Streptomyces -Derived Bioactive Pigments: Ecofriendly Source of Bioactive Compounds

: Pigments have been used since historical times and are currently used in food, cosmetic, pharmaceutical, and other industries. One of the main sources of natural pigments are plants and insects; however, microorganisms are of great interest due to their bioactivities and advantages in their production. Actinobacteria, especially the genus Streptomyces , are biotechnologically valuable, producing specialized metabolites with a broad spectrum of bioactivities, such as antioxidant, anti-cancer, antibioﬁlm, antifouling, and antibiotic activities, as well as pigments, among others. In this review, we identify, summarize, and evaluate the evidence regarding the potential of Streptomyces strains to be biological sources of bioactive pigments. To conclude, future research will include purifying pigmented extracts that have already been reported, studying the puriﬁed compounds in a speciﬁc application, isolating new microorganisms from new isolation sources, improving the production of pigments already identiﬁed, modifying culture media or using new technologies, and developing new extraction techniques and a wide range of solvents that are ecofriendly and efﬁcient.


Introduction
Pigments are used to manufacture various products because they can enhance the natural color or replace color lost during the manufacturing process, generating greater consumer appeal by adding a novel sensory aspect.Since the introduction of synthetic dyes by Perkin in 1856 [1], their production has increased, and natural colorants from plants and animals have decreased due to synthetic pigments being relatively cheaper [2].
In the 20th century, natural organic pigments were almost entirely displaced by synthetic molecules such as phthalocyanines, ranging from blue to green, and quinacridones, ranging from orange to violet [3].Advances in organic chemistry allowed these compounds' mass production to replace the natural ones, which are often more complex to acquire [4].Therefore, the use of synthetic organic dyes has been the most cost-effective approach for years [5].Synthetic dyes are superior to natural pigments in their staining power, ease of application, stability, and cost/effect [6,7].
The production of synthetic pigments in 2019 represents 8 × 10 5 tons per year [6,8]; however, some of them have negative effects in the human health and environment [6].Firstly, they are not biodegradable and are very difficult to remove from effluents or to dispose of due to their stability to oxidation and reduction processes; it is even worse in the case of degradation, their byproducts have been directly or indirectly proven to be health hazards.For example, anionic dye removal is a huge challenge as they produce very bright colors in water and show acidic properties [1,9].Secondly, the synthetic pigments can affect human health, having negative effects on several vital organs, such as the brain, kidneys, liver, and heart; and systems such as respiratory, immune, or reproductive [6].Especially, neoformans, Colletotrichum lagenarium, Alteromonas nigrifaciens, and most of the Streptomyces species [23,40].
On the other hand, characterized pigments from Vibrio spp (prodigiosin) [41], Serratia marcescens [42], and Janthinobacterium lividum [43] have been evaluated in the staining of different fibers, including wool, nylon, acrylics, silk, cotton, and polyester microfiber, obtaining good color shades.In addition, due to their antibacterial activity, they are being used in the development of antimicrobial textiles for hospital infections [44].
Among microbial pigments, one of the most interesting genera is Streptomyces, due to its great reproductive capacity, and also because one of the most produced pigments in the industry, melanin, can be produced by this bacterium [40,45].In addition, this type of actinomycetes has a fascinating genetic distribution, which is attractive for replication in the biotechnology industry [46][47][48].In addition, Streptomyces are well known for their abundant secondary metabolism, which has provided different bioactive compounds, namely antibiotics, anti-inflammatories, antioxidants, and cytotoxins [49][50][51].Several of these compounds are colored [52] and, given the bioactivity potential shown for Streptomyces strains [23], many of the colored Streptomyces-derived compounds could signify an exciting opportunity to find bioactive pigments.
Considering that the need for safe pigments is applicable in different areas, and with the additional beneficial activities and the biotechnological potential of Streptomyces, we accomplished a literature review of pigments produced by Streptomyces.We identified which ones have some bioactivity such as antimicrobial, antioxidant and cytotoxic activities, which are relevant for determining possible future applications.Afterward, we summarize the conditions to optimize the pigment production of the strains on which this study was performed.This review aimed to identify, summarize, and evaluate the evidence regarding the potential of Streptomyces strains as a biological source of bioactive pigments.

General Findings
The literature search identified 3904 articles, of which 176 were not original and 1253 were duplicates, giving a total of 2475 articles.These articles were screened by reading titles and abstracts, following inclusion or exclusion criteria.From this stage, 112 papers were selected for full-text evaluation.Finally, 53 articles were selected for full-text assessment and were used for data extraction (Figure 1).
Even though the study of Streptomyces-derived pigments dates back many years, starting in 1973, most articles (41.7%) were published between 2018 and 2022 (Figure 2a).Thus, it is evident the relevance that the production and evaluation of Streptomyces-derived pigments have taken, given the growth in the number of articles published in recent years (Figure 2b).Even though the study of Streptomyces-derived pigments dates back many ye starting in 1973, most articles (41.7%) were published between 2018 and 2022 (Figure Thus, it is evident the relevance that the production and evaluation of Streptomycesrived pigments have taken, given the growth in the number of articles published in rec years (Figure 2b).[53] (see Table S1).[53] (see Table S1).
Even though the study of Streptomyces-derived pigments dates back many years, starting in 1973, most articles (41.7%) were published between 2018 and 2022 (Figure 2a).Thus, it is evident the relevance that the production and evaluation of Streptomyces-derived pigments have taken, given the growth in the number of articles published in recent years (Figure 2b).This increase in the number of items may be due to several reasons: First, consumers demand natural pigments as they are considered safe, nontoxic, noncarcinogenic and biodegradable [1].Second, the pigment market's exponential growth, which represents USD 36.4 billion in 2021, is projected to expand at a compound annual growth rate (CAGR) of 5.2% from 2022 to 2030 [54].Third, actinobacteria are among the most profitable and biotechnologically valuable [46,55].Streptomyces especially is responsible for the vast majority of specialized metabolites [46][47][48]56].Fourth, the need to investigate unexplored or underexploited habitats as new sources of specialized metabolites [46].
On the other hand, analyzing the map of the countries of the corresponding authors of the articles (Figure 3) showed India and China are the countries with the highest scientific production, while the contribution of articles from Latin America, Europe, and Africa is extremely limited or almost null.In addition, with respect to collection and isolation areas (Figure 4), 20.4% of the articles did not specify the isolation area.India stands out as one of the countries with the highest number of Streptomyces collection areas.In this way, Asia is the main continent where the scientific production and collection sites around Streptomyces-derived pigments are concentrated.
On the other hand, analyzing the map of the countries of the correspondin of the articles (Figure 3) showed India and China are the countries with the high tific production, while the contribution of articles from Latin America, Europe, a is extremely limited or almost null.In addition, with respect to collection and areas (Figure 4), 20.4% of the articles did not specify the isolation area.India stan one of the countries with the highest number of Streptomyces collection areas.In Asia is the main continent where the scientific production and collection site Streptomyces-derived pigments are concentrated.
This may be because Asian countries have numerous exotic places to isolate cies of microorganisms [57]; such an example are the tropical forests of Southea the reefs of the 'coral triangle' and the river basins are unique on Earth [58].Sp India has the Western Ghats, which are one of the thirty-four biodiversity hotsp world [59], and interesting ecosystems for microbiologists, such as the Vellar Est the Gulf of Mannar Biosphere Reserve [61], the Thar Desert [62], and the Sabarim est [63], among others [57].ity of specialized metabolites [46][47][48]56].Fourth, the need to investigate unexp underexploited habitats as new sources of specialized metabolites [46].
On the other hand, analyzing the map of the countries of the corresponding of the articles (Figure 3) showed India and China are the countries with the high tific production, while the contribution of articles from Latin America, Europe, a is extremely limited or almost null.In addition, with respect to collection and areas (Figure 4), 20.4% of the articles did not specify the isolation area.India stan one of the countries with the highest number of Streptomyces collection areas.In Asia is the main continent where the scientific production and collection site Streptomyces-derived pigments are concentrated.
This may be because Asian countries have numerous exotic places to isolate cies of microorganisms [57]; such an example are the tropical forests of Southea the reefs of the 'coral triangle' and the river basins are unique on Earth [58].Spe India has the Western Ghats, which are one of the thirty-four biodiversity hotsp world [59], and interesting ecosystems for microbiologists, such as the Vellar Estu the Gulf of Mannar Biosphere Reserve [61], the Thar Desert [62], and the Sabarim est [63], among others [57].This may be because Asian countries have numerous exotic places to isolate new species of microorganisms [57]; such an example are the tropical forests of Southeast Asia-the reefs of the 'coral triangle' and the river basins are unique on Earth [58].Specifically, India has the Western Ghats, which are one of the thirty-four biodiversity hotspots in the world [59], and interesting ecosystems for microbiologists, such as the Vellar Estuary [60], the Gulf of Mannar Biosphere Reserve [61], the Thar Desert [62], and the Sabarimalai forest [63], among others [57].
Another important aspect that was observed in the literature review is the type of substance with which the articles worked.Most of them (50.9%) reported the identification of the pure compound or a partial purification; 43.4% indicated that they evaluated the extract, and a minor quantity worked with fraction and pure cultures (Figure 5a).According to the type of source, it was found that the most used solvent for extraction and purification of the extract was ethyl acetate; other solvents used were methanol, chloroform, and ethanol.As for the source of isolation, 52.8% belonged to soil (Free-living), 7.5% to marine (Free-living) and marine symbionts, and a minor quantity (3.8%) were terrestrial symbionts and freshwater (Free-living).In addition, 24.6% of the articles did not report the source of isolation (Figure 5b).

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Another important aspect that was observed in the literature review is the type of substance with which the articles worked.Most of them (50.9%) reported the identification of the pure compound or a partial purification; 43.4% indicated that they evaluated the extract, and a minor quantity worked with fraction and pure cultures (Figure 5a).According to the type of source, it was found that the most used solvent for extraction and purification of the extract was ethyl acetate; other solvents used were methanol, chloroform, and ethanol.As for the source of isolation, 52.8% belonged to soil (Free-living), 7.5% to marine (Free-living) and marine symbionts, and a minor quantity (3.8%) were terrestrial symbionts and freshwater (Free-living).In addition, 24.6% of the articles did not report the source of isolation (Figure 5b).S5), pure compounds (see Table S6), fraction, or pure culture.(b) Percentage of articles by the environment where the isolation occurred.
In this way, it is important to keep in mind for future research to purify pigmented extracts that have already been reported, study the purified compounds in a specific application, and isolate new microorganisms from new isolation sources.In addition, it is necessary to study extraction with different solvents and to propose new extraction techniques that are environmentally friendly and have high yields.
Analyzing the number of pigment-producing strains from different isolation sources and the evaluated bioactivities, it was observed that the highest number of reported strains are from the soil (Free-living), with 29 reported strains, and the most evaluated bioactivity was antimicrobial activity.In addition, 10 pigment-producing strains were evaluated for more than 1 bioactivity.On the other hand, the source of isolation that least reported pigment-producing strains is terrestrial symbiont, and 21 of the pigment-producing strains were not evaluated for any bioactivity (see      S5), pure compounds (see Table S6), fraction, or pure culture.(b) Percentage of articles by the environment where the isolation occurred.
In this way, it is important to keep in mind for future research to purify pigmented extracts that have already been reported, study the purified compounds in a specific application, and isolate new microorganisms from new isolation sources.In addition, it is necessary to study extraction with different solvents and to propose new extraction techniques that are environmentally friendly and have high yields.
Analyzing the number of pigment-producing strains from different isolation sources and the evaluated bioactivities, it was observed that the highest number of reported strains are from the soil (Free-living), with 29 reported strains, and the most evaluated bioactivity was antimicrobial activity.In addition, 10 pigment-producing strains were evaluated for more than 1 bioactivity.On the other hand, the source of isolation that least reported pigment-producing strains is terrestrial symbiont, and 21 of the pigment-producing strains were not evaluated for any bioactivity (see Table 1). 1 More than one of the bioactivities (antimicrobial, antioxidant, and cytotoxic) were studied. 2 The compounds responsible for the bioactivity were not elucidated. 3Information not available.
Quinones are aromatic compounds widely present in nature (Figure 7).They can be classified according to their chemical structures into benzoquinones, anthraquinones and naphthoquinones.Specifically, naphthoquinones are structurally related to naphthalene and are characterized by their two carbonyl groups in the 1,4 position or 1,2 position with minor incidence; they are highly reactive organic compounds used as dyes whose colors range from yellow to red [112].Actinomycin is a DNA-targeting antibiotic and anticancer, composed of a chromophore group and two pentapeptide chains with a variable composition of amino acids.The pentapeptide precursors are biosynthesized by a nonribosomal peptide synthetase (NRPS) assembly line, and actinomycins are formed through oxidative condensation of two 3-hydroxy-4-methylanthranilic acid (4-MHA) pentapeptide lactones (PPLs) [80].
Actinomycins X2 (10) are formed through the sequential oxidation of the γ-prolyl carbon by the cytochrome P450 enzyme saAcmM.Actinomycin L (8,9) is formed through the spontaneous reaction of anthranilamide with the 4-oxoproline site of actinomycin X2 (10) prior to the condensation of the two 4-MHA PPLs into actinomycin L (8,9) [80] (Figure 8).Actinomycin is a DNA-targeting antibiotic and anticancer, composed of a chromophore group and two pentapeptide chains with a variable composition of amino acids.The pentapeptide precursors are biosynthesized by a nonribosomal peptide synthetase (NRPS) assembly line, and actinomycins are formed through oxidative condensation of two 3hydroxy-4-methylanthranilic acid (4-MHA) pentapeptide lactones (PPLs) [80].
Actinorhodin ( 13) is a blue pigment whose polyketide backbone must undergo two regiospecific reductions, two intramolecular aldol condensations, hemiketalization, aromatization of two rings, oxidation to form a quinone, hydroxylation, and dimerization [113] (Figure 9).This pigment is a redox-active secondary metabolite and a potent, bacteriostatic, pH-responsive antibiotic.Additionally, it is redox-active and can act in redox-cycling reactions.Moreover, it act as an organocatalyst of oxidative reactions in vitro, which suggests that actinorhodin (13) might kill bacteria via the accumulation of toxic concentrations of H 2 O 2 [114].The pentapeptide precursors are biosynthesized by a nonribosomal peptide synthetase (NRPS) assembly line, and actinomycins are formed through oxidative condensation of two 3-hydroxy-4-methylanthranilic acid (4-MHA) pentapeptide lactones (PPLs) [80].
Actinomycins X2 (10) are formed through the sequential oxidation of the γ-prolyl carbon by the cytochrome P450 enzyme saAcmM.Actinomycin L (8,9) is formed through the spontaneous reaction of anthranilamide with the 4-oxoproline site of actinomycin X2 (10) prior to the condensation of the two 4-MHA PPLs into actinomycin L (8,9) [80] (Figure 8).Actinorhodin ( 13) is a blue pigment whose polyketide backbone must undergo two regiospecific reductions, two intramolecular aldol condensations, hemiketalization, aromatization of two rings, oxidation to form a quinone, hydroxylation, and dimerization Coatings 2022, 12, x FOR PEER REVIEW 9 of 36 [113](Figure 9).This pigment is a redox-active secondary metabolite and a potent, bacteriostatic, pH-responsive antibiotic.Additionally, it is redox-active and can act in redoxcycling reactions.Moreover, it act as an organocatalyst of oxidative reactions in vitro, which suggests that actinorhodin (13) might kill bacteria via the accumulation of toxic concentrations of H2O2 [114].The act PKS includes the minimal PKS components (KS, CLF, and ACP), which together synthesize the octaketide backbone, a C-9 ketoreductase (KR), a didomain aromatase/cyclase (ARO/CYC) which is required for the formation of the first aromatic ring, and a second ring cyclase (CYC2).Additionally, based on the above PKS definition, the C-3 The act PKS includes the minimal PKS components (KS, CLF, and ACP), which together synthesize the octaketide backbone, a C-9 ketoreductase (KR), a didomain aromatase/cyclase (ARO/CYC) which is required for the formation of the first aromatic ring, and a second ring cyclase (CYC2).Additionally, based on the above PKS definition, the C-3 enoyl reductase and the third ring cyclase/dehydratase (if one exists) may also associate with the PKS complex [113].
Grixazone contains a phenoxazinone chromophore.Especially, grixazone A( 14) is a novel compound, and grixazone B (15) has been reported to show a parasiticide activity [115] (Figure 10); expression of the biosynthetic genes for this yellow pigment is probably under the control of A-factor (factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone)), which triggers the synthesis of almost all of the secondary metabolites produced by Streptomyces griseus [115], and controlled by the phosphate concentration of the medium [98].In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded.GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.No study on the precursor(s) or the biosynthetic enzyme for the phenoxazinone skeleton of these compounds has so far been reported.Because grixazones A ( 14) and B (15) contain an aldehyde and a carboxyl group, respectively, at the 8-position, the biosynthesis of the phenoxazinone skeleton in grixazones should be the same as those in michigazone and texazone [115].
Indigoidine ( 16) is a member of the class of pyridone, an extracellular blue pigment from S. aureofaciens CCM 3239 (Figure 11).The bpsA gene, which encodes nonribosomal peptide synthetase, is responsible for the biosynthesis of the blue pigment indigoidine ( 16) [116].Novakoba et al. [95] determined that a deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment and had a positive effect on auricin production, indicating the involvement of the bpsA gene in the biosynthesis of the indigoidine ( 16) blue pigment in S. aureofaciens CCM 3239 [95].Katorazone ( 17) is an alkaloid with a 2-azaquinone-phenylhydrazone structure (Figure 12).Characteristic structural features of 2-azaquinones in nature are the presence of a In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded.GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.No study on the precursor(s) or the biosynthetic enzyme for the phenoxazinone skeleton of these compounds has so far been reported.Because grixazones A ( 14) and B (15) contain an aldehyde and a carboxyl group, respectively, at the 8-position, the biosynthesis of the phenoxazinone skeleton in grixazones should be the same as those in michigazone and texazone [115].
Indigoidine ( 16) is a member of the class of pyridone, an extracellular blue pigment from S. aureofaciens CCM 3239 (Figure 11).The bpsA gene, which encodes nonribosomal peptide synthetase, is responsible for the biosynthesis of the blue pigment indigoidine ( 16) [116].Novakoba et al. [95] determined that a deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment and had a positive effect on auricin production, indicating the involvement of the bpsA gene in the biosynthesis of the indigoidine ( 16) blue pigment in S. aureofaciens CCM 3239 [95].In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded.GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.No study on the precursor(s) or the biosynthetic enzyme for the phenoxazinone skeleton of these compounds has so far been reported.Because grixazones A ( 14) and B (15) contain an aldehyde and a carboxyl group, respectively, at the 8-position, the biosynthesis of the phenoxazinone skeleton in grixazones should be the same as those in michigazone and texazone [115].
Indigoidine ( 16) is a member of the class of pyridone, an extracellular blue pigment from S. aureofaciens CCM 3239 (Figure 11).The bpsA gene, which encodes nonribosomal peptide synthetase, is responsible for the biosynthesis of the blue pigment indigoidine ( 16) [116].Novakoba et al. [95] determined that a deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment and had a positive effect on auricin production, indicating the involvement of the bpsA gene in the biosynthesis of the indigoidine ( 16) blue pigment in S. aureofaciens CCM 3239 [95].Katorazone ( 17) is an alkaloid with a 2-azaquinone-phenylhydrazone structure (Figure 12).Characteristic structural features of 2-azaquinones in nature are the presence of a methyl group at C-3 and different substituents in ring C [87].The biosynthetic pathway of katorazone (17) of bacterial origin is unclear; however, it is a derivative of anthraquinones, which in plants have two main biosynthetic pathways: the polyketide pathway and the chorismate/o-succinylbenzoic acid pathway [117].4,8,13-trihydroxy-6,11-dione-trihydrogranaticins A (TDTA) ( 18) is a type cin, which is a benzoisochromanequinone that is structurally very similar to ac (13) (Figure 13); however, the stereochemistry around the oxygen bridge of the in granaticin is opposite to that of actinorhodin (13) and the presumed tricycli diate undergoes C-glycosylation instead of dimerization.The gra gene cluster e the biosynthesis and transfer of the appropriate deoxysugar group.The gra include the minimal PKS genes, a KR gene, and an ARO/CYC gene.Further s of this gene cluster has led to the identification of several genes involved in d biosynthesis [113].Resistomycin ( 19) is a pentacyclic polyketide metabolite and quinone-rela otic, which has a unique structure-a ring system that differs from other bac matic polyketides [93] (Figure 14).Jakobi et al. [118] identified the entire gene coding resistomycin (19) biosynthesis and determined that the rem gene clust several unusual features of the type II PKS involved, most remarkably a putativ CoA acyltransferase (MCAT) with highest homology to AT domains from mod [118].4,8,13-trihydroxy-6,11-dione-trihydrogranaticins A (TDTA) ( 18) is a type of granaticin, which is a benzoisochromanequinone that is structurally very similar to actinorhodin (13) (Figure 13); however, the stereochemistry around the oxygen bridge of the pyran ring in granaticin is opposite to that of actinorhodin (13) and the presumed tricyclic intermediate undergoes C-glycosylation instead of dimerization.The gra gene cluster encodes for the biosynthesis and transfer of the appropriate deoxysugar group.The gra PKS genes include the minimal PKS genes, a KR gene, and an ARO/CYC gene.Further sequencing of this gene cluster has led to the identification of several genes involved in deoxysugar biosynthesis [113].4,8,13-trihydroxy-6,11-dione-trihydrogranaticins A (TDTA) ( 18) is a type cin, which is a benzoisochromanequinone that is structurally very similar to ac (13) (Figure 13); however, the stereochemistry around the oxygen bridge of the in granaticin is opposite to that of actinorhodin ( 13) and the presumed tricycl diate undergoes C-glycosylation instead of dimerization.The gra gene cluster e the biosynthesis and transfer of the appropriate deoxysugar group.The gra include the minimal PKS genes, a KR gene, and an ARO/CYC gene.Further s of this gene cluster has led to the identification of several genes involved in d biosynthesis [113].Resistomycin (19) is a pentacyclic polyketide metabolite and quinone-rela otic, which has a unique structure-a ring system that differs from other ba matic polyketides [93] (Figure 14).Jakobi et al. [118] identified the entire gene coding resistomycin (19) biosynthesis and determined that the rem gene clust several unusual features of the type II PKS involved, most remarkably a putativ CoA acyltransferase (MCAT) with highest homology to AT domains from mod [118].Resistomycin (19) is a pentacyclic polyketide metabolite and quinone-related antibiotic, which has a unique structure-a ring system that differs from other bacterial aromatic polyketides [93] (Figure 14).Jakobi et al. [118] identified the entire gene cluster encoding resistomycin (19) biosynthesis and determined that the rem gene cluster exhibits several unusual features of the type II PKS involved, most remarkably a putative malonyl CoA acyltransferase (MCAT) with highest homology to AT domains from modular PKSs [118].
matic polyketides [93] (Figure 14).Jakobi et al. [118] identified the entire gene coding resistomycin (19) biosynthesis and determined that the rem gene clust several unusual features of the type II PKS involved, most remarkably a putati CoA acyltransferase (MCAT) with highest homology to AT domains from mo [118].Otherwise, melanins (Figure 15) are polymers with diverse structures and brown to black colorations [116].They have multiple important functions and are formed by the oxidative polymerization of phenol and/or indolic compounds; however, their structures are not well understood [55].Actinobacteria members produce a dark pigment, melanin, which is considered valuable for taxonomic relatedness [116].
Coatings 2022, 12, x FOR PEER REVIEW Otherwise, melanins (Figure 15) are polymers with diverse structures and b black colorations [116].They have multiple important functions and are formed oxidative polymerization of phenol and/or indolic compounds; however, their str are not well understood [55].Actinobacteria members produce a dark pigment, m which is considered valuable for taxonomic relatedness [116].The enzyme tyrosinase is responsible for the first step in the melanin biosy and the gene melC encodes for the tyrosinase operon [116].The regulatory regio mel gene has three unique sites for the SstI, BglII, and SphI restriction endonu which permits the easy recognition of colonies containing the insertion of the D interest [116].
There are five types of melanins (eumelanin, pyomelanin, pheomelanin, allom and neuromelanin), and each of these pigments is synthesized enzymatically or no matically from different precursors by different metabolic pathways [107].
Eumelanin is a polymer of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyin carboxylic acid (DHICA) [55], originating from the tyrosine or phenylalanine amin [107]; however, the detailed polymer structure is undetermined [55].The path eumelanogenesis may be divided into two phases, one proximal and the other dis proximal phase consists of the enzymatic oxidation of tyrosine or L-DOPA to it The enzyme tyrosinase is responsible for the first step in the melanin biosynthesis and the gene melC encodes for the tyrosinase operon [116].The regulatory region of the mel gene has three unique sites for the SstI, BglII, and SphI restriction endonucleases, which permits the easy recognition of colonies containing the insertion of the DNA of interest [116].
There are five types of melanins (eumelanin, pyomelanin, pheomelanin, allomelanin, and neuromelanin), and each of these pigments is synthesized enzymatically or nonenzymatically from different precursors by different metabolic pathways [107].
Eumelanin is a polymer of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [55], originating from the tyrosine or phenylalanine amino acids [107]; however, the detailed polymer structure is undetermined [55].The pathway of eumelanogenesis may be divided into two phases, one proximal and the other distal.The proximal phase consists of the enzymatic oxidation of tyrosine or L-DOPA to its corresponding dopaquinone catalyzed by tyrosinase and the distal phase is represented by chemical and enzymatic reactions which occur after dopachrome formation and lead to the synthesis of eumelanins [55].
Pyomelanin is a natural polymer of homogentisic acid (HGA, 2,5-dihydroxyphenylacetic acid) synthesized through the L-tyrosine pathway, and belongs to the heterogeneous group of allomelanins [120].It is formed by the catabolism of tyrosine and/or phenylalanine [90].

Pigment Purification
The initial process, in most cases, wherein the pigment is secreted into the culture broth, is centrifugation to remove the biomass [66,85].To partially separate the pigment from the other metabolites that generally accompany it, different types of reagents are applied, such as acids or bases.To modify the pH and make the target pigment precipitate [40,66], solid-liquid extraction is used [94,102].To add solvents in different ratios (organic, polar, and nonpolar) [63] or to establish a solvent system to extract the pigment [84], liquidliquid extraction is used [79].In addition, different conditions are used that modify the pressure or the temperature to concentrate the pigment, such as vacuum-freeze-drying or lyophilization [40,66,67], flash evaporation [60,108] or reduction of pressure [61], and rotary evaporation [79,99].Some research has used physical methods to assist in the purification of intracellular pigments, such as using an ultrasonic cell-disrupter system to perform lysis (sonicated) [82,105], filtration using membranes [98] and ultrafiltration membranes [100], commercial products such as a centricon-30 concentrator [100], and a Soxhlet extractor [106].The number of times a specific procedure is repeated on the purification strategy is variable.For example, performing centrifugation two or three times [60,103,104,107], performing a re-extraction many times [61,82], and repeated crystallizations [106].
Once a crude extract containing the pigment is available, different separation techniques are applied, among which the most used is chromatography.Depending on the physicochemical characteristics of the pigment, the most appropriate one will be chosen.
Additionally, it should be clarified that some of the purification processes allow one to concentrate the pigment.These include lyophilization or freeze-drying, which is a dehydration process based on the sublimation of ice contained in the material, the above in order to obtain a final product with little damage caused by thermal and chemical degradation [121].
Additionally, the largest scale-up was from 800 mL (8 Erlenmeyer of 250 mL with 100 mL of medium) to a 5 L bioreactor with 3 L of medium; this scaling up by increasing the agitation from 200 to 300 rpm and adding an air flow of 3 L/min resulted in a significant increase in pigment production yields (from 5030 to 9000 mg/L) [92].

Stability Tests of the Pigments
Even though one of the main disadvantages of pigments is their stability, only 17% of the selected articles evaluated them.The stability of the pigment includes assays under different conditions of pH, temperature, or light [76,97], or, in the presence of metal ions, additives, vitamins, and reducing and oxidizing agents [82,86].
Most of the Streptomyces pigments reported are very stable (see Table 3), which is a relevant fact for its potential applications; however, the condition at which some are most unstable is pH.TDTA (18) pigments from Streptomyces sp.A1013Y and λ-actinorhodin (12) from Streptomyces coelicolor 100, especially, were subjected to multiple stability tests and the only disadvantage is their sensitivity to different pHs [82,86].

Optimization of the Pigment's Production
One of the main concerns in pigment production is yield.For this reason, the optimization of culture media and fermentation conditions becomes important.There are different ways to improve the amount of the pigment: by changing the kind of medium used, including the ISP (International Streptomyces Project) medium, among others; changing the concentration of the components of a base culture medium; changing the main sources of nutrients, such as carbon and nitrogen sources (see Table 4); even using experimental designs such as Plackett-Burman and central composite design with multiple factors and levels [43,73,78].

Antimicrobial Activity
Antibiotics are essential for human health and are one of the pillars of modern medicine; however, we are dealing with the evolution and dissemination of resistance mechanisms that endangers the current arsenal of antibiotics.One of the requirements in this matter is the discovery of new antimicrobial compounds with high efficiency and nontoxicity [80,122,123].In this way, actinobacteria and especially the Streptomyces genus are responsible for most antibiotics in use today [48,80,124].
Many of the Streptomyces strains that produce pigments have antimicrobial activity reporting the measure of the inhibition zone (see Table 5) and, in other cases, were determined to have minimal inhibitory concentrations (MIC) (see Table 6).Thus, the reported Streptomyces strains are promising antibiotic producers against innumerable pathogens. 1This is the average of the extracts with activity in different solvents (methanol, dichloromethane, diethyl ether extract, chloroform extract, and ethyl acetate). 2The original data were reported with standard deviations. 3This is a range; the two forms of melanin (insoluble and soluble) were evaluated at different concentrations. 4The measure presented is the maximum inhibition zone of different concentrations. 5Additionally, there is information about antimicrobial activity of biopigment-assisted synthesized nanoparticles. 1 Only performed the antimicrobial assay once and did not report their respective standard deviations. 2 The original data were reported with standard deviations. 3These compounds are pigment-associated compounds; however, its role as a pigment or its coloration are not clear.
Especially, Streptomyces sp.D25 was evaluated against M. tuberculosis H37Rv using luciferase reporter mycobacteriophage (LRP) assays, and the results were expressed in the percentage reduction in the relative light unit (RLU).In this case, they evaluated extracts in different solvents (methanol, dichloromethane, diethyl ether extract, chloroform extract, and ethyl acetate) and the activity ranged from 84.74 ± 3.60 to 91.59 ± 4.02 [62].
The minimum inhibitory concentration varies widely (0.5-200 µg/mL).Especially, the S. spectabilis strain L20190601 and Streptomyces sp.MBT27 have antimicrobial activity, even at very low concentrations [77] (see Table 6).Of the few that reported an MIC value, only some performed the assay more than once and reported their respective standard deviations.
The prodiginine family are primarily red-pigmented, specialized metabolites that have a tripyrrole structure [125] and include compounds such as undecylprodigiosin (2) and metacycloprodigiosin (1).These compounds are promising antimicrobials against Gram-positive and Gram-negative bacteria and fungi; therefore, this family can be an inexhaustible source of antibiotics [77,88,89].On the other hand, pigments such as the melanin from Streptomyces sp.MVCS13 have a potential effect against the ornamental fish pathogens of Carassius auratus [66].Additionally, even though actinomycin L 1 (8) and L 2 (9) from Streptomyces sp.MBT27 are diastereomers that stem from the aminal formation at C-10 , actinomycin L 1 (8) showed a somewhat higher bioactivity than actinomycin L 2 [80].

Antioxidant Activity
Oxygen is essential for aerobic life [126]; however, it is a potential hazard because it promotes the formation of reactive oxygen species (ROS) [127].An antioxidant is a substance that delays or prevents the oxidation of a substrate.In addition, in the human body they stabilize the generated radical and reduce the oxidative damage [128].
The second bioactivity most evaluated was the antioxidant activity, and some widely used methods include DPPH [82], ABTS [40,90,102], reducing power assays [92], and hydroxyl [67] and superoxide radical scavenging activity [94].Other, less common methods, include the ferric thiocyanate method [88], lipid peroxidation, and the protein oxidation inhibition assay [91].On the other hand, different kinds of results for antioxidant activity have been reported: IC 50 values and equivalence to vitamin C (see Table 7), percentage to a specific concentration (see Table 8), and some descriptive results (see Table 9).Reducing power assay Antioxidant activity of melanin might be due to redox reactions.

Streptomyces sp. JS520 (Undecylprodigiosin (2))
Ferric thiocyanate method Undecylprodigiosin (2) did not perform as well as commercially available antioxidant α-tocopherol; however, it was effective in delaying lipid peroxidation.[88] Hydrogen peroxide disc-diffusion assay Undecylprodigiosin (2) acted as a scavenger of H 2 O 2 that is released through the process of peroxidation.

Streptomyces coelicolor MSIS1 (Extract) Reducing Power Assay
The pigment had positive results for all the concentrations: 10 mg/mL, 50 mg/mL, and 100 mg/mL. [92] DPPH Acid-treated forms of melanin showed much stronger radical scavenging ability than the intact melanin derivatives.
[70] Hydroxyl radical scavenging activity Rapid oxidation and bleaching of the melanin pigment and thus its capacity to scavenge H 2 O 2 out of the environment.

Free radical scavenging activity
The pigment showed increasing free radical scavenging activity and total antioxidant activity with increased concentrations.
[68] Ferric Reducing Antioxidant Power Hydroxyl Radical Scavenging Activity Two of the most used methods for the evaluation of antioxidant activity are DPPH and ABTS assays.In the cases in which the antioxidant capacity of the same sample was evaluated using these two methods (see Tables 4 and 5), it was evident that there was a difference; the DPPH assay is applicable to only hydrophobic systems [129] while the ABTS assay is applicable to both hydrophilic and lipophilic.Additionally, it is suggested that the ABTS assay better reflects the antioxidant contents than the DPPH assay [130] and is considered to be a method of high sensitivity, which is practical, fast, and very stable [131].
Most reported Streptomyces pigments show high antioxidant activity, regardless of the evaluation method or the units of reporting, demonstrating that these pigmented extracts or compounds are promising as antioxidant agents, with potential applications in the cosmetic industry.Additionally, melanin is highlighted as an antioxidant which, regardless of its origin, has already been reported to have other self-protective roles in response to elevated environmental stress conditions, such as antiultraviolet radiation, chelating metal ions, high temperature tolerance [94,132], and bioactivities (e.g., antibiotic and anticancer) [70].

Cytotoxic Activity
Cancer is a major public health threat worldwide as the leading cause of morbidity and mortality [89,93,133,134].Even worse, cancer treatments such as chemotherapy, surgery, and radiotherapy are unsatisfactory.This is due to the high complexity of the disease and its wide variety of molecular mechanisms for attacking cancer cells, the rapid evolution of resistance to today's multiple anticancer drugs, and the drug side effects [84,85].For these reasons, searching for new secondary metabolites for cancer treatment that are more effective and safer is an urgent priority [84,85,89,93].
Red pigments showed promise as anticancer agents.Extracts of Streptomyces sp.PM4 strain [61] and fractions of Streptomyces sp.A 16-1 [85] extract showed activity at very low concentrations in the range of 0.04-18.5 µg/mL (IC 50 value) against cancer lines and are harmless against healthy lines such as PBMCs.Specifically, red compounds such as undecylprodigiosin (2) showed activity against HeLa [89], and prodigiosin (4) is safe against healthy cell lines [83].
On the other hand, the yellow-pigmented extract was tested against cell lines for different exposure times, showing that the longer the exposure time, the lower the IC 50 value.Again, it is safe against a healthy human lymphocyte line [84].Likewise, melanin from different strains showed great cytotoxic activity against different cancer lines, but its toxicity against healthy lines was predominant [40].
In other cases, cytotoxic activity was reported using different measurements such as growth inhibitory activity (GI 50 ), which is the concentration of the evaluated compound required to cause a 50% decrease in net cell growth [136]; and lethal concentration 50 (LC 50 ), which is the concentration of a given agent that obtains a cellular lethality of 50% (see Table 11) [137].
Resistomycin (19), besides being an excellent natural antibiotic, had its cytotoxic activity evaluated by Vijayabharathi et al. [93] in 2011.It was later studied in detail by Han et al. [138], who determined that resistomycin (19) activates the p38 MAPK signaling pathway, causing apoptosis and G2/M phase arrestin.
Another way to evaluate the cytotoxic activity is by in vivo assays, wherein the measurement reported is the median lethal dose (LD 50 ) (see Table 12) [137].Only actinomycin X2 (10) and λ-Actinorhodin (12) pigment toxicities were evaluated in the brine shrimp A. salina and Mouse, respectively.In both cases, the pigment had a good biological safety property [69,86].

Applications of Streptomyces Pigments
Streptomyces pigments have a wide variety of applications, including their application as antimicrobial (26.1%), anticancer (17.4%), and antioxidant (10.1) agents.Additionally, 13% of the Streptomyces pigments do not have a specific application; therefore, they require further study and may have great biotechnological potential (Figure 16).For the specific application of each pigment, see material Supplementary Tables S7-S13.

Applications of Streptomyces Pigments
Streptomyces pigments have a wide variety of applications, including their application as antimicrobial (26.1%), anticancer (17.4%), and antioxidant (10.1) agents.Additionally, 13% of the Streptomyces pigments do not have a specific application; therefore, they require further study and may have great biotechnological potential (Figure 16).For the specific application of each pigment, see material supplementary Tables S7-S13.Especially, Wibowo et al. [70] studied the activity of purified dissolved melanin (PDM), acid-based precipitation of melanin (AM), and synthetic melanin standard (SM) against Alivibrio fischeri, determining that the quorum-sensing activity of A. fischeri was interrupted more clearly by PDM and SM.Additionally, that was the first report of this activity on melanin and it proposed that the melanin from S. cavourensis SV 21 may have an important function for the microbe-host and/or microbe-microbe interaction.In the same way, Wang et al. [94] reported that insoluble and soluble melanin pigments could reduce biofilm formation against the Gram-positive M. smegmatis ATCC 10231 and the Gram-negative P. aeruginosa ATCC 9027 in a dose-dependent manner.
On the other hand, the pink pigment of Streptomyces sp.NS-05 was used to synthesize silver nanoparticles (AgNPs) which showed antimicrobial activity against Gram-positive and Gram-negative bacteria and can be used for the green synthesis of other nanoparticles [81].In addition, Bayram [107] determined that the amorphous organic semiconductor, X-ray, and γ-ray-absorbing properties of pyomelanin polymers require more investigation for use in nanocomposite and biocomposite material production.
Other notable applications are as antituberculars and anti-HIVs.Thus, the pinkishbrown-pigment-producing Streptomyces sp.S45 showed anti-HIV activity with the IC50 value of 8.75 µg/mL [63].In addition, the pigment from Streptomyces sp.SFA5 was evaluated against M. tuberculosis H37Rv and for inhibitory activity against M. tuberculosis lysine aminotransferase, showing activity in both assays and an IC 50 value of 4.5 µg/mL concentration for the last one [79].
Streptomyces Pigments with Antibiofilm/Antifouling Potential Bacterial biofilms have a structural complex architecture and develop on many abiotic surfaces (plastic, glass, metal, and minerals) and biotic surfaces (plants, animals, and humans) [139].Bacteria growing on biofilms is up to 1000-fold more resistant to antibiotics and biocides compared to their planktonic counterparts [140].Biofilms are the root cause of biofouling [141].Specifically, the attachment of micro-and macroorganisms to waterimmersed surfaces is an undesired phenomenon in some cases, and is known as marine biofouling, resulting in severe problems for aquaculture, shipping, and other industries that rely on coastal and off-shore infrastructures [142].
Napyradiomycin (naphthalene quinone) derivatives (Figure 17) that were isolated from Streptomyces from ocean sediments from the Madeira Archipelago presented antifouling activity.Pereira et al. (2020) [142], revealed that napyradiomycins (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) inhibited ≥80% of the marine-biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae.Napyradiomycin derivates (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) disclosed bioactivity against marine micro-and macrofouling organisms and nontoxic effects towards the studied species, displaying potential to be used in the development of antifouling products [142].The first are highly reactive organic compounds used as dyes, whose colors range from yellow to red [112].The second are natural dyes used as mordant, and among these is quercetin (33), which is part of flavonol, one of the main chromophores in flavonoids with a yellow color; and one of its derivatives, the flavonol taxifolin (32) or dihydroquercetin [147].Napyradiomycin (naphthalene quinone) derivatives (Figure 17) that were isolated from Streptomyces from ocean sediments from the Madeira Archipelago presented antifouling activity.Pereira et al. (2020) [142], revealed that napyradiomycins (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) inhibited ≥80% of the marine-biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae.Napyradiomycin derivates (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) disclosed bioactivity against marine micro-and macrofouling organisms and nontoxic effects towards the studied species, displaying potential to be used in the development of antifouling products [142].Gopikrishnan et al. ( 2019) [145], reported the isolation, characterization, and potential antifouling activity of taxifolin (32), a flavonoid compound from Streptomyces sp.PM33 isolated from mangrove sediments (Figure 18).Toxicity assays based on zebra fish models revealed the less or moderate toxicity of this metabolite.Taxifolin (32) showed significant potential to fight against biofilm formation.It inhibited algal spore germination and mollusk foot adherences were the main mechanism of antibiofilm activities of the metabolite.Taxifolin (32) in the field experiments revealed good antifouling activity when tested on wooden surfaces and PVC panels [145].Sheir and Hafez, in 2017 [148], demonstrated that S. toxytricini fz94 crude pigmented extract was an effective and safe anti-Candida biofilm at concentrations in prevention and destruction modes.It was similar to ketoconazole against clinical Candida isolates and it was more potent than ketoconazole in the destruction of C.albicans biofilm [148].Gopikrishnan et al., in 2016 [149], reported on quercetin (33) from marine-derived Streptomyces sp.PE7 with antibiofouling activity (Figure 19).It was active against 18 biofouling bacteria with an MIC range between 1.6 and 25 μg/mL and had algal spore germination and mollusk foot adherence found at 100 μg/mL and 306 ± 19.6 μg mL −1 , respectively.Previous research by the same research group [146] obtained a crude pigment from the Streptomyces sp D25, produced by agar surface fermentation using yeast extract and malt extract agar and extracted using ethyl acetate.The pigmented extract exhibited antioxidant potential in DPPH and nitric oxide assays and antimicrobial activity against the biofilm-forming bacteria in the disc-diffusion method.Further in vivo studies on this Streptomyces pigment pave the way for its biomedical applications.With the above-Figure 17.Chemical structures of napyradiomycins isolated from marine-derived S. aculeolatus strains PTM-029 [142].Gopikrishnan et al. (2019) [145], reported the isolation, characterization, and potential antifouling activity of taxifolin (32), a flavonoid compound from Streptomyces sp.PM33 isolated from mangrove sediments (Figure 18).Toxicity assays based on zebra fish models revealed the less or moderate toxicity of this metabolite.Taxifolin (32) showed significant potential to fight against biofilm formation.It inhibited algal spore germination and mollusk foot adherences were the main mechanism of antibiofilm activities of the metabolite.Taxifolin (32) in the field experiments revealed good antifouling activity when tested on wooden surfaces and PVC panels [145].[145], reported the isolation, characterization, and poten tial antifouling activity of taxifolin (32), a flavonoid compound from Streptomyces sp PM33 isolated from mangrove sediments (Figure 18).Toxicity assays based on zebra fish models revealed the less or moderate toxicity of this metabolite.Taxifolin (32) showed significant potential to fight against biofilm formation.It inhibited algal spore germina tion and mollusk foot adherences were the main mechanism of antibiofilm activities o the metabolite.Taxifolin (32) in the field experiments revealed good antifouling activity when tested on wooden surfaces and PVC panels [145].Sheir and Hafez, in 2017 [148], demonstrated that S. toxytricini fz94 crude pigmented extract was an effective and safe anti-Candida biofilm at concentrations in prevention and destruction modes.It was similar to ketoconazole against clinical Candida isolates and i was more potent than ketoconazole in the destruction of C.albicans biofilm [148].Gopikrishnan et al., in 2016 [149], reported on quercetin (33) from marine-derived Streptomyces sp.PE7 with antibiofouling activity (Figure 19).It was active against 18 bio fouling bacteria with an MIC range between 1.6 and 25 μg/mL and had algal spore germi nation and mollusk foot adherence found at 100 μg/mL and 306 ± 19.6 μg mL −1 , respec tively.Previous research by the same research group [146] obtained a crude pigment from the Streptomyces sp D25, produced by agar surface fermentation using yeast extract and malt extract agar and extracted using ethyl acetate.The pigmented extract exhibited anti oxidant potential in DPPH and nitric oxide assays and antimicrobial activity against the biofilm-forming bacteria in the disc-diffusion method.Further in vivo studies on this Streptomyces pigment pave the way for its biomedical applications.With the above Sheir and Hafez, in 2017 [148], demonstrated that S. toxytricini fz94 crude pigmented extract was an effective and safe anti-Candida biofilm at concentrations in prevention and destruction modes.It was similar to ketoconazole against clinical Candida isolates and it was more potent than ketoconazole in the destruction of C.albicans biofilm [148].Gopikrishnan et al., in 2016 [149], reported on quercetin (33) from marine-derived Streptomyces sp.PE7 with antibiofouling activity (Figure 19).It was active against 18 biofouling bacteria with an MIC range between 1.6 and 25 µg/mL and had algal spore germination and mollusk foot adherence found at 100 µg/mL and 306 ± 19.6 µg mL −1 , respectively.Previous research by the same research group [146] obtained a crude pigment from the Streptomyces sp D25, produced by agar surface fermentation using yeast extract and malt extract agar and extracted using ethyl acetate.The pigmented extract exhibited antioxidant potential in DPPH and nitric oxide assays and antimicrobial activity against the biofilmforming bacteria in the disc-diffusion method.Further in vivo studies on this Streptomyces pigment pave the way for its biomedical applications.With the above-mentioned research, it is possible to propose the use of pigmented extracts or metabolites of Streptomyces that are ecofriendly and can be the basis for the preparation of biocidal paints to coat different  To our knowledge, most research has evaluated the pigmented compound against biofilm-forming microorganisms; however, studies with Streptomyces pigments have been limited only to biocidal activity and quorum sensing inhibitors, two fundamental bioactivities in biofouling.Future research is required to evaluate these Streptomyces pigments that have already demonstrated these bioactivities in antifouling assays, and this may be the beginning to expand the repertoire of candidates useful in the development of surface coatings that need to be protected from the complex bioprocess of biofouling.

Future Perspectives
Only 50.9% of the articles reported partial or complete purification, 13% had no determined application, and others had potential applications without specific tests and poorly studied isolation sources, such as freshwater (Free-living) or terrestrial symbionts.Future research includes purifying pigmented extracts that have already been reported, studying the purified compounds in a specific application, and isolating new microorganisms from new or poorly studied isolation sources.
However, research has also focused on improving the production of pigments already identified with applications, using different optimization methods to achieve largescale production, changing the culture media, the main sources of nutrients, and their culture conditions.In addition, taking into account the economy, the large amount of agroindustrial waste, and the Sustainable Development Goals Fund, the use of new sources of nutrients, such as sugar cane waste, rice bran, wheat bran, coconut cake, and rice flour, is a new subject of study (as performed by Vasanthabharathi et al. [60]).
Not only the culture media have been the subject of study, but also the use of new technologies such as solid-state fermentation (SSF) using a novel PolyHIPE Polymer (PHP) matrix in a microbioreactor for improving the production of antibiotics from S. coelicolor A3(2) [101].In other ways, it is required to recognize how conditions influence product formation, and online monitoring emerges as a great and valuable tool.For example, Finger et al. [109] determined that oxygen transfer rate and autofluorescence are key features in understanding the cultivation of the model organism Streptomyces coelicolor A3(2) [109].
On the other hand, pigment production has focused on downstream processes, improving existing extraction techniques or developing new techniques, including vacuumfreeze-drying or lyophilization [48,49,73], flash evaporation [60,108] or pressure reduction To our knowledge, most research has evaluated the pigmented compound against biofilm-forming microorganisms; however, studies with Streptomyces pigments have been limited only to biocidal activity and quorum sensing inhibitors, two fundamental bioactivities in biofouling.Future research is required to evaluate these Streptomyces pigments that have already demonstrated these bioactivities in antifouling assays, and this may be the beginning to expand the repertoire of candidates useful in the development of surface coatings that need to be protected from the complex bioprocess of biofouling.

Future Perspectives
Only 50.9% of the articles reported partial or complete purification, 13% had no determined application, and others had potential applications without specific tests and poorly studied isolation sources, such as freshwater (Free-living) or terrestrial symbionts.Future research includes purifying pigmented extracts that have already been reported, studying the purified compounds in a specific application, and isolating new microorganisms from new or poorly studied isolation sources.
However, research has also focused on improving the production of pigments already identified with applications, using different optimization methods to achieve large-scale production, changing the culture media, the main sources of nutrients, and their culture conditions.In addition, taking into account the economy, the large amount of agroindustrial waste, and the Sustainable Development Goals Fund, the use of new sources of nutrients, such as sugar cane waste, rice bran, wheat bran, coconut cake, and rice flour, is a new subject of study (as performed by Vasanthabharathi et al. [60]).
Not only the culture media have been the subject of study, but also the use of new technologies such as solid-state fermentation (SSF) using a novel PolyHIPE Polymer (PHP) matrix in a microbioreactor for improving the production of antibiotics from S. coelicolor A3(2) [101].In other ways, it is required to recognize how conditions influence product formation, and online monitoring emerges as a great and valuable tool.On the other hand, pigment production has focused on downstream processes, improving existing extraction techniques or developing new techniques, including vacuum-freezedrying or lyophilization [48,49,73], flash evaporation [60,108] or pressure reduction [61], ultrasonic cell-disrupter system to perform lysis [67] or sonication [91], and filtration using membranes [98] and ultrafiltration membranes [100].Likewise, a wide range of ecofriendly and efficient solvents have been studied and the extraction processes improved using solvents in different ratio [63], using different solvent systems [84], or performing a re-extraction many times [61,82].As an example of this, Wibowo et al. [70] studied the two forms of melanin from Streptomyces cavourensis SV 21 and proposed a novel acid-free purification protocol of purified particulate melanin (PPM) and purified dissolved melanin (PDM) [70].
Finally, considering the great variety of Streptomyces pigment bioactivities (antioxidant, antimicrobial, and cytotoxic), the great variety of colorations, and the relative safety against healthy cell lines, Streptomyces pigments may be a valuable biotechnological resource with potential applications as nutraceuticals and a potential replacement for synthetic dyes.Additionally, it is possible to propose the use of pigments of Streptomyces that are ecofriendly and can be the basis for the preparation of biocidal paints to coat different surfaces and thus protect them from biofilm or biofouling attacks.Further studies of Streptomyces pigments will be necessary to optimize the bioprocesses and scale-up production, as will preclinical studies and in situ trials to fully establish their feasibility.

Databases and Search Strategy
For a review of the literature as complete as possible, the search was performed using the following databases: Scopus, Web of Science, and PubMed.The terms (including synonyms and related words) and boolean operators used for all searching were defined as follows: Streptomyces AND (pigment OR colorant OR stain OR dye OR coloring OR tint).

Selection Procedure
The selection of the articles was based on the following inclusion criteria: (a) original research articles; (b) studies on extracts, compounds, fractions, or pure cultures derived from Streptomyces strains; and (c) studies related to pigment production (evaluation of bioactivities, purification or/and elucidation, and optimization of the production).
The following were considered exclusion criteria: (a) articles were written in a language other than English, and (b) articles whose full-text version could not be accessed.
The article selection process was subdivided into two stages as follows: in the first stage, four researchers separately assessed each title and abstract in a blind process.At this time, each article was marked as potentially eligible to be included in the review when at least two studies indicated that it met the inclusion/exclusion criteria.When an article was indicated as eligible by only one researcher, a discussion within the research team was carried out to solve disagreement.In the second stage, potentially eligible articles were examined at the full-text level.Thus, those articles that complied with the inclusion/exclusion criteria were finally selected for data extraction.

Data Collection and Tabulation
To guarantee careful and cautious data collection and avoid the risk of bias, a pilot data acquisition form was prepared.The form was evaluated and improved through an exercise including ten randomly selected articles.In this manner, having defined the final version, the form was used for the data acquisition of the complete set of selected articles.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/coatings12121858/s1,Table S1.PRISMA checklist.Table S2.Gram-negative Bacteria evaluated in the antimicrobial test.Table S3.Gram-positive Bacteria evaluated in the antimicrobial test.Table S4.Mushrooms and Yeast evaluated in the antimicrobial test.Table S5.Streptomyces strains source of bioactive crude extracts.Table S6.List of compounds retrieved from the included papers.Table S7.Streptomyces pigments without any specific application.Table S8.Streptomyces pigments belonging to the melanin family.Table S9.Streptomyces pigments belonging to the actinomycin family.Table S10.Streptomyces pigments belonging to the actinorhodin and prodigiosin family.Table S11.Yellow Streptomyces pigments and its applications.Table S12.Red and pink Streptomyces pigments and its applications.Table S13.Other Streptomyces pigments and its applications.Molecules from compounds retrieved from the included papers.Table S14.List of molecules retrieved from the included papers.

Figure 2 .
Figure 2. General findings around the journals of the selected articles.(a) Publication distribution over the time.(b) Cumulative frequency (%) distribution.

Figure 2 .
Figure 2. General findings around the journals of the selected articles.(a) Publication distribution over the time.(b) Cumulative frequency (%) distribution.

Figure 3 .
Figure 3. World map showing where the articles included in this review were produced, sponding author affiliation.

Figure 4 .
Figure 4. World map showing the countries where the Streptomyces strains were isolated

Figure 3 .
Figure 3. World map showing where the articles included in this review were produced, the corresponding author affiliation.

Figure 3 .
Figure 3. World map showing where the articles included in this review were produced, sponding author affiliation.

Figure 4 .
Figure 4. World map showing the countries where the Streptomyces strains were isolated.

Figure 4 .
Figure 4. World map showing the countries where the Streptomyces strains were isolated.

Figure 5 .
Figure 5. Characteristics of the information registered in the selected articles.(a) Percentage of articles that evaluated either crude extracts (see TableS5), pure compounds (see TableS6), fraction, or pure culture.(b) Percentage of articles by the environment where the isolation occurred.

Figure 5 .
Figure 5. Characteristics of the information registered in the selected articles.(a) Percentage of articles that evaluated either crude extracts (see TableS5), pure compounds (see TableS6), fraction, or pure culture.(b) Percentage of articles by the environment where the isolation occurred.

Figure 15 .
Figure 15.Melanin-related structures and some properties.(a) Basic structure (indolic ring) onance structures that are probably involved in the process of color.The arrows show th and sense of polymerization [119].

Figure 15 .
Figure 15.Melanin-related structures and some properties.(a) Basic structure (indolic ring).(b) Resonance structures that are probably involved in the process of color.The arrows show the points and sense of polymerization [119].
protect them from biofilm or biofouling attack, which could replace the available chemical preparations with antibiofilm or antifouling potential.Coatings 2022, 12, x FOR PEER REVIEW 28 of 36 mentioned research, it is possible to propose the use of pigmented extracts or metabolites of Streptomyces that are ecofriendly and can be the basis for the preparation of biocidal paints to coat different surfaces and thus protect them from biofilm or biofouling attack, which could replace the available chemical preparations with antibiofilm or antifouling potential.
For example, Finger et al. [109] determined that oxygen transfer rate and autofluorescence are key features in understanding the cultivation of the model organism Streptomyces coelicolor A3(2) [109].

Table 1 .
Number of strains and compounds by bioactivity and type of source.

Table 1 .
Number of strains and compounds by bioactivity and type of source.

Table 2 .
Yield reported for each type of pigment and the Streptomyces strain that produces it.

Table 3 .
Stability reported for each type of pigment and the Streptomyces strain that produces it.

Table 4 .
Optimization conditions of pigments produced by each Streptomyces strain.

Table 5 .
Streptomyces strains with antimicrobial activity.

Table 6 .
Streptomyces strains with antimicrobial activity and its MIC values.

Table 7 .
Results (IC 50 and equivalence to vitamin C) of the Streptomyces strains with antioxidant activity reported.

Table 8 .
Results (concentration evaluated and percentage) of the Streptomyces strains with antioxidant activity reported.

Table 9 .
Descriptive results of the Streptomyces strains with antioxidant activity reported.

Table 10 .
IC 50 (µg/mL) of Streptomyces-derived pigments and their conditions (cell lines and cell density, among others).

Table 11 .
GI 50 and LC 50 of Streptomyces-derived pigments and their conditions (cell lines and cell density, among others).

Table 12 .
LD 50 of Streptomyces-derived pigments and the in vivo conditions.