*2.2. Biosynthetic Potential of Leptothoe*

The genomic repertoire for secondary metabolism of seven *Leptothoe* genomes was predicted using antiSMASH (Figure 4a,b). *Leptothoe* genomes were found to harbor a considerable number of BGCs (121), the majority of which have no known end product. BGCs with unknown end products are present in almost all cyanobacterial genomes [42], and on the other hand, there are still natural products for which a biosynthetic origin

is unknown [43]. The vast majority of BGCs in *Leptothoe* genomes were predicted to encode non-ribosomal peptide synthetases (NRPS) (24 BGCs), followed by type I polyketide synthase (T1PKS) (20 BGCs) and bacteriocin (17 BGCs) (Figure 4, Table S2). Previously, genome-mining efforts have revealed that a major fraction of cyanobacterial natural products is produced using NRPS or PKS enzymes systems [43,44]. Bacteriocin BGCs, which are widespread in cyanobacterial genomes [45], were detected in almost all *Leptothoe* genomes (except for *Le. kymatousa* TAU-MAC 1615). Bacteriocins have been mainly reported to exhibit antimicrobial activity [46], but are also promising as antivirals, plant protection agents, and anticancer agents [47]. Further, it is suggested that bacteriocins may be involved in shaping bacterial communities through inter- and intra-specific interactions [47]. In addition, lassopeptide and terpene synthase BGCs were detected with high relative abundance in almost all the *Leptothoe* genomes, while cyanobactin and arylpolyene BGCs were rarely found in some of the genomes. Terpene BGCs, reported in a wide variety of bacteria including cyanobacteria [48], were also present in all *Leptothoe* genomes. The considerably similar *Leptothoe* strains CCMR0081 and CCMR0081 (96% average nucleotide identity) associated with corals and macroalgae (isolated from turfs) showed the highest number of natural product BGCs (Figure 4a,b, Table S2). In contrast, the two spongeassociated *Leptothoe* species (sharing ≈84% average nucleotide identity) showed the lowest number of natural product BGCs. Interestingly *Le. spongobia* strain harbored a BGC encoding for a lanthipeptide (Figure 4b). Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities [49], while their detection and isolation are restricted to bacteria [43]. Lanthipeptide BGCs are particularly found in the genomes of many genera of Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, and Cyanobacteria [50].

**Table 2.** Eukaryotic-like protein repeats across symbiotic and free-living cyanobacterial genomes.


We assigned producers of known natural products to the *Leptothoe* lineage by combining 16S rRNA phylogenetic analysis with the "Comprehensive database of secondary metabolites from cyanobacteria 'CyanoMetDB'" [51]. We searched the database for entries attributed to *Leptolyngbya*, *Pseudanabaena* (*Pseudanabaena persicina* = *Leptolyngbya ectocarpi*), *Phormidium* (*Phormidium ectocarpi* = *Leptolyngbya ectocarpi*), or thin filamentous strains of pinkish color, and where a sequence was available, it was included in our phylogenetic analysis (Table S3, Figure 5). This analysis demonstrated that two strains previously assigned to *Leptolyngbya*, *Leptolyngbya ectocarpi* SAG 60.90, and *Leptolyngbya* sp. RS03, reported to produce compounds such as hierridin B, grassypeptolides D and E, a lyngbyastatin analogue, and dolastatin 12, belong to the *Leptothoe* genus (Table S3, Figure 5). However, the natural product BGCs involved in the biosynthesis of the abovementioned compounds have not been studied as the genomes of these *Leptothoe* strains have not been sequenced yet. Our phylogenetic analysis revealed that *Leptothoe* was closely affiliated with three other marine

benthic cyanobacteria, *Salileptolyngbya* and two strains with unknown taxonomic status (Cyanobacterium csf1 and Filamentous cyanobacterium FLK9); csf1 was found to produce two new cyclic depsipeptides, companeramides A and B (Figure 5; CyanoMetDB). Further, in our analysis, other genera of marine origin with benthic lifestyle and often with a reddish to pinkish thallus color, known for the production of natural products such as linear and cyclic peptides, linear and cyclic non-peptides, and linear and cyclic depsipeptides, were extracted from the CyanoMetDB database. These chemically rich genera—*Moorea*, *Caldora*, *Symploca*, *Okeania*, and *Hormoscilla*—were placed in separate clades inside Oscillatoriales and were found to be distantly related to *Leptothoe* (Figure 5).

**Figure 5.** Phylogenetic relationships of *Leptothoe* strains based on the 16S rRNA gene sequence, in relationship to representative strains of other marine filamentous cyanobacteria with benthic lifestyle, with *Gloeobacter* violaceus as outgroup. The tree was constructed with the Bayesian inference (BI) method and the maximum-likelihood (ML) method; BI topology is demonstrated. Support values are indicated as posterior probability for Bayesian inference and bootstrap support for maximum likelihood analysis. The bar represents 0.04 nucleotide substitutions per site.

Most of the natural products from marine cyanobacteria have been isolated from *Moorea* [52], which occur in high densities in marine environments of tropical and subtropical regions, making the harvest of biomass easily accessible [52]. Similarly, *Symploca*, *Caldora,* and *Okeania* form large populations attached to hard substrates in marine habitats [53,54] and yield a great number of natural products. Novel compounds with strong anticancer properties such as apratoxins, grassypeptolides, wewakazole B, odoamide, and caldoramide are isolated from these marine genera [55–59]. Interestingly, cyclic depsipeptides are the main peptides with cytotoxic effects isolated from marine cyanobacteria, including 76 compounds [60]. Genome-mining analysis conducted in the present study for >70 marine cyanobacteria also highlighted the high metabolic potential of the well-studied Oscillatoriales; numerous BGCs were identified in their genomes (Figure 4c). On the other hand, other marine filamentous cyanobacteria with smaller trichomes and a slower growth rate, such as *Leptolyngbya*-like or *Pseudanabaena*-like, have been overlooked [52], as well as the members of *Leptothoe* genus according to our analysis. Herein, we revealed another promising benthic marine cyanobacterium for novel natural products biosynthesis, *Leptothoe*, that warrants further exploration.
