Terrestrial Microorganisms: Cell Factories of Bioactive Molecules with Skin Protecting Applications

It is well known that terrestrial environments host an immense microbial biodiversity. Exposed to different types of stress, such as UV radiation, temperature fluctuations, water availability and the inter- / intra-specific competition for resources, terrestrial microorganisms have been evolved to produce a large spectrum of bioactive molecules. Bacteria, archaea, protists, fungi and algae have shown a high potential of producing biomolecules for pharmaceutical or other industrial purposes as they combine a sustainable, relatively low-cost and fast-production process. Herein, we provide an overview of the different bioactive molecules produced by terrestrial microorganisms with skin protecting applications. The high content in polyphenolic and carotenoid compounds produced by several strains, as well as the presence of exopolysaccharides, melanins, indole and pyrrole derivatives, mycosporines, carboxylic acids and other molecules, are discussed in the context of their antioxidant, photo-protective and skin-whitening activity. Relevant biotechnological tools developed for the enhanced production of high added value natural products, as well as the protecting effect of some antioxidant, hydrolytic and degrading enzymes are also discussed. Furthermore, we describe classes of microbial compounds that are used or have the potential to be used as antimicrobials, moisturizers, biosurfactants, pigments, flavorings and fragrances.


Introduction
Microorganisms are extremely diverse organisms, including bacteria, archaea, protists, fungi and algae. In recent decades, there has been great progress on exploiting the immense chemical diversity available from the abundant microbial world [1,2]. After the discovery of the fungal metabolite penicillin in 1928, which was the beginning of the golden age of microbial-derived natural products and pharmaceuticals, treatments for fungal and parasitic infections as well as for several types of cancers followed [3]. In the forties and early fifties, almost all groups of important antibacterial antibiotics (tetracyclines, cephalosporins, aminoglycosides, macrolides) were discovered, while in the fifties and sixties, antitumor, antiviral and non-antibiotic-enzyme-inhibitory-metabolites were isolated, mainly from Streptomyces species [4].
The successful and wide utilization of microbial metabolites in various therapeutic areas (e.g., cyclosporine as immunosuppressant, doxorubicin as anticancer, and statins as cardio protective agents), Figure 1. During lifetime, skin is exposed to numerous environmental stressors such as UVA and 88 UVB, ionizing radiation and air pollutants, as well as to stressors that may originate from diet (e.g., 89 high fat diets), internal sources (e.g., metabolism or tissue inflammation) and/or lifestyle (e.g.,

Antioxidants 96
Oxidative stress is one of the prevailing causes of skin aging due to increased production 97 and/or accumulation of Reactive Oxygen (ROS) and Nitrogen Species (RNS). Imbalance between 98 their production and the endogenous antioxidant defense mechanisms may result in cellular 99 oxidative stress, causing wrinkling, drying, photo-aging, pigmentation and elastosis of the skin. In 100 addition, accumulation of free radicals may be responsible for cutaneous inflammation and skin 101 cancer [15]. 102 Reactive oxygen species (ROS) are formed as either by-products of normal metabolism (e.g., 103 mitochondrial oxidative phosphorylation), as well by NAD(P)H oxidases, or by exogenous sources 104 such as atmospheric pollutants, UV light, X-or gamma-rays [16]; if their concentration exceeds the 105 cellular antioxidant capacity, ROS cause oxidative stress and damage to all cellular biomolecules 106 [10]. 107 Topical antioxidant products could act as scavengers of reactive species, inhibiting the 108 initiation of chain reactions, responsible for cellular oxidative stress [17]. Many reports have 109 demonstrated the ability of marine microorganisms to biosynthesize antioxidant compounds [2]. 110 Concerning terrestrial microbes, compounds with a significant inhibition of oxidation reactions, like 111 polyphenols, carotenoids or exopolysaccharides, are extensively discussed in the following sections. 112 Bioassays involving the neutralization of different radicals such as the stable radical 2,2-113 diphenyl-1-picrylhydrazyl (DPPH), the cation radical 2,2′-azino-bis-3-ethylbenzotiazolin-6-sulfonic 114 acid (ABTS), as well as the hydroxyl and nitric oxide radicals are widely applied for determining 115 the in vitro antioxidant potential. Even if the relation to the in vivo antioxidant efficacy was not 116 Figure 1. During lifetime, skin is exposed to numerous environmental stressors such as UVA and UVB, ionizing radiation and air pollutants, as well as to stressors that may originate from diet (e.g., high fat diets), internal sources (e.g., metabolism or tissue inflammation) and/or lifestyle (e.g., smoking). While young, these stressors are effectively neutralized by cell protective mechanisms (e.g., the antioxidant transcription factor Nrf2). During aging, defenses are compromised, resulting in accumulating ROS, genome and proteome damage; damage of biomolecules then disrupts normal cell signaling and homeodynamics, resulting in (among others) coarse wrinkles, hyperpigmentation and skin shallowness.

Antioxidants
Oxidative stress is one of the prevailing causes of skin aging due to increased production and/or accumulation of Reactive Oxygen (ROS) and Nitrogen Species (RNS). Imbalance between their production and the endogenous antioxidant defense mechanisms may result in cellular oxidative stress, causing wrinkling, drying, photo-aging, pigmentation and elastosis of the skin. In addition, accumulation of free radicals may be responsible for cutaneous inflammation and skin cancer [15].
Reactive oxygen species (ROS) are formed as either by-products of normal metabolism (e.g., mitochondrial oxidative phosphorylation), as well by NAD(P)H oxidases, or by exogenous sources such as atmospheric pollutants, UV light, X-or gamma-rays [16]; if their concentration exceeds the cellular antioxidant capacity, ROS cause oxidative stress and damage to all cellular biomolecules [10].
Topical antioxidant products could act as scavengers of reactive species, inhibiting the initiation of chain reactions, responsible for cellular oxidative stress [17]. Many reports have demonstrated the ability of marine microorganisms to biosynthesize antioxidant compounds [2]. Concerning terrestrial microbes, compounds with a significant inhibition of oxidation reactions, like polyphenols, carotenoids or exopolysaccharides, are extensively discussed in the following sections.
Bioassays involving the neutralization of different radicals such as the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), the cation radical 2,2 -azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTS), as well as the hydroxyl and nitric oxide radicals are widely applied for determining the in vitro antioxidant potential. Even if the relation to the in vivo antioxidant efficacy was not clearly described, the measured antioxidant activity can give an estimation of the amount of the compounds that can be oxidized under conditions of the assays [18].

Phenolic Compounds
Phenolic compounds are well known for their strong antioxidant and radical scavenging activity, as well as for their interaction with different pharmacological targets. The strong correlation between the microbial phenolic content and the antioxidant activity has been shown by several authors using different microorganisms. Huang et al. confirmed this positive correlation during their investigation of fungal endophytes isolated from medicinal Chinese plants [19]. The strong contribution to the antioxidant activity was also confirmed in Aspergillus austroafricanus, an endophytic fungus isolated from Zingiber officinale rhizome. HPLC analysis of the crude extract showed mainly the presence of hydroxycinnamic acids such as ferulic acid (1), p-coumaric acid (2) and cinnamic acid (3) [20] (Figure 2). Those molecules are well known in the plant kingdom and have been extensively studied for their antioxidant capacity (Table 1).
Resveratrol (4), another well-known natural compound produced by plants, has recently been reported from endophytes isolated from grapevine varieties [27,28]. It is considered one of the most famous compounds for its unique anti-aging properties ( Figure 2). It has been widely reported to be a strong inhibitor of ROS production and protein oxidation and a more effective agent than vitamins E and C against lipid peroxidation [29]. Microorganisms have been successfully considered for the production of resveratrol, since its synthesis and/or its extraction from plants is considered inefficient due to high requirements of organic solvents, biomass and low final yield. Resveratrol was first industrially produced in 2009, using Saccharomyces cerevisiae. Since this development, different methods such as bioconversion and genetic engineering have been used in order to obtain higher yields. For instance, resveratrol has been produced by Alternaria sp. (1.4 µg/L), and by genetically modified S. cerevisiae (531.4 mg/L), and E. coli (2370 mg/L) [30]. The molecules discussed, as well as additional microbial phenolic compounds that have antioxidant or other related biological activities, are presented in Table 1.

Carotenoids
Carotenoids are the most common natural pigments; they are well known for their powerful antioxidant activity, as they are very efficient physical quenchers of singlet oxygen and scavengers of other ROS. Carotenoids are also well known for their capacity to act as quenchers of photosensitization products, giving them photo-protective properties [109].
In the last decade, the interest in microbial fermentation for the production of natural carotenoids has increased. Carotenoid production by bacteria, asporogenous yeasts, filamentous fungi [110] and microalgae [111] has been extensively reported, with cyanobacteria to be the most prominent source [112]. Accordingly, carotenogenic microbes Xanthophyllomyces dendrorhous, Blakeslea trispora, and Haematococcus pluvialis have been widely used in large-scale processes. Furthermore, the transformation of the non-carotenogenic microbes E. coli, S. cerevisiae, Candida utilis, and Zymomonas mobilis, with carotenoid genes from selected microbes has been successfully applied for the production of carotenoids [113]. In fact, E. coli in fed-batch fermentation produced 72.6 mg/g cdw (cell dry weight) of β-carotene [1] and 1.44 g/L of lycopene [49], while astaxanthin production was enhanced 1.4-fold compared to the X. dendrorhous parental strain, reaching 1.25 mg/L (Table 1) [46]. Astaxanthin (5), β-carotene (6) and lutein are the carotenoids with the highest added value (Figure 2) [114]. The oxycarotenoid lutein is mainly produced by microalgae of the genus Chlorella, Dunaliella, and Haematococcus [114]. Its profound effect on the antioxidant defense system is attributed to its chemical structure. In in vitro systems, it significantly scavenged the superoxide (IC50: 21 µg/mL), the hydroxyl (IC50: 1.75 µg/mL), the nitric oxide (IC50: 3.8 µg/mL), and the DPPH (IC50: 35 µg/mL) radical and inhibited lipid peroxidation (2.2 µg/mL). In in vivo systems, it has been proved to be an effective scavenger of superoxide radical (IC50: 21 µg/mL) [51].

Exopolysaccharides (EPSs)
EPSs are high-molecular-weight carbohydrate polymers demonstrating strong scavenging activities, metal chelating ability and lipid peroxidation inhibition. These compounds are among the most exploited bioactive substances for their anti-aging capacity [115].

Carotenoids
Carotenoids are the most common natural pigments; they are well known for their powerful antioxidant activity, as they are very efficient physical quenchers of singlet oxygen and scavengers of other ROS. Carotenoids are also well known for their capacity to act as quenchers of photosensitization products, giving them photo-protective properties [109].
In the last decade, the interest in microbial fermentation for the production of natural carotenoids has increased. Carotenoid production by bacteria, asporogenous yeasts, filamentous fungi [110] and microalgae [111] has been extensively reported, with cyanobacteria to be the most prominent source [112]. Accordingly, carotenogenic microbes Xanthophyllomyces dendrorhous, Blakeslea trispora, and Haematococcus pluvialis have been widely used in large-scale processes. Furthermore, the transformation of the non-carotenogenic microbes E. coli, S. cerevisiae, Candida utilis, and Zymomonas mobilis, with carotenoid genes from selected microbes has been successfully applied for the production of carotenoids [113]. In fact, E. coli in fed-batch fermentation produced 72.6 mg/g cdw (cell dry weight) of β-carotene [1] and 1.44 g/L of lycopene [49], while astaxanthin production was enhanced 1.4-fold compared to the X. dendrorhous parental strain, reaching 1.25 mg/L (Table 1) [46]. Astaxanthin (5), β-carotene (6) and lutein are the carotenoids with the highest added value (Figure 2) [114]. The oxycarotenoid lutein is mainly produced by microalgae of the genus Chlorella, Dunaliella, and Haematococcus [114]. Its profound effect on the antioxidant defense system is attributed to its chemical structure. In in vitro systems, it significantly scavenged the superoxide (IC 50 : 21 µg/mL), the hydroxyl (IC 50 : 1.75 µg/mL), the nitric oxide (IC 50 : 3.8 µg/mL), and the DPPH (IC 50 : 35 µg/mL) radical and inhibited lipid peroxidation (2.2 µg/mL). In in vivo systems, it has been proved to be an effective scavenger of superoxide radical (IC 50 : 21 µg/mL) [51].

Exopolysaccharides (EPSs)
EPSs are high-molecular-weight carbohydrate polymers demonstrating strong scavenging activities, metal chelating ability and lipid peroxidation inhibition. These compounds are among the most exploited bioactive substances for their anti-aging capacity [115].
EPSs are mainly biosynthesized by bacteria and fungi. The ability of a microorganism to produce antioxidant EPSs was first introduced with the study of Paenibacillus polymyxa. This endophytic bacterium, isolated from the root of Stemona japonica, produces different EPSs with strong scavenging activity against the superoxide and the hydroxyl radical [116,117] (Table 1). When tested at a concentration of 1 mg/mL, the scavenging effect of the crude EPS against the superoxide radical was 74.38%, while the activity of the purified EPS-1 and EPS-2 was higher than ascorbic acid. At the same concentration, EPS, EPS-1, EPS-2 were also very effective against the hydroxyl radical [56]. EPS-1 and EPS-2 were composed of mannose, fructose and glucose in molar ratios of 2.6:29.8:1 and 4.2:36.6:1, respectively. Since this discovery, many endophytes were found to produce antioxidant EPSs. A characteristic case is the purified rhamno-galactan fraction of Fusarium solani and Bacillus cereus isolated from Alstonia scholaris and Artemisia annua L., respectively. This EPS fraction showed a significant scavenging activity against the DPPH, (IC 50 :0.6 mg/mL), the superoxide (IC 50 : 2.6 mg/mL) and the hydroxyl radical (IC 50 : 3.1 mg/mL) [54,55].
Optimization of cultivation parameters of P. polymyxa using sucrose, yeast extract and CaCl 2 showed an EPSs yield of 35.26 g/L (18.74%), which was 1.55-fold higher compared to the original medium [57]. EPSs structures are in a great variety. EPSs isolated from the culture medium of the endophytic fungus Aspergillus sp. were mainly composed of mannose and galactose (89.4:10.6) [59], while EPSs isolated from the endophytic bacteria Burkholderia tropica were mainly composed of rhamnose, glucose and glucuronic acid (2:2:1) [60]. Antioxidant EPSs have also been isolated from the terrestrial microalgae Rhodella reticulata. Its extracellular polysaccharides showed strong antioxidant activity, significantly higher than α-tocopherol. The radical scavenging ability against the superoxide radical of the deproteinized extracellular polysaccharide reached 328.48 U/L, compared to 174.03 U/L of α-tocopherol [118].

Enzymes
Enzymes are produced by microorganisms as a primary cell protective detoxification mechanism (e.g., from ROS) as they catalyze the removal of ROS through the formation of less reactive molecules such as oxygen or water. Superoxide dismutases, catalases, and peroxidases are involved in these mechanisms.
Superoxide dismutases (SODs) catalyze the neutralization of two superoxide radicals by the addition of two hydrogen ions to form hydrogen peroxide and oxygen. Belonging to the family of metalloisozymes, SODs are differentiated in their metal cofactor: Ni-SOD, CuZn-SOD, Fe-SOD and Mn-SOD; the last three are commonly found in microalgae. SOD biosynthesis is directly correlated with level of cellular ROS. In fact, a study carried out on microalgae Scenedesmus vacuolatus and Pinnularia viridis showed that the concentration and SOD activity is correlated with ROS related stress [119,120]. Similarly, the elimination of ROS in most Streptococcus and Lactococcus bacterial spp., conforms to this general antioxidant defense system since both genera express MnSOD. However, these bacteria possess only one type of SOD, namely the Mn-containing enzyme (MnSOD), rendering this enzyme an essential part of the antioxidant cell machinery [121].
Catalases contain porphyrin heme active sites that degrade hydrogen peroxide into water and oxygen [119]. One molecule of catalase is able to convert six billion molecules of hydrogen peroxide each minute [122]. In the yeast S. cerevisiae, the overexpression of catalase reduces lactic acid-induced oxidative stress [123]. Furthermore, a study involving the single-cell green alga Chlamydomonas reinhardtii showed that hydrogen peroxide from the media was faster degraded when the catalase inhibitor aminotriazole was absent; thus, catalase is one of the major enzymes involved in ROS detoxification [124].
Finally, peroxidases catalyze the oxidation of several substrates by hydrogen peroxide. Ascorbate, cytochrome C, pyrogallol, and glutathione are examples of these substrates. As for the other antioxidant enzymes, the induction of peroxidases activity upon ROS accumulation seem to be concentration-and time-dependent [119].

Photo-Protective Agents
Ultraviolet A (UVA, 315-400 nm) and ultraviolet B (UVB, 280-315 nm) play a major role in skin cell damage. UVA is mainly involved in the creation of ROS while UVB heavily affects DNA and proteins integrity. To protect themselves against UV radiation, terrestrial microorganisms have developed several strategies, one of which is the accumulation of photo-protective compounds [2].
Despite the evidence that several compounds from microorganisms have photo-protective activities, there has been surprisingly little work carried out involving in vivo skin models. This might be partially explained by the fact that the EU has banned the in vivo testing of cosmetics since 2013. Thus, potential skin protecting effects have been established based on existing in vitro studies [125].

Melanins
Bacteria, fungi and protists are able to produce a diverse group of pigments. Melanized fungi are mostly black yeasts, and melanized bacteria belong mainly to Actinobacteria [126].
The basic role of melanins in microorganisms are still a matter of controversy and speculation. The fact that these compounds are interceptors of UV photons leads to a lower vulnerability of micro-ecosystems to UV radiation. Melanins are also involved in energy production due to their ability to accept electrons. Finally, in some pathogenic microorganisms, these compounds act like virulence factors, lowering the defense mechanisms of the host [127].

Indole and Pyrrole Derivatives
Scytonemin (7) is a yellow to brown alkaloid pigment composed of an indolic and a phenolic subunit. Until now, only four different derivatives have been reported: dimethoxyscytonemin (8), scytonin (9), scytonemin-3a-imine (10) and tetramethoxyscytonemin (11) (Figure 3). Known for their strong UV-absorbing function and free radical scavenging capacity, scytonemin and its derivatives are excellent candidates for skin protecting purposes. Scytonemin prevents up to 90% of solar UV radiations from entering the cell. The strong radical scavenging activity of this compound (IC 50 : 36 µM against the ABTS radical), combined with its localization in the bacterial cell wall explains its protective role and the inability of UV-A radiation to cross the cellular envelope [130,131].
Prodigiosin (12) is characterized by a common pyrrolyl dipyrromethene skeleton containing a 4-methoxy-2,2 -bipyrrole ring system (Figure 3). This red pigment is mainly produced by strains belonging to the bacterial genus Serratia [75]. Well known for its antimalarial, antibacterial, and anticancer activity, prodigiosin has also demonstrated UV protective activity. When used as an additive in commercial sunscreens (4% w/w prodigiosin), the sunscreen protection factors (SPF) increased by 20-65%. In the same study, addition of 4% (w/w) of prodigiosin in photo-protective leaf extracts of Aloe vera and Cucumis sativus fruits showed an increasing of SPFs up to 3.5 orders of magnitude [133]. Bacteria Pseudomonas magneslorubra, Vibrio psychroerythrous, Vibrio gazogenes, Alteromonas rubra, and Rugamonas rubra, along with actinomycetes, such as Streptomyces rubrireticuli and S. longisporus ruber, have been studied for their capacity to produce prodigiosin or its derivatives [133]. Improvement in the production of prodigiosin (277 mg/L) was reported by the addition of a ram horn peptone (RHP, 0.4% w/v) in the culture media of S. marcescens MO-1 [75] (Table 1).

Mycosporines and Mycosporine-Like Amino Acids (MAAs)
Originally detected in the mycelia of terrestrial basidiomycetes, mycosporines present a central cyclohexenone or cyclohexenimine ring and a wide variety of substitutions. Mycosporine-like amino acids are imine derivate of mycosporines. The ring absorbs UV light and dissipates energy as heat, without generating ROS. Cyanobacteria and microalgae can synthesize mycosporines and MAAs, while fungi produce only mycosporines [126] (Table 1).
Mainly known for their photo-protective activity, MAAs are also efficient antioxidants and scavengers of ROS. These activities have led to several patents in the research of natural UV filters [135].
As in other cases, the production of microbial MAAs can be optimized following modification of culturing parameters. Khosravi et al. showed that the combination of UV irradiation and Violacein (13) is a purple pigment that presents an unusual structure consisting of a 2-pyrollidone and an oxindole ring system connected by a double bond, and a 5-hydroxyindole unit (Figure 3) [134]. Known to possess antibacterial effects against Staphylococcus aureus and other Gram-positive pathogens, violacein can also act as a photo-protective agent against UV irradiation. This compound absorbs at visible wavelengths and presents a broad absorption band extended out to 700 nm [69]. When used as an additive in commercial sunscreens (4% w/w violacein), the SPFs increased by 10-22%.
Furthermore, the addition at 4% (w/w) of violacein in photo-protective extracts of A. vera leaves and C. sativus fruits, showed an increasing of SPFs up to 3.5 orders of magnitude [133]. Violacein is mainly produced by the bacterial strains Janthinobacterium lividum, Pseudoalteromonas sp. and Chromobacterium violaceum (Table 1). It is worth mentioning that the medium pH, culture volume, concentration of potassium nitrate, and L-tryptophan, affect significantly violacein production. In fact, the cultivation of C. violaceum, isolated from various plant waste sources, in a medium supplemented with sugar bagasse and L-tryptophan 10% (v/v), increased the final yield production of violacein to 0.82 g/L [70]. Similarly, optimized cultivation parameters of Duganella sp. increased by 4.8 folds the final yield of crude violacein (1.62 g/L) [71].

Mycosporines and Mycosporine-Like Amino Acids (MAAs)
Originally detected in the mycelia of terrestrial basidiomycetes, mycosporines present a central cyclohexenone or cyclohexenimine ring and a wide variety of substitutions. Mycosporine-like amino acids are imine derivate of mycosporines. The ring absorbs UV light and dissipates energy as heat, without generating ROS. Cyanobacteria and microalgae can synthesize mycosporines and MAAs, while fungi produce only mycosporines [126] (Table 1).
Mainly known for their photo-protective activity, MAAs are also efficient antioxidants and scavengers of ROS. These activities have led to several patents in the research of natural UV filters [135].
As in other cases, the production of microbial MAAs can be optimized following modification of culturing parameters. Khosravi et al. showed that the combination of UV irradiation and elevated salinity significantly increase the bioaccumulation of MAAs [136]. Indeed, the exposure of terrestrial fungi to UV radiation, desiccation and nutrient scarcity significantly increased the production of the UV-absorbing compound mycosporine-glutaminol-glucoside (14) (Figure 3) [137].

Skin-Whitening Agents
Skin-whitening agents are commercially available for cosmetic and clinical purposes, to obtain lighter skin complexion and treat hyperpigmentary disorders [138]. Uneven pigmentation of the skin may lead to blotches, patches of brown to grey discoloration or freckling which may require cosmetic interventions [13]. Whitening agents act at various levels of melanin production of the skin, either by inhibiting the activity of tyrosinase, the key enzyme in melanogenesis in plants and animals, or by inhibiting the transport of melanosomes from melanocytes to surrounding keratinocytes [139][140][141].

Pyrones
Kojic acid (15) is an inexpensive water-soluble fungal secondary metabolite (Figure 4). It has two OH-groups, the primary at C-7 and the secondary at C-5, which is essential to the radical scavenging and tyrosinase interference activity (IC 50 : 14 µM) [142,143]. The skin depigmenting activity of kojic acid results from the inhibition of the creolase and catecholase activities of tyrosinase. It prevents the conversion of the O-quinone to DL-DOPA and dopamine to its corresponding melanin. Decreased melanin content is demonstrated in melanocytes after their treatment with kojic acid [143]. This compound has been extensively used for skin depigmentation (and consequently as a cosmetic agent) with an excellent whitening effect, due to its ability to inhibit tyrosinase activity.
Mainly produced by Penicillium sp. and Acetobacter sp., kojic acid has also been isolated from other terrestrial microorganisms, such as Aspergillus flavus, an endophytic fungus of Vigna unguiculata [81]. To produce this compound, fermentation of Aspergillus sp., is widely used. Other strains are also commonly employed, such as A. oryzae (0.26 g kojic acid/g glucose), A. parasiticus (0.089 g/g glucose) and A. candidus (0.3 g/g sucrose). A high yield of 0.453 g/g glucose was obtained with the culture of A. flavus [82,83,144] (Table 1).
Mainly produced by Penicillium sp. and Acetobacter sp., kojic acid has also been isolated from other terrestrial microorganisms, such as Aspergillus flavus, an endophytic fungus of Vigna unguiculata [81]. To produce this compound, fermentation of Aspergillus sp., is widely used. Other strains are also commonly employed, such as A. oryzae (0.26 g kojic acid/g glucose), A. parasiticus (0.089 g/g glucose) and A. candidus (0.3 g/g sucrose). A high yield of 0.453 g/g glucose was obtained with the culture of A. flavus [82,83,144] (Table 1).

Phenolic Lactones
Ellagic acid (16) is an antioxidant polyphenol that has generated commercial interest due to recommendations for topical use as a skin-whitening agent (Figure 4). This compound inhibits melanogenesis via the chemical reduction of O-quinones (O-dopaquinone) and semiquinones [145].
Ellagic acid can be produced from plant tannins via fermentation using different A. niger strains [146,147]. A yield of 6.3 and 4.6 mg of ellagic acid/g of dried pomegranate husk were obtained by converting of pomegranate ellagitannins into ellagic acid in a solid state fermentation [85] (Table 1).

Carboxylic Acids
Azelaic acid (17) is a saturated dicarboxylic acid which is produced by Malassezia furfur (also known as Pityrosporum ovale), a yeast that lives on normal skin [91] (Figure 4) (Table 1). It is effective in treating several skin conditions, such as acne, inflammation and hyperpigmentation. As a competitive inhibitor of tyrosinase in vitro, it has been used to treat melasma, Lentigo maligna and

Phenolic Lactones
Ellagic acid (16) is an antioxidant polyphenol that has generated commercial interest due to recommendations for topical use as a skin-whitening agent (Figure 4). This compound inhibits melanogenesis via the chemical reduction of O-quinones (O-dopaquinone) and semiquinones [145].
Ellagic acid can be produced from plant tannins via fermentation using different A. niger strains [146,147]. A yield of 6.3 and 4.6 mg of ellagic acid/g of dried pomegranate husk were obtained by converting of pomegranate ellagitannins into ellagic acid in a solid state fermentation [85] (Table 1).

Carboxylic Acids
Azelaic acid (17) is a saturated dicarboxylic acid which is produced by Malassezia furfur (also known as Pityrosporum ovale), a yeast that lives on normal skin [91] (Figure 4) (Table 1). It is effective in treating several skin conditions, such as acne, inflammation and hyperpigmentation. As a competitive inhibitor of tyrosinase in vitro, it has been used to treat melasma, Lentigo maligna and post-inflammatory hyperpigmentation. The minimum concentration at which azelaic acid demonstrates its anti-enzymatic activity is 10 −3 mol/L and it is approximately equal to the 20% content of azelaic acid in a cream applied topically [148,149]. Furthermore, the efficacy of 20% azelaic acid cream is superior than a 2% hydroquinone (HQ) cream while severe side effects were not reported [90,150]. Clinical trials demonstrated that this cream was also effective against melasma when used in parallel with a broad-spectrum sunscreen. Thus, the ability of azelaic acid to reduce the amount of melanin in a specific region of skin tissue as well as the lack of side effects makes it widely used in cosmetic formulations.
Lactic acid is also used as skin whitener (Table 1). At a dose of 500 µg/mL it inhibits melanin formation in a dose-dependent manner without affecting cell growth [151]. Recent studies have shown that species of Rhizopus could offer a valuable alternative source for lactic acid production [152]. The filamentous fungus R. oryzae converts both glucose and xylose under aerobic conditions into l(+)-lactic acid with yields varying between 0.55 and 0.8 g/g [87].
Poly γ-glutamic acid (γ-PGA) is a natural polymer produced by different species of Bacillus (yields vary from 10 to 50 g/L depending on the species) [88] (Table 1). Studies related to the inhibitory effect against mushroom tyrosinase and tyrosinase in B16 melanoma cells reported a dose dependent activity. γ-PGAs, and especially the low molecular weight polymers, has attracted much attention owing to its great potential in cosmetics as skin-whitening agents [153].

Enzymes and Derived Products
The possibility of using melanolytic enzymes in skin lightening was examined by screening the potential melanolytic activity of wild fungal isolates. Among them, Sporotrichum pruinosum was the most promising from the very limited number of fungi that decolorize synthetic melanin [154]. As described in the US 20030077236 Patent, compositions containing melanin-degrading enzymes derived from Aspergillus fumigatus or S. cerevisiae were twice as effective as kojic acid in producing a whitening effect on the skin.
A large variety of compounds with potential skin protecting applications can be obtained through biotechnological processes by using enzymes isolated from terrestrial microorganisms. This is the case of retinol, the most active form of Vitamin A, a skin-whitening agent that has been synthesized by the esterification of palmitic acid using a modified lipase B from Candida antarctica (CALB) and a modified lipase from Pseudomonas fluorescens, in order to maximize its solubility in water and minimize skin irritation. Other Vitamin A modifications include the esterification with oleic, lactic, succinic or methylsuccinic, catalyzed by CALB or by Rhizomucor miehei lipase [155].
A better dermal absorption and a 10% higher skin-whitening activity, as compared to the well-known tyrosinase inhibitor arbutin, was demonstrated by its derivative arbutin undecylenic acid ester which has been enzymatically synthesized using an alkaline protease from Bacillus subtilis [94,155]. In addition, α-arbutin glycosides were synthesized by the trans glycosylation reaction of cyclomaltodextrin glucanotransferase from Bacillus macerans. Synthesized glucosides exhibited higher inhibition on human tyrosinase than α-arbutin [156].

Additives and Other Active Ingredients
Additive products provide long-term physical stability, inhibit germination and influence the sensory perception. Recently, the cosmetic industry has been strongly criticized for the addition of chemicals such as formaldehyde, dioxane, parabens, and phthalates. Controversies regarding the human health impact of those synthetic molecules and their analogues has encouraged the research of new additives from natural sources.

Antimicrobial Agents
One of the most widely used antimicrobial agents against bacteria, viruses and fungi contamination in cosmetics is chitosan (18) (Figure 5). This polysaccharide is composed mostly of glucosamine and a variable number of N-acetylglucosamine residues. Although chitosan is present in large amounts in the exoskeleton of crustaceans, insects, crabs, and shrimps, its production is limited due to factors such as seasonality, production sustainability and processing cost. To face these difficulties, chitosan can be produced by an alternative and more effective sources of microbial origin since 22-44% of the cell wall of fungi is composed of chitosan [2]. An optimal production was found in Rhizopus oryzae (0.5 g/L), R. japonicus (0.6 g/L) and Mucor indicus (0.75 g/L) ( Table 1) [63], while A. niger, isolated from the lichen Roccella montagnei, showed a higher yield of 1.3 g/L, which was further increased to 1.93 g/L when glucose was added [65]. In addition to the antimicrobial activity, chitosan is known for its emulsifying and delivering properties. This compound has a better water-binding capacity than methyl-cellulose, which is commonly used in cosmetics [2]. Consequently, chitosan and its derivatives, like the copolymer chitin-glucan, can present potential candidates for cosmetic and cosmeceutical formulations. Other examples with anti-aging activity that also combine antimicrobial activity are presented in Table 1.

19
antimicrobial activity, chitosan is known for its emulsifying and delivering properties. This compound has a better water-binding capacity than methyl-cellulose, which is commonly used in cosmetics [2]. Consequently, chitosan and its derivatives, like the copolymer chitin-glucan, can present potential candidates for cosmetic and cosmeceutical formulations. Other examples with anti-aging activity that also combine antimicrobial activity are presented in Table 1.

Moisturizers and Biosurfactants
Concerning moisturizing care, ectoine (19) is commonly used for its strong hydration properties ( Figure 5). This cyclic amino acid is produced by several bacterial species in response to osmotic stress. Corynebacterium glutamicum is widely studied as a microbial cell factory for the biotechnological production of ectoine. The optimization of some cultivation parameters led to the production of 6.7 g/L/day of ectoine [98] (Table 1).
Glycolipids represent an important class of biosurfactants. Among them, sophorolipids and trehalolipids are efficient biosurfactants. Sophorolipids are mainly produced by yeasts belonging to the genus Candida (formerly called Torulopsis), like C. bombicola, C. petrophilum and C. apicola, while trehalolipids by Rhodococcus sp., Mycobacterium sp., Nocardia sp., and Corynebacterium sp. Trehalolipids represent structures with a variation in the number of carbon atoms and the degree of unsaturation.
Rhamnolipids are commonly used in cosmetics as moisturizers and biosurfactants [108].
Rhamnolipids, primarily crystalline acids, are composed of a -hydroxy fatty acid attached by the carboxyl end to a rhamnose sugar molecule and are classified as mono and di-rhamnolipids [157]. Compared to chemical surfactants, biosurfactants have several advantages, because of their better compatibility, lower toxicity and higher biodegradability [158]. Rhamnolipids are mainly produced by Pseudomonas aeruginosa as well as by other Pseudomonas sp. They are also used in the

Moisturizers and Biosurfactants
Concerning moisturizing care, ectoine (19) is commonly used for its strong hydration properties ( Figure 5). This cyclic amino acid is produced by several bacterial species in response to osmotic stress. Corynebacterium glutamicum is widely studied as a microbial cell factory for the biotechnological production of ectoine. The optimization of some cultivation parameters led to the production of 6.7 g/L/day of ectoine [98] (Table 1).
Glycolipids represent an important class of biosurfactants. Among them, sophorolipids and trehalolipids are efficient biosurfactants. Sophorolipids are mainly produced by yeasts belonging to the genus Candida (formerly called Torulopsis), like C. bombicola, C. petrophilum and C. apicola, while trehalolipids by Rhodococcus sp., Mycobacterium sp., Nocardia sp., and Corynebacterium sp. Trehalolipids represent structures with a variation in the number of carbon atoms and the degree of unsaturation.
Rhamnolipids are commonly used in cosmetics as moisturizers and biosurfactants [108]. Rhamnolipids, primarily crystalline acids, are composed of a β-hydroxy fatty acid attached by the carboxyl end to a rhamnose sugar molecule and are classified as mono and di-rhamnolipids [157]. Compared to chemical surfactants, biosurfactants have several advantages, because of their better compatibility, lower toxicity and higher biodegradability [158]. Rhamnolipids are mainly produced by Pseudomonas aeruginosa as well as by other Pseudomonas sp. They are also used in the pharmaceutical industry for their antiviral and antimicrobial properties [159,160] and for others targets related to skin regeneration such as wound healing with reduced fibrosis, cure of burn shock and treatment of wrinkles [161].

Pigments
Microorganisms produce several compounds that can be used as natural pigments. A lot of synthetic dyes have been commercialized, but few of them are eligible in cosmetics. Natural pigments are more stable and less allergenic compared to synthetics [162]. Pigments commonly biosynthesized by fungi include aromatic polyketides such as quinones, anthraquinones, naphthoquinones, melanins, flavins and ankaflavins. Purpurogenone (20) and mitorubrin (21) are two characteristic examples, produced by the fungus Penicillium purpurogenum [95] (Figure 5) (Table 1). Recently, the potential use of terrestrial fungi as a source of natural pigments has been considerably investigated [163][164][165].
Cyanobacteria are an interesting source of pigments, that have the ability to produce phycobiliproteins, which are brilliantly colored fluorescent proteins. Among phycobiliproteins, phycocyanins are already used in diagnostic assays such as flow cytometry, fluorescence activated cell sorting, histochemistry, etc. Their intense blue color allows their use in cosmetics as natural dyes [166]. Phycocyanins are mainly produced by the photoautotrophic cyanobacterium Arthrospira platensis (3.2 g/L) [167]. However, the unicellular rhodophyte Galdieria sulphuraria showed excellent results; this red alga, growing usually in acidic springs, produced c-phycocyanin with a yield of 2.9 g/L [168] ( Table 1).

Flavoring and Fragrances
Many flavoring and fragrance compounds on the market are still produced through plant and animal sources. However, a rapid and sustainable alternative is given as such high value compounds can be also produced by microorganisms [169]. Numerous yeasts and terrestrial fungal and bacterial strains are able to synthesize potentially valuable fragrance compounds, including alcohols, aldehydes, esters, fatty acids, ketones, lactones, aromatic compounds and pyrazines [170]. In support, several articles and reviews have been published and offer sufficient information regarding the use of microbial cultures or enzyme preparations for the production of flavor compounds valuable for the cosmetic industry [171][172][173][174]. Vanillin (22) is a very good example of a natural fragrance where the increasing demand and value have led to the development of alternative strategies for its production [175] (Figure 5). Strains including Pseudomonas putida, Aspergillus niger, Corynebacterium glutamicum, Corynebacterium sp., Arthrobacter globiformis and Serratia marcescens were successfully introduced for its production by converting eugenol or isoeugenol to vanillin [170].
Benzaldehyde (23) is among the most commonly used flavoring agent, with a strong cherry and almond-like aroma. An E. coli strain was successfully engineered to produce this aromatic [100,176], while the fungus Ashbya gossypii has been tested for its ability to synthetize the rose flavour 2-phenylethanol (24) [104]. Among terpenes, limonene (25) is one of the most widely used terpene due to its unique citrus scent [169]. Optimization of the expression pathway in E. coli led to a yield of 435 mg/L with 1% of glucose as carbon source [177]. When the impact of a different carbon source have been explored, the fermentation using glycerol led to the titers of 2.7 g/L [106] (Figure 5) (Table 1).

Other Targets of Skin Protecting Interest
Elastase and collagenase inhibitors of microbial origin are promising cosmeceutical agents that worth to be further explored. Elastase, a member of the chymotrypsin family of serine proteases, is responsible primarily for the breakdown of elastin, which is an important protein found within the extracellular matrix of the skin, whose damage has a significant impact in skin ageing. Nostopeptins A and B isolated from the freshwater cyanobacterium Nostoc minutum are the only reported inhibitors of elastase (IC 50 : 1.3 and 11.0 µg/mL) [178]. On the other hand, collagen, the major constituent of the skin (80% of skin dry weight), is responsible for the tensile strength. The metalloproteinases named collagenases are capable of cleaving collagen and elastin. To the best of our knowledge, terrestrial microorganisms, apart from the aforementioned example of nostopeptins A and B, have not been investigated thoroughly yet for their ability to produce metabolites with elastase and collagenase inhibitory effects although that large screening programs on terrestrial microorganism and endophytes have been recently presented [179,180].

Targets for Future Developments
Beyond the above applications of microbial-derived natural products, it is worth mentioning some new cosmeceutical targets with great potential for future development.
It is well known that skin retains its young-looking appearance for many years due to numerous cell genome and proteome protective mechanisms; these are mostly driven by protein machines that execute both DNA and proteome damage responses. Proteome quality control is carried out through the curating activity of the proteostasis network (PN) and is critical for cellular functionality [7,10,11]. Key components of the PN are the two main degradation machineries, namely the autophagy-lysosome and the ubiquitin-proteasome pathways; several short-lived transcription factors are also considered to be part of the PN as they mobilize genomic cytoprotective responses [11]. These, among many others, include Nrf2, which responds to oxidative, electrophilic, and/or proteotoxic stress [11,181,182]. Deregulation of the PN functionality is associated with ageing and it is considered a major risk factor for a wide spectrum of age-related protein conformational diseases such as immunological and metabolic disorders, cardiovascular and neurodegenerative diseases and cancer [11,183]. On the other hand, several studies have shown that the activation of proteostatic modules by genetic, dietary and/or pharmacological interventions increases organismal health-and/or life-span and delays cellular senescence [7,182].
Concomitantly, natural compounds that activate the PN have also been reported to possess anti-aging properties at either cell-based or in vivo models [7,11,182]; likewise, natural products significantly delay the appearance of the aged skin hallmarks. To the best of our knowledge, only few molecules of microbial origin were reported to activate proteostatic modules. Betulinic acid was recently isolated from the endophytic fungi Phomopsis sp. and its preferentially activating the chymotrypsin-like proteasomal activity with no or minimal effects on trypsin-like and caspase-like activities [184,185]. The second case of a microbial natural product, that is well known for its anti-aging proprieties is rapamycin. This molecule isolated from Streptomyces hygroscopicus, delay cellular senescence through (among others) the inhibition of the TOR pathway and the downstream induced alterations to both autophagy and the rate of protein synthesis [7].

Conclusions
Naturally derived molecules are traditionally used in skin protection products (Table 1, CosIng inventory). Consequently, natural compounds isolated and/or produced using biotechnological tools from microorganisms are already used for dermatologic purposes in topical cosmetic formulations. These products can aesthetically improve the skin's appearance but can also prevent and/or treat age-related skin disorders. Beyond the "established" molecules, there are several small molecules and/or enzymes derived from microorganisms that have great potential to be used in cosmetics or cosmeceutical formulations (Table 1).
Interestingly, several biomolecules that are already included in the European Inventory of accepted cosmetic ingredients (CosIng inventory) [14] are registered for one of their biological activities, but are used differently in cosmetic applications. A characteristic example is kojic acid, which is registered as "antioxidant", while the main application in cosmetics is its strong anti-tyrosinase activity, and thus its application as a skin whitening agent (Table 1).
Considering the immense microbial biodiversity and microbial adaptation to virtually any environment on earth, it is to be expected that microbes represent an extraordinary inventory of highly diverse structural scaffolds of biomolecules with potential skin protective activities. Although research on marine environment has started match later that the terrestrial environment, we have several cases where cosmetic applications and patents are in favor of marine-derived microorganisms. As mentioned in the case of MAAs known for their photo-protective activity, they are included in several patents for natural UV filters. However most of them were developed with microorganisms from marine environments (72.2%), while patents developed on terrestrial and fresh water microorganisms have not exceeded 21.4% and 2.4%, respectively [135]. This study reflects that to date, in some cases the terrestrial environment has been neglected.
Overall, taking into consideration that most of the world's microbial terrestrial biodiversity remains largely uninvestigated and that microorganisms offer a sustainable, relatively low-cost and fast production process, we remain confident that in the near future, systematic research will reveal additional microorganisms that can be used as cell factories for producing high added value biomolecules with applications in the cosmetic industry as active ingredients.