Natural Products and Biological Activity from Actinomycetes Associated with Marine Algae

Marine natural products have been recognized as the most promising source of bioactive substances for drug discovery research. This review illustrates the diversity of culturable actinobacteria associated with marine algae, their bioactivity and metabolites, and approaches to their isolation and determination of their biological properties. Furthermore, actinobacteria associated with marine algae are presented as a new subject for an extensive investigation to find novel and active natural products, which make them a potentially rich and innovative source for new drug development deserving more attention and exploration.


Introduction
More than 50% of new drug discoveries are derived from natural products and their derivatives, and natural products play an important role in drug discovery [1]. Over recent decades, natural marine products have been a fruitful field for organic chemistry research; more than 39,845 publications and 40,218 compounds have been deposited in the database of marine natural products research (http://pubs.rsc.org/marinlit, accessed on 5 June 2023). Marine organisms are excellent producers of secondary metabolites with diverse structures and bioactivities due to their special habitations and unique ecological conditions, such as low or high temperatures, low pH, high pressures, and high salt concentrations [2,3]. Marine microorganisms have taken the limelight as a producer of active natural products, including anti-bacterial, anti-fungal, anti-viral, enzyme, antibiofilm, anti-cancer, anti-oxidant, and anti-inflammation substances [4]. Due to co-existing with their host and the possible production of compounds with novel structures and diverse activities, marine microorganisms associated with marine animals and plants have attracted much attention [5,6].
Marine algae have attracted attention as a source of new bioactive molecules of biomedical interest, and they provide suitable living conditions and abundant nutrition for different microorganisms, while in return, microorganisms may provide protection and ultimately survival value to their hosts by producing bioactive molecules, or by affecting the growth and evolution process via the signal transduction pathway [5][6][7][8][9]. Although algae-associated microorganisms have great potential for secondary metabolite synthesis, some related studies deserve more attention [10,11]. At present, the research on algaeassociated microorganisms focuses on macroalgae-associated fungi, and more than 400 new natural products have been obtained from them, which have anti-tumor, anti-bacterial, anti-oxidant, and insecticidal activities, providing a rich and innovative source for new Algae are one of the major contributors to marine ecosystems, and are found in almost all parts of the coastal regions around the globe [24]. According to size, marine algae, also known as seaweed, can be divided into macroalgae and microalgae. Furthermore, macroalgae can be classified into brown algae (phaeophyceae), red algae (rhodophyceae), and green algae (chlorophyceae), due to their different pigmentations [25]. Additionally, algae can provide a relatively stable and nutrient-rich habitat for microorganisms that live on their surface, and harbor diverse groups of bacteria, depending on the species and season [26,27].
To obtain actinomycetes strains associated with marine algae, fresh algal samples should be placed in individual sterile plastic bags and transported at 4 • C; these samples should then be processed immediately. Firstly, fresh samples should generally be rinsed at least three times with sterile seawater and undergo surface sterilization with 70% EtOH for a few seconds, before being aseptically cut into small pieces and homogenized with a sterile pestle in moderate sterile seawater [15]. Secondly, the polished samples should be serially diluted and plated onto the isolation media. These prepared samples may be heat-treated (such as at 55 • C for 5 min) and selective culture media that contain antibiotics to inhibit the growth of Gram-negative bacteria and fungi may be chosen [28][29][30][31]. Thirdly, along with the prepared plates incubated at 28 • C for 2-8 weeks, the emergence of actinomycetes colonies should be assessed every week [15,28]. Then, colonies are selected, and pure cultures are obtained by repeated streaking on agar plates. Finally, actinomycetes-like strains are selected based on the colony morphology: solid density of colonies, growth inside the agar media, and steady border of the colonies [32].
To identify the isolated actinomycetes, the 16S rRNA gene sequencing method should be employed. In detail, the 16S rRNA gene of these isolated actinomycetes should be amplified by PCR with the universal primers 27F and 1492R, using their genomic DNA as templates. Then, these PCR products should be sequenced and submitted to blast the NCBI Gen-Bank or the EMBL database using Basic Local Alignment Search Tool (BLAST) [28,[31][32][33]. Lastly, these 16S rRNA sequences should be aligned and subjected to a phylogenetic analysis using MEGA software (version 11) [28].

Abundance of Actinomycetes Associated with Marine Algae
Marine algae harbor a diverse group of bacteria, depending on the season, species, and thallus structure [26,27], and the actinomycetes associated with marine algae are less studied. Ulfah et al. reported that a total of 15 actinobacteria were isolated from the red algae Gelidiella acerosa collected from Drini Gunungkidul Yogyakarta [34]. Rajivgandhi et al. reported that 50 endophytic actinomycetes were isolated from green algae Cauler pataxifolia [35] and 100 actinomycetes strains were isolated from brown macroalgae Turbinaria ornata and Sargassum wightii, collected from the southeast coast of Tamil Nadu, India [36]. Four actinomycetes strains associated with the brown algae Sargassum cinereum and three actinomycetes strains associated with the green algae Codium dwarkense were obtained by Majithiya et al. in 2022 [37]. Ninety actinomycetes strains were isolated from the brown algae Laminaria ochroleuca by Girão et al. [29]. Thirty-six actinomycetes were obtained from the marine brown algae Laminaria saccharina by Wiese et al. from the Baltic Sea, Germany [15].

Biological Activities of the Actinomycetes Associated with Marine Algae
The most studied biological activity of actinomycetes associated with marine algae is anti-bacterial activity. As the report from Wiese et al. in 2009 showed [15], 36 actinobacteria, obtained from the marine brown algae Laminaria saccharina, showed different inhibition capacities of Bacillus subtilis, Escherichia coli, Staphylococcus lentus and/or Candida albicans. Of 100 actinomycetes, 40 isolated from brown macroalgae Turbinaria ornata and Sargassum wightii were active in antagonistic activity against various clinical pathogens [36]. Of a total of 15 actinobacteria, isolated from the red algae Gelidiella acerosa, 8 showed inhibition against Vibrio alginolyticus [34]. Of 50 endophytic actinomycetes, 20 isolates isolated from green algae Cauler pataxifolia showed antimicrobial activity against urinary tract infections bacteria (including E. coli, Proteus mirabilis, Pseudomonas aeruginosa, Kilebsiella pneumonia, and Enterobacter sp.) and the strain DMS 3 showed the best anti-bacterial activity among them [35]. Girão et al. (2019) also obtained 90 actinobacterial strains from brown algae Laminaria ochroleuca; 45 isolates inhibited the growth of C. albicans and/or Staphylococcus aureus, and 28 extracts among them affected the viability of at least one human cancer cell line (breast carcinoma T-47D or neuroblastoma SH-SY5Y) and non-carcinogenic endothelial cell line (hCMEC/D3) [29]. The crude extract and partially purified compounds from Nocardiopsis sp. DMS 2 were shown to have high inhibition activities against biofilmforming K. pneumoniae [45].
Some actinomycetes associated with marine algae have been reported to show special enzyme activities, flocculating activity, and heavy metal sorption. Streptomyces sp. SNA-JSM6 not only produced 56 U/mL of α-amylase, but also showed excellent anti-bacterial activity against selected pathogenic bacteria (P. aeruginosa, Enterobacter sp., Salmonella sp., and Micrococcus luteus) [46]. Nocardiopsis sp. GRG 3 showed a maximum flocculating activity of 80.90% with glucose, and the yield was 4.52 g/L. Furthermore, its heavy metal sorption effectively removed 55.90% Cd, 85.90% Cr, 74.7% Pb, and 51.90% Hg [47]. Micrococcus sp. GNUM-08124 could use agar as the sole carbon source, and showed higher agarase activity when cultured in an oligotrophic culture medium than in a rich media [48]. Streptomyces sp. SNJASM6 not only showed significant emulsification activities with tween 20, coconut oil, and xylene (which are the subsequent substrates of surfactant, oils, and hydrocarbons respectively), but also showed activity against bacterial pathogens including E. coli, Bacillus cereus, P. aeruginosa, Klebsiella pneumoniae and C. albicans [49].

Bioactive Metabolites from Actinomycetes Associated with Marine Algae
There are 82 compounds that have been isolated from 20 actinomycetes associated with marine algae. Additionally, 35 new metabolites have also been isolated from these actinobacteria. Depending on their chemical structure, the metabolites are classified into polyketides, peptides, glycoglycerolipids, alkaloids, and pyrones. These compounds also showed diverse biological activities, and they are described below in the order of the Latin names of their producers.

Bioactive Metabolites from Actinomycetes Associated with Marine Algae
There are 82 compounds that have been isolated from 20 actinomycetes associated with marine algae. Additionally, 35 new metabolites have also been isolated from these actinobacteria. Depending on their chemical structure, the metabolites are classified into polyketides, peptides, glycoglycerolipids, alkaloids, and pyrones. These compounds also showed diverse biological activities, and they are described below in the order of the Latin names of their producers.
Streptomyces althioticus MSM3 was isolated from intertidal macroalgae brown algae Ulva sp. Collected from the Cantabrian Sea in Pedreña. A new compound desertomycin G (16) (Figure 1) was separated from its liquid fermentation with an R5A medium. Compound 16 exhibited inhibitory activities against clinical infection pathogens, including the strong inhibition of Gram-positive bacteria (Corynebacterium urealyticum, S. aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, Clostridium perfringens and Mycobacterium tuberculosis) and the moderate inhibition of Gram-negative bacteria (Bacteroides fragilis, Haemophilus influenzae and Neisseria meningitidis). Additionally, it can decrease the viability of tumor cell lines MCF-7 (human breast adenocarcinoma) and DLD-1 (colon carcinoma) [67].
Molecules 2023, 28, x FOR PEER REVIEW 8 of 17 As regards Streptomyces albidoflavus KC180, isolated from the marine brown algea Carpodesmia tamariscifolia, collected from the Atlantic coast of Morocco, the organic extracts of fermentation broths showed anti-bacterial activity to methicillin-resistant Staphylococcus aureus (MRSA), imipenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. Further research showed that it produced the active metabolite desferrioxamine B (14) and its new derivative desferrioxamine B2 (15) (Figure 1) against multidrug-resistant bacteria [51].
Streptomyces sundarbansensis WR1L1S8 was associated with brown algae Fucus sp., collected along the Bejaia coastline, Algeria. A new polyketide (31) with three known phaeochromycins (32-34) (Figure 3) was obtained from agar solid fermentation. The new compound 31 was the major metabolite under culture conditions, and its activity against the pathogenic MRSA was prominent, with an MIC of 6 µΜ. In addition, the compounds 31, 33, and 34 also showed potent activity against E. coli and P. aeruginosa [53].
Streptomyces violaceoruber SCH-09 was isolated from brown algae Undaria pinnatifida (collected from the coast of Korea) and screened out for its anti-fouling activities from culture extracts. Two furanone derivatives, omF (35) and omF2 (36) (Figure 3), were obtained as active compounds from its culture extracts, and they showed anti-fouling activities against zoospores of U. pertusa, mussel M. edulis, and diatom N. annexa, with an EC50 range of 0.02-0.1 µM [60].
Streptomyces sundarbansensis WR1L1S8 was associated with brown algae Fucus sp., collected along the Bejaia coastline, Algeria. A new polyketide (31) with three known phaeochromycins (32-34) (Figure 3) was obtained from agar solid fermentation. The new compound 31 was the major metabolite under culture conditions, and its activity against the pathogenic MRSA was prominent, with an MIC of 6 µM. In addition, the compounds 31, 33, and 34 also showed potent activity against E. coli and P. aeruginosa [53].
Streptomyces violaceoruber SCH-09 was isolated from brown algae Undaria pinnatifida (collected from the coast of Korea) and screened out for its anti-fouling activities from culture extracts. Two furanone derivatives, omF (35) and omF2 (36) (Figure 3), were obtained as active compounds from its culture extracts, and they showed anti-fouling activities against zoospores of U. pertusa, mussel M. edulis, and diatom N. annexa, with an EC 50      Streptomyces sp. OUCMDZ-3434 was isolated from the marine green algae Enteromorpha prolifera, collected from Zhanqiao Beach, Qingdao, Shandong Province, China. The EtOAc extract of its fermentation broth exhibited significant α-glucosidase inhibitory activity at 50 µg/mL. In addition, two new epimeric polyketides (wailupemycins H (46) and I (47)) with an unusual carbon skeleton, along with the three known compounds 5, 6, and wailupemycins G (48) (Figure 5), were obtained in the chemical study. Furthermore, the five compounds 5, 6, and 46-48 showd stronger inhibition of α-glucosidase and lower cytotoxicity than acarbose, with the IC 50    Streptomyces sp. OUCMDZ-3434 was isolated from the marine green algae Enteromorpha prolifera, collected from Zhanqiao Beach, Qingdao, Shandong Province, China. The EtOAc extract of its fermentation broth exhibited significant α-glucosidase inhibitory activity at 50 µg/mL. In addition, two new epimeric polyketides (wailupemycins H (46) and I (47)) with an unusual carbon skeleton, along with the three known compounds 5, 6, and wailupemycins G (48) (Figure 5), were obtained in the chemical study. Furthermore, the five compounds 5, 6, and 46-48 showd stronger inhibition of α-glucosidase and lower cytotoxicity than acarbose, with the IC50/CC50 values of 19.    The continuous study of the remaining part of the EtOAc extract led to the isolation and identification of five new polyketides, 3-O-methylwailupemycin G (49), wailupemycin J (50), R-wailupemycin K (51), S-wailupemycin K (52) and wailupemycin L (53) (Figure 5), along with the known compounds 7 and 8. In addition, compound 49 showed a-glucosidase inhibition with an IC 50 863.6 µM, and 52 was cytotoxic on the HeLa cell with an IC 50 8.2 mM. Furthermore, 8, 51, and 52 showed inhibitory activities against the H1N1 virus, with inhibition rates of 47.8%, 42.5%, and 60.6% at a concentration of 50 µM, respectively [62].
Streptomyces sp. PNM-9 isolated from the brown algae Dictyota sp. exhibited the ability to inhibit the in vitro growth of phytopathogens Burkholderia glumae and Burkholderia gladioli. Two known compounds (66, 67) ( Figure 5) were identified from the organic extract of a 15-day LB media culture, and were active against the rice pathogenic bacteria B. glumae with MICs of 2.43 mM and 1.21 mM, respectively [52].
Streptomyces sp. PNM-9 isolated from the brown algae Dictyota sp. exhibited the ability to inhibit the in vitro growth of phytopathogens Burkholderia glumae and Burkholderia gladioli. Two known compounds (66, 67) ( Figure 5) were identified from the organic extract of a 15-day LB media culture, and were active against the rice pathogenic bacteria B. glumae with MICs of 2.43 mM and 1.21 mM, respectively [52].
Streptomyces sp. YM5-799 was isolated from the surface of brown algae, collected from Hokkaido in north Japan. Three new catechol-type siderophores, streptobactin (68), dibenarthin (69), and tribenarthin (70), along with a known benarthin (71) (Figure 6), were obtained from the culture broth (ASG medium containing 0.1 µM FeCl3) of the strain. Compounds 68, 69, and 71 shoed an Fe-chelating activity, with the ED50 values 156, 117, and 937 µM, comparable to that of deferoxamine mesylate (ED50 = 195 µM) using a CAS assay [50].  Streptomyces sp. ZZ502 is associated with the green algae Ulva conglobatea growing on rocks on the coast of Zhoushan Archipelago in the East China Sea. Three new compounds, 72-74, together with three known benzamide derivatives, 75-77 ( Figure 6), were isolated from the solid culture extract. None of these isolated compounds showed activity in inhibiting the proliferation of glioma cells or the growth of MRSA, E. coli, or C. albicans [64].
Streptomyces sp. ZZ502 is associated with the green algae Ulva conglobatea growing on rocks on the coast of Zhoushan Archipelago in the East China Sea. Three new compounds, 72-74, together with three known benzamide derivatives, 75-77 ( Figure 6), were isolated from the solid culture extract. None of these isolated compounds showed activity in inhibiting the proliferation of glioma cells or the growth of MRSA, E. coli, or C. albicans [64].
The unidentified actinomycete CNC-837 was isolated from the surface inoculum of brown algae Lobophora variegate, which was collected from the Caribbean and produced two new macrolides. Two new compounds, 23 and 24 (Figure 2), showed anti-inflammatory activity, inhibiting topical PMA-induced edema in the mouse ear assay [55].
Natural products are a large resource for the development of drugs, and also a promising area for therapeutic agents [1,12,72]. Combining the microbial versatility and particularities of the marine environment, marine microorganisms have been considered to be the most promising natural source for drug discovery [13,72]. Marine actinomyces have shown an excellent biosynthetic ability to generate bioactive metabolites [23]. The most studied marine actinomyces are Streptomyces. Up to 2016, 547 new compounds had been isolated from marine Streptomyces [73]. Thereafter, more than 100 new compounds were Micromonospora sp. CNY-010 was isolated from the surface of the brown algae Stypopodium zonale, collected from the Bahamas Islands. A new 28-membered macrolide containing 19 chiral centers named neomycin B (79) (Figure 7) was obtained from liquid fermentation. Compound 79 showed potent cytotoxicity, and was moderately active against RPMI-8226, a myeloma cell line involved in multiple myeloma [33].
The unidentified actinomycete CNC-837 was isolated from the surface inoculum of brown algae Lobophora variegate, which was collected from the Caribbean and produced two new macrolides. Two new compounds, 23 and 24 (Figure 2), showed anti-inflammatory activity, inhibiting topical PMA-induced edema in the mouse ear assay [55].
Natural products are a large resource for the development of drugs, and also a promising area for therapeutic agents [1,12,72]. Combining the microbial versatility and particularities of the marine environment, marine microorganisms have been considered to be the most promising natural source for drug discovery [13,72]. Marine actinomyces have shown an excellent biosynthetic ability to generate bioactive metabolites [23]. The most studied marine actinomyces are Streptomyces. Up to 2016, 547 new compounds had been isolated from marine Streptomyces [73]. Thereafter, more than 100 new compounds were added every year (except for 80 new compounds in 2020), and by 2021, more than 1196 new compounds had been obtained from marine Streptomyces [12,[74][75][76][77]. These compounds included alkaloids, polyketides, halogens, terpenoids, and peptides, among which most compounds exhibited tumor cytotoxicity, anti-bacterial, anti-malarial and anti-parasitic activates, glycosidase inhibition, and other biological activities. Furthermore, Nocardiopsis was also an important source of secondary metabolites of marine actinomyces. According to the statistics, 67 natural products had been obtained from marine Nocardiopsis by 2019, with structures including pyranone, diketopiperazine, polypeptide, and so on [78]. The compounds summarised in this review were mainly derived from Streptomyces, followed by Nocardiopsis and Micromonospora, which is consistent with the study of marine actinomyces. In addition, the secondary metabolites of the first obligate marine actinomycete genus Salinispora have been found with 30 different structures, including Salinosporamide A [79], which was approved by the U.S. Food and Drug Administration (FDA) as an orphan drug for the treatment of multiple myeloma (Marizomib). Salinispora was mainly distributed in tropical and subtropical marine sedimentary environments, and was also found in marine sponges, sea squirts, and corals [80]. Salinispora recently proved to be abundant in Hainan Xisha marine algae, and may provide a rich and innovative source for new drug candidates [28].
Related to the source of actinomycetes associated with algae, it is clear that the abundance of actinomycetes that are associated with algae was less than that of those associated with sponges or corals [21,44]. However, 82 naturally occurring products, including 35 new ones, have been obtained from only 20 strains of the actinomycetes associated with marine algae. Many strains isolated from marine algae related to this review have not yet studied for their secondary metabolites, especially the seven remaining active strains summarized in Table 2. On the other hand, marine algae are broadly distributed in the ocean, with a great diversity of between 30,000 and more than 1 million different species [81]; in other words, there is much more scope for the resources of actinomycetes associated with marine algae to be studied. Furthermore, new actinomycetes resources and their biosynthetic potential are an untapped source of novel molecules and natural products. In conclusion, actinomycetes associated with marine algae are a good source for isolating novel and bioactive natural products deserving more attention and investment.

Conclusions
Marine algae have emerged as a vast source of bioactive metabolites and unique structures since marine resources have been paid attention to. The interesting ecological relationship between algae and associated microorganisms has since been addressed. In addition, the research on new natural products derived from algae-associated fungi is focused, and a large number of natural products with anti-tumor, anti-bacterial, anti-oxidant, and insecticidal activities have been obtained to provide a rich resource for new drug candidates. In this review, we summarized the abundance and bioactivity of actinomycetes associated with marine algae, and assessed the secondary metabolites for the chemistry and bioactivity of the natural products found in them. In total, 22 genera in 11 families of cultivable actinomycetes were obtained from marine algae, and they exhibit diverse biological activities, such as anti-bacterial activity, anti-fungal activity, anti-inflammatory, anti-tuberculosis, cytotoxicity, herbicidal activity, special enzyme activities, flocculating activity, and heavy metal sorption. From these actinomycetes, 82 naturally occurring products, including 35 new ones, have been obtained, and most of them show a variety of bioactivities. It is noteworthy that brown algae are the most representative samples from which actinomycetes are isolated, and Streptomyces spp. are the main producers of these metabolites so far. The actinomycetes associated with marine algae represent a new structure and a new source of bioactive natural products; however, they are still underexplored. Optimistically, future research on actinomycetes associated with marine algae may yield new developments and even more amazing breakthroughs.
Author Contributions: Conceptualization, Z.G. and Z.X.; writing-original draft preparation, Z.X. and T.X.; writing-review and editing, Z.G., R.W., T.X. and Z.X.; supervision, S.Z. and S.M.; project administration, S.Z. and Z.X.; funding acquisition, Z.G. and S.M. All authors have read and agreed to the published version of the manuscript.