Cyanobacteria and Eukaryotic Microalgae as Emerging Sources of Antibacterial Peptides

Cyanobacteria and microalgae are oxygen-producing photosynthetic unicellular organisms encompassing a great diversity of species, which are able to grow under all types of extreme environments and exposed to a wide variety of predators and microbial pathogens. The antibacterial compounds described for these organisms include alkaloids, fatty acids, indoles, macrolides, peptides, phenols, pigments and terpenes, among others. This review presents an overview of antibacterial peptides isolated from cyanobacteria and microalgae, as well as their synergism and mechanisms of action described so far. Antibacterial cyanopeptides belong to different orders, but mainly from Oscillatoriales and Nostocales. Cyanopeptides have different structures but are mainly cyclic peptides. This vast peptide repertoire includes ribosomal and abundant non-ribosomal peptides, evaluated by standard conventional methodologies against pathogenic Gram-negative and Gram-positive bacteria. The antibacterial activity described for microalgal peptides is considerably scarcer, and limited to protein hydrolysates from two Chlorella species, and few peptides from Tetraselmis suecica. Despite the promising applications of antibacterial peptides and the importance of searching for new natural sources of antibiotics, limitations still persist for their pharmaceutical applications.


Introduction
The advent of the antimicrobial era are, together with sanitation and increasing access to safe drinking water, among the greatest milestones in public health [1]. Antibiotic therapy affords the control of many infectious diseases, otherwise highly lethal. In addition, it pushes forward the boundaries of many other medical treatments, such as immunosuppressive treatments or successful surgical procedures.
The notion of total elimination of infectious diseases by antibiotic therapies soon turned out to be utopic. Today, the world is facing a deep global antimicrobial resistance (AMR) crisis, with an alarming decrease of effectiveness in antibiotic treatments due to the rising resistance acquired by pathogens [2,3]. The overuse, and often misuse, of antimicrobials in clinics [3], is one of the main reasons of AMR, but not the only one. The induction of antibiotic resistance outside nosocomial settings, strongly associated to the antibiotic use in livestock farming [4], aquaculture [5], and the uncontrolled dumping of antibiotics into the environment [6,7], account for the horizontal transmission of antibiotic-resistance traits out of the nosocomial setting. Furthermore, zoonosis may act as a reservoir for resistant organisms. Altogether, the term "One Health" was coined as a common umbrella to Table 1. Antibacterial compounds from cyanobacteria.

Antibacterial Peptides from Cyanobacteria
Cyanobacteria are an almost endless source of new peptide scaffolds. The peptides are synthesized as secondary metabolites required for a successful strive with other microorganisms, as well as for their astonishing environmental adaptation [118]. The biotechnological and medical potential of cyanobacterial peptides has been frequently reviewed and updated in the literature [88,[119][120][121][122][123][124][125].
Since the first description of a cyanobacterial peptide with antibacterial activity, the cyclic peptide schizotrim A isolated from a culture of Schizothrix sp. many other peptides have been described. Table 3 summarizes the main cyanobacterial peptides, the producer species, their structure and their effect on known pathogenic target bacteria.
The antibacterial peptides from cyanobacteria are of different types, although those with a cyclic structure are more frequent (Figure 1). The antibacterial peptides identified peptides belong to different orders of cyanobacteria, being Oscillatoriales and Nostocales the most prolific ones. Inside Oscillatoriales, members of the genus Lyngbya are important producers of bioactive peptides with a potential therapeutic use. Four cyclic undecapeptides named lyngbyazothrins A, B, C, and D were identified from the freshwater strain Lyngbya sp. The mixtures A/B showed antimicrobial activity only against the Gram-positive bacteria Micrococcus flavus, while lyngbyazothrins C/D were active against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens), and the Gram-positive Bacillus subtilis, although not on methicillin susceptible Staphylococcus aureus [92,[125][126][127].
structure are more frequent (Figure 1). The antibacterial peptides identified peptides belong to different orders of cyanobacteria, being Oscillatoriales and Nostocales the most prolific ones. Inside Oscillatoriales, members of the genus Lyngbya are important producers of bioactive peptides with a potential therapeutic use. Four cyclic undecapeptides named lyngbyazothrins A, B, C, and D were identified from the freshwater strain Lyngbya sp. The mixtures A/B showed antimicrobial activity only against the Gram-positive bacteria Micrococcus flavus, while lyngbyazothrins C/D were active against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens), and the Gram-positive Bacillus subtilis, although not on methicillin susceptible Staphylococcus aureus [92,[125][126][127]. Other antibacterial peptides described for the Lyngbya genus are the lipopeptide pahayokolide A, and the depsipeptides pitipeptolide A-F. Furthermore, within the Oscillatoriales order, the Oscillatoria and the Phormidium genus are known producers of antibacterial peptides although with much lower representation than Lyngbya. All of them are cyclic peptides. Concerning the Nostocales order, the Nostoc genus stands out, with a variety of cyclic and linear antibacterial peptides; Anabaena and Scytonema genera produced depsipeptides and lipopeptides, respectively [88,94,125,[128][129][130][131][132].
The marine cyanobacterium Prochlorococcus marinus produces the lantipeptide prochlorosin, with encoding genes distributed throughout its genome. Lantipeptides are a large family of linear and cyclic peptides, ribosomally synthesized as precursor peptides that underwent post-translational modifications, including the formation of lanthionine bridges, heterocyclization, oxidation, methylation, prenylation, and cyclization. The formation of lanthionine bridges in prochlorosin is catalyzed by LanM, a lanthionine synthetase C enzyme [133].
This vast spectrum of peptides produced by cyanobacteria comprises not only the ribosomal synthesis of peptides, including their posttranslational modifications, but also abundant nonribosomal peptides (NRPs) synthesized by non-ribosomal peptide synthetases (NRPSs) [85,125,134], modular multienzymatic complexes working as an assembly line for amino acid incorporation into the polypeptide chain and, frequently, to their in situ modification. In addition NRPSs may appear associated to polyketide synthases (PKS), forming NRPS-PKS clusters [135,136] broadening even further the variety of chemical motifs incorporated into the polypeptide chain. Other antibacterial peptides described for the Lyngbya genus are the lipopeptide pahayokolide A, and the depsipeptides pitipeptolide A-F. Furthermore, within the Oscillatoriales order, the Oscillatoria and the Phormidium genus are known producers of antibacterial peptides although with much lower representation than Lyngbya. All of them are cyclic peptides. Concerning the Nostocales order, the Nostoc genus stands out, with a variety of cyclic and linear antibacterial peptides; Anabaena and Scytonema genera produced depsipeptides and lipopeptides, respectively [88,94,125,[128][129][130][131][132].
The marine cyanobacterium Prochlorococcus marinus produces the lantipeptide prochlorosin, with encoding genes distributed throughout its genome. Lantipeptides are a large family of linear and cyclic peptides, ribosomally synthesized as precursor peptides that underwent post-translational modifications, including the formation of lanthionine bridges, heterocyclization, oxidation, methylation, prenylation, and cyclization. The formation of lanthionine bridges in prochlorosin is catalyzed by LanM, a lanthionine synthetase C enzyme [133].
This vast spectrum of peptides produced by cyanobacteria comprises not only the ribosomal synthesis of peptides, including their posttranslational modifications, but also abundant non-ribosomal peptides (NRPs) synthesized by non-ribosomal peptide synthetases (NRPSs) [85,125,134], modular multienzymatic complexes working as an assembly line for amino acid incorporation into the polypeptide chain and, frequently, to their in situ modification. In addition NRPSs may appear associated to polyketide synthases (PKS), forming NRPS-PKS clusters [135,136] broadening even further the variety of chemical motifs incorporated into the polypeptide chain.  NRPSs represent a major class of secondary metabolites in cyanobacteria, with a broad range of biological and pharmacological properties, mostly as antibiotics. It can been speculated that blue-green algae acquired nrps genes after the first endosymbiotic process that led to the formation of algae, or algae may subsequent have lost nrps genes [101].
Microcystins are non-ribosomal cyclic heptapeptides; these toxins are the most commonly found in blooms produced by cyanobacterial genera, such as Microcystis, Anabaena, Nodularia, Oscillatoria, Nostoc, Cylindrospermopsis, Aphanizomenon, Planktothrix, Anabaenopsis, Synechocystis, and Lyngbya. Aside from them, the strain Synechocystis aqualitis M62C produces a microcystin active against S. aureus, but devoid of the mcyB gene, one of the genes related to the mycrocystin synthesis, whereas the strain S. aqualitis M204BG showed microcystin production and mcyB gene in its genome, but was inactive against S. aureus and P. aeruginosa [138].

Antibacterial Peptides from Microalgae
When compared with the number of cyanobacterial peptides, the number of antibacterial peptides from microalgae is considerably lower. The first report was an antibacterial 30mer peptide purified from the culture of Stichochrysis immobilis Pringsheim, active against marine bacteria [145].
The following reports correspond to protein hydrolysates. Sedighi et al. [146], evaluated the antibacterial activity of peptide fractions of the microalga Chlorella vulgaris. Protein fraction with 62 kDa were hydrolysates by pepsic digestion and antibacterial activity was determined against E. coli CECT 434. The effect of hydrolysate was 8.5 and 1.6 times greater than Chlorella biomass and its proteins, respectively, suggesting that Chlorella peptides provoked the cell wall destruction and cell growth inhibition. Furthermore, pepsin hydrolysates and peptide fractions from Chlorella sorokiniana displayed antibacterial activity against E. coli and S. aureus using the agar well diffusion method [147].
On the other hand, microalgae were also used for transgenic production of alien AMPs, as the bovine AMP lactoferricin by transgenic Nannochloropsis oculata. This transfected alga was used as biofunctional food for the medaka fish (Oryzias latipes) with an improved survival when challenged with Vibrio parahaemolyticus infection [149].
The limited knowledge of antibacterial peptides from microalgae will increase through the use of biotechnological tools such as transcriptomics that will help to understand its genome and its pharmacological interactions with bacteria. Transcriptomic sequencing will provide useful data to identify species with antibiotic potential and pathways for the synthesis of new functional metabolites. The transcriptome of the microalgae Chrysochromulina tobin revealed the expression of genes involved in the defense of the algae that encode potential antibiotics, antibiotic extrusion proteins, and novel antibacterial peptides [107].

Mechanism of Antibacterial Action of Peptides and Compounds of Cyanobacteria and Microalgae
In general, the mechanism of action of cyanobacterial and microalgae peptides against bacterial cells has not yet been established, and further studies are needed to elucidate the biological activity of these antimicrobial peptides [90,147]. For those antibacterial peptides with a clear cationic character, e.g., from microalgae, we may surmise a mechanism of action rather similar to those peptides described in higher eukaryotes; that is, the disruption of the cell membrane after specific insertion into the bacterial cell membrane. Specificity is mostly achieved by the different electrical charge of the external hemilayer of the cell membrane, negative for prokaryotes and lower eukaryotes, zwitterionic in higher eukaryotes. This mechanism has two important consequences; first, the negative electrical charge of the membrane is considered as a pathogen associated molecular pattern, as such, common to many microorganisms, that makes them susceptible to a given peptide. Secondly, as the bactericidal mechanism is based on the stoichiometric interaction of the peptide with the phospholipids of the lipid bilayer, physicochemical characteristics of the peptide, such as charge, size, amphipaticity, and even secondary structure, are more important than the primary sequence of the peptide. For others, their mechanism of action differs from membrane disruption, with involvement of intracellular targets, and specificity achieved by subtle recognition between the peptide and its target. This is the case for some cyanopeptides, as the cyclic peptides brunsvicamides B and C from Tychonema sp., reported as inhibitors of phosphatase B of Mycobacterium tuberculosis, or the cyclic depsipeptide scyptolin A, isolated from Scytonema hofmanni, an inhibitor of a serine protease working as a transpeptidase involved in the bacterial cell wall biosynthesis for certain pathogenic bacteria [88].
Antibiofilm activity is an appealing asset for an antibacterial candidate, as infections in clinical devices are frequently associated to biofilm formation and higher resistance against antibiotics. The cyclic peptides portoamides produced by Phormidium sp. display this activity against marine bacteria such as Cobetia marina, Halomonas aquamarina and Pseudoalteromonas atlantica, by inhibition of ATPase H + -transport activity [132]. This activity has a straightforward application as antifouling agents, and their test as antibiofilm compounds for relevant clinical bacteria is pending.
In some cases, structure-activity relationships were obtained with a variable degree of success, either by sequence comparison among similar cyanopeptides from the same or different cyanobacteria, by genetic mutation, or by chemical synthesis. The antibacterial activity of the lipopeptide schizotrin A against B. subtilis has been associated to the presence of a proline linked to the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Aound), and their uptake into the bacterial cell facilitated by the presence of the fatty acid [94,126]. The dipeptide motif formed by a proline residue bound to the amino group of a 2-hydroxy-3 amino-long chain acid residue is shared for other cyanobacterial cyclopeptides, such as scytonemin A from Scytonema sp [88,143]. The presence of this fatty acid was also identified in lyngbyazothrins A-D, and this acyl chain at position C-5 is relevant for the antibacterial activity of the peptide.
The amino acid analyses of the cyanopeptides lyngbyazothrins A-D reveal three unusual amino acids identified as 4-methoxyhomophenylalanine in A and C, homophenylalanine in B and D, and 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Aound) in A-D; moreover, C and D have an additional N-acetyl-N-methyltyrosine unit and it seems that the acyl residue at C-5 plays an important role in antimicrobial activity. Schizotrin A and pahayokolides A and B have sequence similarity to lyngbyazothrins. Schizotrin A presents a 4-methoxyhomophenylalanine (Htm) residue, similar to lyngbyazothrins A and C, bound to the Pro-Aound-Gln-Gly-Pro sequence, common to all of the lyngbyazothrins. The same sequential motif is also found in pahayokolides A and B but, in contrast to schizotrin A, it is linked to an homophenylalanine. Significant differences were found for the remaining five residues of the cyclic systems among the three classes of peptides: Phe-Val-Ser-DeHThr-Ser in schizotrin A, Phe-Z-Dhb-Ser-E-DhB-Thr in pahayokolides and Pro-allo-Ile-Ser-DeHThr-Thr in lyngbyazothrins. The free hydroxyl group at C-5 of the Aound residue in lyngbyazothrins A and B is substituted by N-acetyl-N-methyltyrosine in C and D instead of the N-butyryl-Nmethylalanine residue in schizotrin A. On the other hand, schizotrin A and pahayokolide A contain the aliphatic amino acids alanine and leucine, while lyngbyazothrins C and D include the aromatic amino acid tyrosine; it has been proposed that the nature of these amino acids may also account for the activity of lyngbyazothrins against the Gram-negative bacterium E. coli, absent in schizotrin and pahayokolide [126].
The ribosomal cyclopeptide aeruginazole A isolated from the cyanobacterium Microcystis sp. (IL-323), inhibits the growth of B. subtilis and S. aureus; in its cyclic structure it contains three subsequent glycine residues plus l-val, l-phe, thiazole-l-val, thiazole-d-leu, d-tyr and thiazole-l-asp. Similarly, aeruginazole DA1497 isolated from M. aeruginosa, is a large cyclopeptide with four thiazole (tzl) moieties, having a cyclo-structure of (-(tzl-phe)-gly-ala-ile-(tzl-ala)-ser-(tzl-val)-pro-gly-val-(tzl-leu)-pro-gly-). It seems that the larger size and the greater number of thiazole groups of these compounds may be associated with their bioactivity. Only DA1497 out of the one of five aeruginazole peptides tested (DA1304, DA1274, DA1338 and DA1372) was active against S. aureus, even when minor differential sequential variations occurred among the five peptides [92]. The cyclic lipopeptide Trichormamide C from Oscillatoria sp. UIC 10,045 is characterized by the presence of three non-proteinogenic α-amino acid residues (N-methyl-Ile and two 3-hydroxy-Leu) and one β-amino acid, with a key role on its anti-M. tuberculosis activity [125].
The antibacterial mechanisms of microalgal peptides have been scarcely reported to date. The few references on the subject refer to extracts or protein hydrolysates, and not to specific peptides. Microalgal extracts from the species Leptocylindrus danicus (FE322) and L. aporus (FE332) strongly inhibited the biofilm formation by the bacteria Staphylococcus epidermidis, but did not show cytotoxicity by standard antibacterial tests [150]. Tejano et al. [151], reported a higher antibacterial activity on Gram-positive than on Gram-negative bacteria for the pepsin hydrolysate and the peptide fractions from Chlorella sorokiniana, likely associated to a hindered penetration of the peptide by the outer membrane.
It has been proposed that microalgal compounds with antibacterial activity are released after the loss of algal integrity, or alternatively induced by the presence of bacteria. For other microalgal compounds involved in a defense mechanism against predators and pathogenic bacteria, it appears that the bacterial cell membrane would be the main site of action. There is some evidence of deleterious effects of fatty acids on the bacterial membrane, causing cell leakage, a reduction in nutrient intake and a reduction in cellular respiration. The antibacterial action of fatty acids can also be mediated by the inhibition of the synthesis of bacterial fatty acids; this effect could be bactericidal or bacteriostatic preventing bacterial multiplication. It has also been reported that antibacterial exometabolites released by T. suecica inhibited several Vibrio species in vitro causing a rapid decrease in bacterial mobility with cells elongation and vacuolization [84].
Advances in the knowledge of the mechanisms of action underlying the bactericidal activity of peptides from cyanobacteria and microalgae will contribute to the development of these peptides as novel drugs. The role of "omics" techniques in this process, more specially proteomics and peptidomics, will push forward the boundaries for this field [90,147].

Synergy of Cyanobacterial Peptides
Synergy among AMPs in nature is an important asset in evolution, as it ensures a proper microbicidal function with a spare of components. The issue of microbicidal synergy was extensively addressed for AMPs from higher eukaryotes [152][153][154], as well as for bacteriocins [155]. This synergism may occur under different modalities. A specific molecular recognition between two different AMPs from Xenopus laevis, magainin and PGLa, led to the formation of a functional heterodimer as the forming unit of the pore on the bacterial membrane, with an increased lethal permeation over those formed exclusively by a single AMP species [156]. In this case, the two synergic partners shared a single target, but synergy may also arise from two different AMPs each one acting on their own and specific target in a concerted manner. The disruption of bacterial membranes by the human AMP LL-37 allows the histone H2A to gain access into the bacterial cytoplasm to interact with the bacterial genome, and to halt transcription [157]. Synergism is not limited to AMPs as exclusive partners; other actions or compounds may synergize with a given AMP to improve the final microbicidal aftermath. Any AMPs, regardless of its biological origin, able to disrupt the outer membrane of Gram-negative bacteria, which is a strong permeability barrier for small size antibiotics, may likely synergize with them, as described for the bacterial polymyxins B and other AMPS from higher eukaryotes. In addition, small antibiotics may also synergize with AMPs acting mostly through host immunomodulation, as the IDR-1018 peptide [158].
Compared to the synergism described for AMPs, there is a dearth of reports on synergy among cyanobacterial peptides, especially those concerning activity against pathogens of putative clinical interest. Synergism in cyanobacteria was mainly approached from an environmental perspective, where peptides were used by cyanobacteria as allelochemicals for a successful striving for survival among other environmental competitors or plankton grazers.
Portoamides A and B, produced by the mat forming Oscillatoria, act synergistically against the microalgae C. vulgaris, Ankistrodesmus falcatus, and Chlamydomonas reinhardtii, but also against the cyanobacteria Cylindrospermopsis raciborskii [159]. Planktothrix sp, other cyanobacteria, produced the ribosomal microviridins by ribosomal synthesis, and microcystin-LR, a peptide inhibitor for protein phosphatases 1 and 2A, as well as anabaenopeptins through the non-ribosomal pathway. When cyanobacterial knock-outs for any of these three peptide families were infected with the chytrid parasitic fungus pNIVA-CYA126/8, its virulence was higher on knockouts than on the parental strain [160].
Beyond this environmental frame, synergy among cyanobacterial peptides has also been reported for antifungal activity on species with clinical or phytological importance. Lobocyclamides A and B from Lyngbya confervoides act synergistically against Candida spp [161], the cyclic undecapeptides laxaphycins A and B from Anabaena torulosa against Aspergillus oryzae and Candida albicans [162], and the same peptides isolated from Anabaena laxa against A. oryzae [163].
Interestingly, the synergism of cyanobacterial peptides for antitumoral activity has been also reported on human tumoral cell lines for laxaphycins A and B from A. torulosa, and for laxaphycins A and B4 from Hormothamnion enteromorphoides [162,164]. Protoamides A and B showed synergism on the human cancer cell line H460 [159].

Other Relevant Functions of Peptides from Cyanobacteria and Microalgae
In addition to their antibacterial activity, the peptides produced by cyanobacteria and microalgae also have other fields of application, being one of the most prominent treatments of cancer. Some representative examples follow:

Antitumoral Activity
The NRPS and NRPS-PKS described on the cyanobacteria of the genus Nostoc, were responsible for the production of bioactive peptides (reviewed by Fidor et al. [120]). Among the extensive peptide armamentarium of this genus are cryptophycins; cyclic 16mer depsipeptides are almost exclusively produced by this genus, with activity against different cancer lines. Other peptides of the genus Nostoc are the nostocyclopeptides, cyclic heptapeptides characterized by an imino linkage between the first residue (Tyr) and the aldehyde hydrate of the seventh residues. They induce apoptosis in tumoral cell lines, endorsing their feasible use as anticancer compounds.
The heptapeptide LLAPPER (MW = 796.4) was obtained by in vitro gastrointestinal digestion of P. lutheri [165]. After its chemical synthesis, the peptide was tested on HT1080 fibrosarcoma cells, with a decrease of the transcript encoding the matrix metalloproteinase B/MMP9, an enzyme involved in the degradation of collagen type IV, through inactivation of the NFκB pathway, as well as blocking of JNK and p38 phosphorylation. Matrix metalloproteinases are involved in the degradation of the extracellular matrix, to promote metastasis in cancer.
Interestingly other cyanobacterial peptides promotes differentiation, as the peptide dubbed PPLF obtained from P. lutheri fermentation by Qian et al. [166]. PPLF induced osteoblastic differentiation in the human cell line MG-63 at 50 and 100 µg/mL.

Antihypertensive Activity
Heo et al. [167] hydrolyzed Spirulina with a mixture of digestive enzymes (pepsin, trypsin and chymotrypsin). Afterwards, the heptapeptide TMEPGKP (MW = 759) was isolated from a fraction with inhibitory activity on the angiotensin I converting enzyme (ACE). By molecular modelling and functional assays, this peptide resulted as a non-competitive inhibitor of ACE, and decreased the phosphorylation of p38 MAP Kinase (MAPK), of the expression of the inducible nitric oxide synthase (iNOS) with the concomitant decrease of nitric oxide (NO) production. The levels of reactive oxygen species (ROS), and endothelin-1 (ET-1), also decreased after peptide treatment. Altogether, these results make this heptapeptide a good candidate as antihypertensive compound. In addition, the peptide showed an inhibitory effect on the proliferation of EA.hy926 cells at 125 and 250 µM.
Carrizzo et al. [168] described the fractionation of Spirulina lysates by a treatment that simulates gastrointestinal digestion (GID). From these extracts, four peptides were obtained and characterized by mass spectrometry (SP(3-6)). In a further step, these peptides were synthesized by Fmoc chemistry and tested for vasorelaxation in an ex vivo model consisting of mouse mesenteric arteria. SP6 peptide (GIVAGDVTPI, MW = 940.52), showed a dose dependent vasorelaxation effect and antihypertensive activity through the activation of endothelial nitric oxide synthase and NO production.
Suetsuna and Chen [169] searched for peptide fractions from the peptic digest of the microalgae S. platensis and C. vulgaris and to be tested for their antihypertensive activity through ACE inhibition. A total of ten peptides (three to five residues) were isolated, two of them shared by both algae and the other four sequences specific for each species. The two shared tripeptides FAL and AEL showed IC 50 on ACE of 26.3 and 57.1 µM, respectively. The peptides specific for S. platensis: IAE, IAPG, VAF have IC 50 of 34.7, 11.4 and 35.8 µM, respectively, and the IC 50 for those exclusively found in C. vulgaris: IVVE, AFL, VVPPA, were 315.3, 63.8 and 79.5 µM, respectively.
Hot water extract of the microalgae Chlorella sorokiniana was hydrolyzed with proteinase N, and tested for ACE inhibition [170]. Four dipeptides were isolated and sequenced by automated Edman degradation. In a further step, the four peptides were synthesized by solid phase peptide synthesis (SPPS) and tested for their ACE inhibitory activity: WV, VW, IW, and LW with IC 50 on ACE of 307.6 µM, 0.58 µM, 0.50 µM, and 1.11 µM. Their low IC 50 values plus their small size make them extremely appealing candidates as hypertensive agents. Furthermore, they were impervious to in vitro gastric digestion.
The hydrolysate of the marine microalga, N. oculata obtained by digestion with several enzymes, such as pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutralase were used by Samarakoon et al. [172] in the search for ACE inhibitory peptides. The pepsin hydrolysate exhibited the highest ACE inhibitory activity. From this lysate, two ACE inhibitory peptides were purified: GMNNLTP (MW = 728; IC 50 = 123 µM) and LEQ (MW = 369; IC 50 = 173 µM). These peptides were proposed as novel inhibitory agents in the functional food industry.

Anti-Inflammatory Activity
The group of Qian and Jung sought to find active compounds from the marine microalga Pavlova lutheri after fermentation with the yeast Hansenula polymorpha. They reported the in vitro antioxidant activity of the fermented microalga [173]. In subsequent works [174], reduction of oxidative stress was achieved by the tetrapeptide MGRY (MW = 526), that works as a scavenger for free radicals, with an IC 50 of 0.285 mM for the DPPH antioxidant test, 0.068 mM for hydroxyl radicals and 0.988 mM for hydrogen peroxide. The peptide also showed inhibitory properties in melanogenesis process when tested in B16F10 melanoma cells. As such, its use was proposed in cosmeceutical and pharmaceutical applications.
To note, the inhibition of ACE activity described for some cyanopeptides, make them putative candidates for competitive inhibition of the SARS-CoV-2 virus into ACE, the main receptor used for this pandemic virus to infect human cells.

Antifouling Activity
Portoamides are cyclic dodecapeptides from the cyanobacterium Phormidium sp. They were used for their antifouling activity with an EC 50 of 3.16 µM against the settlement of the larvae of the mussel Mytilus galloprovincialis [132]. Portoamides were nontoxic against the mussel larvae, therefore a deterrent effect towards surface colonization was instead proposed. The results of previous reports of portoamides' activity against the microalgae C. vulgaris (IC 50 12.8 µg/mL), Ankistrodesmus falcatus (IC 50 24.7 µg/mL) and C. reinhardtii (IC 50 12.6 µg/mL), and the cyanobacterium Cylindrospermopsis raciborskii (IC 50 28.4 µg/mL) are also described.

Synthetic Approach
Synthetic approach has become an almost mandatory companion to the discovery of new peptides. The synthesis of peptides in particular is an area of wide development in the current pharmacology. The main goal of this technique for cyanobacterial and microalgal peptides is to overcome the frequent extremely low amount of isolated natural peptides, that limits further studies on their mechanism of action, on the definition of SAR studies, and of a feasible pharmacological development. Peptides have become valuable drug candidates, largely driven by improvements in their synthetic methodology.
Peptide chemistry has developed on two main areas: biosynthesis and chemical synthesis. A special number of Chemical Reviews dealt with the first strategy, with excellent reviews on the subject and some resulting applications [175][176][177][178].
However, the chemical synthesis is the first-choice approach as it allows to obtain pure products with good yield, needed to establish its activity in an unambiguous way, and pursue the establishment of its mechanism of action.
Solid phase peptide synthesis is a well-established methodology developed more than 50 years ago [179,180]. Nowadays, the Fmoc/tBu strategy is the most used [181,182]. Several approaches have been carried out to optimize the process, to improve its efficiency, and more recently to develop an ecofriendly approach, more compliant with the green chemistry principles [41,42,183,184].
Peptide synthesis can be carried out manually, with the possibility of simultaneously synthesizing many sequences by using the "tea bag" protocol [185,186], or also in an automated way where developments such as microwave [187,188], and ultrasonication [189] procedures shortened the time of synthesis and reduced solvent consumption as well as waste generation, additionally allowing the synthesis of longer peptides.
These methodologies were straightforwardly applied to peptides derived from microalgae, with a lower complexity, and in general devoid of posttranslational modifications, due to their ribosomal origin. In contrast, non-ribosomal peptides from cyanobacteria have a much higher structural and compositional complexity, broadening their chemical space, due to the versatility and diversity provided by non-ribosomal synthesis. Although the synthesis of cyanobacterial peptides requires morecomplex strategies, it has been successfully approached for some peptides.
Inguimbert's group performed the chemical synthesis of cyclic lipopeptides of the laxaphycin family, isolated from several cyanobacterial species. In a first step, the dodecapeptide laxaphycin B was synthesized [190], and assayed for cytotoxicity against a wide range of cancer cell lines (IC 50 ranging from 0.2 to 6.0 µM). An automated SPPS with an on-resin "head-to-tail" cyclization of the linear precursor of laxaphycin B (laxaB) was used. Synthesis optimization was achieved by the use of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluororophosphate (HATU) as a coupling reagent. Finally, the head to tail cyclization was carried out with DIC/Oxyma to prevent epimerization. This strategy was also applied for the synthesis of lyngbyacyclamide A. NMR analysis confirmed that the structures of natural and synthetic peptides were identical. In a later work, the same group synthesized other laxaB analogues [191], by replacement of the non-natural amino acids with commercially available counterparts (2-aminodecanoic acid (Ade): β-alanine, the 3-hydroxyleucines (Hle): threonine, and the 3-hydroxyasparagine (HAsn): asparagine. They also implemented an automated microwave SPPS protocol using the same reagents and conditions determined in the previous work. The cyclization residues were selected according to retrosynthetic analysis, and the final product was purified and characterized as before. Although the peptide obtained did not exactly match the expected results, a synthesis and characterization protocol was established.
The synthesis of the peptide mozamide A, produced by a marine sponge of the genus Theonella was approached by Junk and Kazamaier [192]. This peptide is a hydroxylated brunsvicamide, a family of peptides isolated from the cyanobacteria Tychonema, but differing in the configuration of Val, Lys and Ile amino acids. Interestingly, the right configuration of these residues was confirmed in the synthetic peptide, and established a synthetic scheme for these types of compounds.
Synthesis of lipocyclopeptides, such as the cyclic undecapeptide trichromamide A (TcA) from the cyanobacteria Trichormus sp., was performed by Gaillard et al. [193]. SPPS was carried out on a clorotrytil resin to which the uncommon residue aminodecanoic acid (Ada) was anchored and the cyclization step was made in solution with PyOxym/Oxyma. They reported the synthesis of TcA and a second compound, possibly a diastereomer, with a good yield.
Additionally, multiple approaches can be applied to improve the biological performance of peptides, with a special focus to improve their biological stability. This is achieved, by replacement of amino acids that are not susceptible to enzymatic hydrolysis [194], by structural restriction, by cyclization, or by stapling [46,195].
An interesting research area is related with the use of metallic elements in association with peptides to enhance their activity for applications in biomedicine [196][197][198].
In addition to the development in synthesis, there are also advances in peptide purification and analysis methodologies that include techniques, such as HPLC, MS, circular dichroism (CD), and nuclear magnetic resonance (NMR), which can be helpful in studies of structure-activity relationship or in the determination of mechanisms of action [184,[199][200][201].

Conclusions
The deep global crisis produced by antimicrobial resistance (AMR) has led to the search for new compounds to provide alternative ways for the development of antibacterial agents that exploits mechanisms of action different from traditional antibiotics. This report has reviewed the wide variety of peptide compounds produced by cyanobacteria and microalgae, both extremely versatile microorganisms, with activity against human and aquatic pathogens, their structural diversity, and their mechanisms of action.
Cyanobacteria produce a wide variety of peptides, due to their extraordinary synthetic plasticity, endowed by their capacity to synthesize not only ribosomal peptides, but also non-ribosomal and polyketide-associated peptides. Additionally, their adaptability makes them easily cultured in the laboratory, requiring a low amount of inorganic nutrients, producing different compounds under different experimental conditions.
In contrast, there is a scarce knowledge on how microalgae face bacterial infection and their antibacterial peptides involved. Despite the wide diversity of microalgal species, only few of them have been cultivated and explored for their biotechnological potential. The insight on their exploitation as an appealing source for novel antimicrobial peptides has just started.
A major advantage for many antimicrobial peptides is their low propensity to generate resistance, due to their multiple mechanisms of action, and the synergy among them. The implementation of cyanobacterial peptides as candidates for antibacterial drugs, required a careful and rational planning where several factors are involved; among these, the selection of the target bacteria, and MIC or MBC expected or required are essential. Concerning the first one, an especial effort is addressed for those species responsible for a higher a health risk against human health, mostly due to the rise of resistance and depletion of alternative antibiotics, according to WHO criteria [202] as those included in the group called ESKAPE [203]. Particular attention should be given for those peptides with a broad spectrum of activity. Concerning activity, an ideal compound should have an IC 50 25 µM or 10 µg/mL, whereas for extracts at the initial steps of purification, a standard cut-off should be below 100 µg/mL, similar to the criteria established for natural compounds [204][205][206][207]. In this review, we explored the peptides produced by cyanobacteria and microalgae, mainly as antibacterial, with a special focus on compounds with MIC values below those aforementioned (Table 3), as the most appealing candidates for their further pharmacological development and clinical implementation.
The impressive advances both in isolation and characterization of peptides runs parallel to the development of technologies devoted to these tasks. Among them, new and improved chemical synthesis of peptides, mass spectrometry, "omics" techniques, and bioinformatics tools for in silico selection and identification of feasible starting candidates are crucial. Furthermore, concerning antibacterial assays, the implementation of microfluidics and robotized assays, allow high-throughput screenings with a spare of the peptide required. Likewise, new strategies to improve the stability and bioavailability of peptides were reported, to curb these major shortcuts that jeopardizes the effectiveness of peptides as new drugs. Nowadays, very few peptides derived from cyanobacteria or microalgae overcome the initial steps of the pharmacological development, but it is likely that this is only tip of iceberg for a massive exploitation of these organisms as a new source of antibacterial compounds and a promising alternative for current antibiotics in the future.
Author Contributions: Conceptualization, V.R. and L.R; formal analysis, V.R., L.R., C.C. and F.G.; writing-original draft preparation V.R., L.R., C.C. and F.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by FONDECYT 1170379. LR was supported by a grant (RICET RD16/0027/0010) from the Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER.