomycetes. For instance, the bioinformatics analysis of all sequenced Micromonospora isolates revealed TS genes, which differ significantly from other groups of characterized bacterial TSs and may be useful as markers of the genus, while Mycobacterium tuberculosis H37Rvн produced specific diterpene nucleosides, 1-and N 6 -tuberculosinyladenosines, promising for development as specific diagnostic markers of tuberculosis.

Conclusions
Thus, the synthesis of terpenes and terpenoids is an important pathway in the secondary metabolism of actinomycetes. The compounds produced may be promising therapeutic agents for the treatment of viral, inflammatory, cancerous, and other diseases in the future. Terpenoids and meroterpenoids synthesized by actinomycetes and possessing high antibacterial activity against drug-resistant pathogenic microorganisms may be useful for the development of new antibiotics. Further study of actinomycetes, accumulation of genetic information about this group of microorganisms, and employment of modern and development of novel tools of synthetic biology and genetic engineering will open prospects for creation of ideal "cell factories" using actinomycetes.
Author Contributions: All authors have read and agreed to the published version of the manuscript.