Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments

Pyomelanin is a polymer of homogentisic acid synthesized by microorganisms. This work aimed to develop a production process and evaluate the quality of the pigment. Three procedures have been elaborated and optimized, (1) an HGA-Mn2+ chemical autoxidation (PyoCHEM yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (PyoBACT, 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (PyoENZ, 1.25 g/g substrate) and all the criteria for a large-scale prototype. The chemical structures had been investigated by 13C solid-state NMR (CP-MAS) and FTIR. Car–Car bindings predominated in the three polymers, Car–O–Car (ether) linkages being absent, proposing mainly C3-C6 (α-bindings) and C4-C6 (β-bindings) configurations. This work highlighted a biological decarboxylation by the laccase or bacterial oxidase(s), leading to the partly formation of gentisyl alcohol and gentisaldehyde that are integral parts of the polymer. By comparison, PyoENZ exhibited an Mw of 5,400 Da, was hyperthermostable, non-cytotoxic even after irradiation, scavenged ROS induced by keratinocytes, and had a highly DPPH-antioxidant and Fe3+-reducing activity. As a representative pigment of living cells and an available standard, PyoENZ might also be useful for applications in extreme conditions and skin protection.


Production of pyomelanin (Pyo BACT ) by an induced wild Halomonas strain. A bacterial strain able
to compete with the enzymatic process (Pyo ENZ ) in terms of production yield, was sought. The strategy consisted to select a Halomonas species among a large collection, like our previous studies [32][33][34] . Phenolic compounds in the medium were identified and controlled along with the growth of the induced cultures, the strains preferentially utilized the aromatic over glucose. These halophile bacteria easily grow and have been shown to produce dihydroxy phenols from 4-hydroxyphenylacetic acid (4-HPA), such as HGA in H. olivaria 32,34 , H. venusta, H alkaliphila, and 3,4-dihydroxyphenylacetic acid (3, in H. alkaliantartica, H. neptunia, H. sulfaedris (this work), and H. sp. HTB24 35 , through routes 2b and 3, respectively (Fig. 1S). H. titanicae is a γ-proteobacterium isolated on the Titanic wreck, its genome now entirely available 36 , and has been selected for the most intense brown-black color from 5 mM 4-HPA supplemented cultures measured by the A 400 nm (this work). The suspected presence of pyomelanin was first confirmed by identification of HGA (λ max 290 nm) only in the exponential phase, but not 3,4-DHPA or other dihydroxyphenylacetic derived compounds, by RP-HPLC-DAD and GC-MS www.nature.com/scientificreports/ of the TMS-derived metabolites, showing a matched fragmentation spectrum with that of the HGA standard. The strain could not grow in the presence of L-Tyr and was unable to metabolize 2-HPA or 3-HPA, hence suggesting that a 4-HPA-5-hydroxylase or a 4-HPA-6-hydroxylase were not implied, respectively. We concluded that H. titanicae was able to produce pyomelanin by direct conversion of 4-HPA to HGA through a 4-HPA-1-hydroxylase (4-HPAH-1, route 2b, Fig. 1S). Following this, pyomelanin production has been optimized. Because 5 mM 4-HPA was rapidly consumed in 2 days and served as an inducer of 4-HPAH enzymes 34,35 , successive additions of well-defined amounts of 4-HPA at different culture times were carried out by following an experimental design procedure (see Methods). Finally, pyomelanin was overproduced in a 500 mL medium by adding 5 mM 4-HPA at starting, then 10 mM after 3 days, in a total culture time of 6 days. In these conditions, H. titanicae was able to furnish 0.55 ± 0.09 g Pyo BACT per Liter of culture, a mean of three independent experiments. Relative to the total amount of 4-HPA added, the recalculated yield was 0.241 ± 0.04 g Pyo BACT per g of 4-HPA.
Production by chemical autoxidation (Pyo CHEM ). While pyomelanin issued from the HGA autoxidation has been commonly used 2,28 , the reaction has never been optimized to date. In presence of the transition metal Mn 2+ to enhance the catalysis 26 , an (HGA)/(Mn 2+ ) ratio of 20 for an optimal pyomelanin yield was obtained (see Methods). By applying this ratio, the developed on-line process (Fig. 1, steps 1-2′-3′-4) provided 0.317 ± 0.031 g Pyo CHEM per g of 2,5-DMPA (mean of 5 experiments), a yield four times lower than that of the enzymatic (Pyo ENZ ) and higher than the bacterial (Pyo BACT ) process (summarized in Table 2). Despite this, the production of Pyo CHEM remains interesting because the cheapest and easy to implement for use on a laboratory scale.

Figure 1.
Schematic diagram of the Pyo ENZ process (in blue) that uses a laccase and showing the associated decarboxylation mechanism and the two resulting products identified in the polymer. Comparison to the Pyo CHEM synthesis (abiotic autoxidation, in red). The common precursor 2,5-DMPA could also be synthesized from 2,5-dimethoxyacetophenone by a Willgerodt-Kindler reaction type 25 .
Step 4 is the final HCl precipitation followed by washing and drying. Doubling the amount of HBr (step 1) led to incomplete demethylation and the extra formation of 2,5-dihydroxyphenylacetaldehyde (~ 6%) identified from the HPLC-DAD spectrum (λ max 292 nm) and the EI-MS profile (molecular ion [M + 2TMS] at m/z 294, characteristic fragments [M -CHO] at m/z 265, and [M -CH 2 CHO] at m/z 251), similarly to the NIST data bank and previous data 34 . In step 2, the addition of sulfite (Na 2 SO 3 ) 25 was unnecessary because the solution was immediately buffered to 6.8 and the polymerization by the laccase followed (step 3). The alkaline opening of the lactone was essential, indeed the rMt laccase was unable to open the lactone nor demethylate 2,5-DMPA at pH 6.8, even after several days of agitation. BQA, 1,4-benzoquinone acetic acid; gentisaldehyde, 2,5-dihydroxybenzaldehyde; gentisyl alcohol, 2,5-dihydroxybenzyl alcohol; 2,5-DMPA, 2,5-dimethoxyphenylacetic acid; 2,5-DMAPO, 2,5-dimethoxyacetophenone. Solid-state 13 C CP-MAS NMR. Spectra were cumulated in Fig. 2, and chemical shifts summarized in Table 1 along with those of pure HGA analyzed in the same conditions. The three spectra exhibited common, typical, and prominent signals in slightly varied positions, at δ 172-173.4 ppm that corresponded to the unprotonated carbon in C-O/C = O of the carboxylic group, then at 149.3-149.4 of the unprotonated carbon (suggested C 5 ) of the ring bearing the -OH group, with shoulders at 143.4 for the three pyomelanin (suggested C 2 ), and at 118-119 ppm provided by the ethylenic and protonated carbons of the ring (-CH = C-) together in broadband. Less high signals at 33.0-34.8 ppm observed on the three structures represented the saturated aliphatic carbons (-CH 2 -) of the acetic acid moiety. From the three 13 C solid-state NMR spectra, the main differences are in the region around δ 45-78 ppm, precisely at 52.5 and 67.9 (larger) ppm in Pyo ENZ , and 52.5 ppm alone in Pyo BACT , whereas these two shifts are absent in Pyo CHEM (Fig. 2). They suggested secondary reactions during the biological BQA polymerization that did not occur during autoxidation of HGA in abiotic and alkaline conditions. By comparison to standard molecules analyzed in parallel, the δ 52.5 ppm shift corresponds to the bi-protonated carbon of the ethanolic moiety (-CH 2 -O-) from 2,5-dihydroxybenzyl alcohol (gentisyl alcohol). Besides gentisyl alcohol, we also noted minor peaks at δ 190.5 and 191 ppm in Pyo BACT and Pyo ENZ respectively, absent in Pyo CHEM , and ascribed to an aldehyde group of the end-product 2,5-dihydroxybenzaldehyde (gentisaldehyde) ( Table 1). Gentisyl alcohol and gentisaldehyde resulted from a decarboxylation reaction extensively detailed in Fig. 1. With a lot of precautions because solid-state NMR was a semi-quantitative tool, the relative level of the decarboxylation products in the polymers was deduced from areas of the corresponding peaks (Fig. 2), and approximately evaluated at 11-13% (gentisyl alcohol 9-10% + gentisaldehyde 2-3%), these two compounds could not be identified by FTIR. Besides, low signals visualized at δ 17.2-23.1 (Pyo ENZ ) and 17.4-23.9 ppm (Pyo BACT ) were attributed to lipid residues provided by the enzyme extract and the culture medium, respectively. The broad signal at δ 67.9 ppm present in Pyo ENZ only had been assigned to the hydroxylated 13 C of a saccharide moiety (> C-OH) also brought by the laccase extract, whereas it was absent in Pyo BACT probably because the H. titanicae medium was not supplemented with glucose. Importantly, the area ratio of the 170/118 ppm resonances for each pyomelanin The peaks area allowed us to estimate gentisyl alcohol at ~ 9 to 10% and gentisaldehyde at ~ 2 to 3% in Pyo ENZ and Pyo BACT . The loss of carboxylic moieties observed on the Pyo CHEM structure (ratio 1/10, black line) was explained by a partial degradation due to the alkaline conditions during the polymerization. Spectra were recorded on an Avance III spectrometer (BRUKER BIOSPIN GmbH, Germany) operated at the Larmor frequencies of 400.43 MHz and 100.70 MHz on 1 H and 13 C nuclei, respectively, by using a 4 mm crosspolarization magic angle spinning (CP-MAS) probe head. The zirconia rotors were filled with 80 mg of fine powder polymer or standards. 2048 scans were used to acquire the 13 C spectra, the acquisition time was 28.7 ms. The 1 H 90° pulse length and the power level were 3.8 s and 80 kHz, respectively. The spectral width was 35.7 kHz and 2048 points were acquired to describe the free induction decay. A two-pulse phase modulation (TPPM) proton decoupling (75 kHz) was used during the 13 C acquisition. Spectra were externally referenced to the carbonyl peak of glycine at 176.03 ppm downfield of TMS. No line broadening was applied. All the spectra were acquired using the same receiver gain. sh, shoulder. www.nature.com/scientificreports/ ( Fig. 2, data framed) indicates a correct -CH 2 -COOH substitution for Pyo ENZ and Pyo BACT with a value of 1/6, whereas a loss of the carboxylic moiety on Pyo CHEM structure (ratio 1/10) has occurred. More information on the polymer assembly was necessary to elucidate the mechanism of polymerization and the types of linkage between the rings, i.e. C ar -C ar (aryl carbon) or/and C ar -O-C ar (aryl ether) linkages.

FTIR analyses.
Pyo BACT and Pyo ENZ exhibited very similar FTIR and 13 C-NMR spectra, hence the study was focused on the Pyo ENZ and Pyo CHEM absorptions (Fig. 5S) noting that the spectrum of Pyo CHEM was better resolved in reason to its less high M w (2,300 Da, Table 2). The peaks at the following wavenumbers and their corresponding structures included the bands for Pyo ENZ and Pyo CHEM , respectively at (i) 3401 and 3278 cm −1 (broad) indicative of the -OH stretch of polymeric structures; (ii) two smaller bands for each compound at 2960 (Pyo ENZ ) and much more intense at 2925-2927 cm −1 (Pyo CHEM ), which corresponded to stretching vibrations of the aliphatic C ar -H groups; (iii) 1711 and 1720 cm −1 quite resolved here and ascribed to carbonyl stretching (C=O) of the -COOH group, these bands were however absent on other microbial pyomelanin 29 Presence of N in Pyo ENZ and Pyo BACT . Especially, a reaction of substitution on the C 4 position of the BQA ring by primary and secondary amines had been reported 38 , such substitutions might occur in biological systems. Here, the volume of the laccase extract added for the Pyo ENZ synthesis seemed insignificant, hence it remained difficult to look for amide or amine bonds from polymers, especially when they are minor. These C-N absorptions were generally encountered at δ 155-180 ppm (amides formed from the carboxylic moiety) and 135-145 ppm (aromatic amines) in 13 C NMR, mainly at 3000-3500 cm -1 (N-H stretching vibrations of aromatic www.nature.com/scientificreports/ amines) in FTIR, thus drowned in those of the major functional groups. Faced with this inability to detect traces of nitrogenous derivatives by NMR and FTIR, elemental analyses of the three pyomelanin were carried out and showed the presence of N in Pyo ENZ (2.75%) and Pyo BACT (3.65%), as expected none in Pyo CHEM , and higher in the indole-based melanin Mel SYNTH (6.34%) and Mel SEPIA (6.31%) ( Table 2). The presence of N in Pyo ENZ and Pyo BACT is due to amino acids and amines linked on C 4 of the HGA rings and provided by the rich laccase extract and the components of the H. titanicae culture medium, respectively.
Linkage determination. Interestingly, the FTIR spectra showed absorption at 1534 cm -1 strongly present in Pyo CHEM (Fig. 5S, red) and absent in Pyo ENZ (blue) and Pyo BACT . This resonance did not correspond to amides and was rather ascribed to aromatic C ar -H. From this remarkable difference, it has been established that Pyo ENZ Table 2. Summarized chemical and biological properties of pyomelanin and commercial melanin. Since the A 400 nm value of solubilized pyomelanin depends on the size of the pigment, a spectrophotometric quantification by surrogate melanin for calibration will not be correct. Weighing precisely the final purified pigment remains the only valuable technique for the quantification of pyomelanin as well as other melanin. a Identified by 13 C solid-state NMR ( Fig. 2 and Table 1). b To confirm the global structure of Mel SYNTH and Mel SEPIA , assays on their degradation by alkaline-H 2 O 2 were conducted similarly to those on human melanin 41 . After centrifugation, 10 μL of the reaction volume was injected into an RP-HPLC(DAD)-QToF system in negative mode and confirmed the L-Dopa melanin structure by the presence of the two markers PTCA and PDCA. c Elemental analysis (C, H, N, S) of the pigments was performed by combustion on a Thermo Finnigan EA 1112 analyzer equipped with an autosampler, all managed by the Eager Xperience software (THERMO SCIENTIFIC, France). The oven was set at 970 °C and the flash combustion at 1800 °C. In the formula C x H y N z , each index was deduced by (x, y, z) = %atom (data from elemental analysis) x M w /M atom . d The index w (for oxygen O w ) was deduced from 100% -C -H -N. e Standard deviations were < 5%. Composition (C, H, N) of Mel SYNTH was similar to those described 63 , that of Mel SEPIA close to the reported values 42 (see sample Com). f Molecular weight of the S. officinalis melanin could not be determined on the MCX column eluted in the same conditions (see Fig. 6S). GA, gentisyl alcohol; GALD, gentisaldehyde; ND, not determined. www.nature.com/scientificreports/ contains much less C ar -H free, which means much more C ar -C ar linkages than Pyo CHEM . As an important finding from the three pyomelanin 13 C NMR spectra, C ar -O-C ar (aryl ether) linkages were absent (Fig. 2), the related signal generally resonates at around δ 160-167 ppm 39,40 . Hence, the three HGA polymers were assembled by C ar -C ar linkages only.
Alkaline-H 2 O 2 oxidation assays. This treatment has also been tried on the three pyomelanin and the commercial melanin 41 (Table 2). While hydrolyzed Mel SYNTH and Mel SEPIA melanin led to the two expected degradation products similarly to the literature 41,42 , pyrrole-2,3-dicarboxylic acid (PDCA, an indicator of DHIderived units) and pyrrole-2,5,5-tricarboxylic acid (PTCA, of DHICA-derived units), any compound has been detected from the three hydrolyzed pyomelanin by LC(DAD)-MS analyses (see Table 2). Thus, pyomelanin could not be hydrolyzed by such peroxide treatment, even after doubling or lowering the peroxide concentration. A pyrolysis-GC-MS coupling method had been developed to analyze pyomelanin from Penicillium chrysogenum 43 but reported too much heterogeneity to obtain uniform results between samples. This method utilizes heat to break the polymer into smaller fragments, such as 4-methoxybenzene acetic acid, 4-methoxybenzene propanoic acid, and other minor phenolic compounds, but not HGA. Unfortunately, this technique failed in Pyo ENZ , Pyo BACT , and Pyo CHEM with any identifiable compound.
Physicochemical properties (summarized in Table 2). All pigments (3 pyomelanin, 2 commercial L-Dopa melanin Mel SYNTH and Mel SEPIA ) are insoluble in neutral or acidic water as well as many usual organic solvents, entirely soluble in alkaline media such as NaOH (0.05 N minimal conc.  6S, Table 2), explaining why Pyo CHEM and Mel SYNTH were more rapidly solubilized in DMSO than the others. These M w data were very close to those resulting from the elemental analyses ( Table 2), indicating that these pigments were sufficiently purified by successive water and ethanol-washings.
Antiradical properties. The scavenging ROS activity was studied for Pyo ENZ , comparatively to the standards Mel SEPIA and Mel SYNTH . UVA induces damage by directly transferring energy or indirectly through ROS generated as primary and secondary radiolytic products 44 . Therefore, the protection by melanin pigment against UVA may be due to their ability to scavenging ROS in the cells. To prove this, a fluorescein-derived compound (DCFH-DA) was used to detect the generation and change of ROS in UVA-visible irradiated keratinocyte cells. Indeed, keratinocytes are a source of ROS that may affect neighboring skin cells, such as melanocytes, and influence the process of melanogenesis or contribute to the progression of vitiliginous lesions. Fluorescence measurements showed that Pyo ENZ effectively scavenged ROS generated by UVA-visible light in the test system with an IC 50 of 82.2 ± 5.6 µg/mL, while IC 50 of Mel SYNTH (284.1 ± 12.3 µg/mL) was higher and that of Mel SEPIA very far (Table 3). Thus, the amount of ROS in the cells decreased as the concentration of Pyo ENZ increased, much more efficiently than the concurrent pigment Mel SYNTH . The DPPH-antioxidant activity was rarely reported due to the insolubility of the pyomelanin in organic solvents, and because the stable DPPH reagent reacts at slightly alkaline pH values. The assays were carried out on the three HGA-pigments, along with the two standards (Mel SEPIA , Mel SYNTH ) and common antiradical agents such as Trolox, ascorbic acid, and propyl gallate, all prepared in DMSO. Figure 7S-A and 7S-B indicated that Pyo ENZ (EC 50 27.5 µg/mL) and Mel SYNTH (EC 50 25.9 µg/mL) have an antioxidant activity equivalent to that of ascorbic acid (29 µg/mL), as already reported for pyomelanin isolated from Pseudomonas stutzeri strain BTCZ10 and Pseudoalteromonas lipolytica BTCZ28 45,46 . The degradation of pyomelanin by enzymes or microorganisms has never been described to date, whereas ascorbic acid is rapidly metabolized and was thought to act as a prooxidant when the glutathione pool is depleted 47 , a feature that must also be controlled in the case of pyomelanin. Barely better than Pyo ENZ , Pyo CHEM EC 50 was 20.0 µg/mL, while EC 50 Pyo BACT was found much higher at 130.0 µg/ mL (Fig. 7S-A). Whatever, Pyo ENZ , Pyo CHEM , and Pyo BACT exhibited much higher DPPH-antioxidant activities than pyomelanin isolated from the Yarrowia lipolytica strain W29 (EC 50 230 μg/mL 48 ), and far from eumelanin from Sepia officinalis (Mel SEPIA , > 300 µg/mL, this work) and the synthetic butylated hydroxytoluene (BHT, EC 50 722 μg/mL 49 ). Although the trihydroxylated benzoic ester, propyl gallate (EC 50 4.2 µg/mL) (Fig. 7S-B), was one of the leading dietary antioxidants, it induced DNA damages 50 ; BHT was also found cytotoxic 51 , hence their use notably in the food industry became restricted.
Electron-transfer efficacy. By an adapted ferrozine assay, Pyo ENZ , Pyo CHEM , and Mel SYNTH exhibited equivalent and highest Fe 3+ -reducing activity among the five polymers tested (Fig. 3). From these data, the equivalent Fe 3+ -reducing activity of Pyo ENZ and Pyo CHEM could not be explained, while Pyo ENZ and Pyo CHEM have a different M w (Table 2), thus none the same number of -OH and carboxylic groups, and even if gentisyl alcohol and gentisaldehyde (at ~ 11 to 13%) are present in Pyo ENZ structure only. Comparatively to Mel SYNTH (100%), the reducing activity in decreasing order was Pyo ENZ (96), Pyo CHEM (95), and to a less extent Pyo BACT (54) and Mel SEPIA (34). Because Pyo ENZ has the best production yield and is dedicated to potent applications, its Fe 3+ -reducing activity was evaluated at 1.73 µM per hour related to 50 µg of pigment, i.e. 5 www.nature.com/scientificreports/ Cytotoxicity. For applications with pyomelanin as an ingredient for cosmetics or pharmaceutical preparations, cytotoxicity toward human keratinocytes has been evaluated by the vital dye NR penetration technique, from pigment prepared in alkaline solutions at dilutions which in no way modified the pH of the assay. Keratinocytes are the most abundant cells of the epithelial layer of the skin and are used as a part of the 3D skin model for the assessment of the toxic hazard of cosmetic ingredients. No reduction of the metabolic activity of the cells was observed as compared to the non-treated cells, thus formally postulating the absence of toxic effect on skin cell metabolic activity for Pyo ENZ , Pyo BACT , Pyo CHEM , Mel SYNTH , and Mel SEPIA , until 500 µg/mL (Table 2). Furthermore, using the normalized OECD protocol commonly used for cosmetology product evaluation, the three pyomelanin and the two standard melanins were found non-phototoxic (PIF < 2) ( Table 2).

Discussion
The laccase process is the most efficient provider of pyomelanin. Comparison of three realistic strategies by optimized production of HGA autoxidation (Pyo CHEM ), induced bacterial culture (Pyo BACT ), and for the first time using a recombinant laccase (Pyo ENZ ) was undertaken. Pyo ENZ has been obtained at the highest level, 1.25 g per g 2,5-DMPA, a yield > 1 g/g due to compounds linked and brought by the concentrated enzyme extract. This procedure meets all the criteria to design a large-scale prototype, high-efficient, cheapest, with mild conditions, and without sterility constraints that are essential in the case of microbial cultures. HGA-lactone was easily prepared from 2,5-DMPA, or even from 2,5-dimethoxyacetophenone to reduce the costs by an additional reaction of Willgerodt-Kindler 25 . Despite the great number of extensive works on pyomelanin-producing microorganisms, to date there have been only three reported quantifications of the pigment, first with the wild yeast Yarrowia lipolytica that furnished 0.035 g/L of culture 52 , second 0.173 g/L culture of the Shewanella algae BrY strain supplemented by 2 g of L-Tyr/L 14 , and third 0.35 g/L by random mutagenesis of Pseudomonas putida 19 . In this work, an induced culture of H. titanicae was shown to convert 4-HPA to HGA by a 4-HPA-1-hydroxylase (4-HPAH-1) at the best microbial yield to date, 0.55 g/L culture, a feature confirmed by the presence in its genome of the related hpaH/C genes (unpublished). Such bioconversion generally occurred with less energy consumption, and for these reasons more efficiently. It seems reasonable to assume that the bacterial (Pyo BACT ) and the chemical (Pyo CHEM ) processes will never be able to compete with the laccase process (Pyo ENZ ) in terms of production, except maybe by developing a recombinant overproducing microorganism. From these results,   www.nature.com/scientificreports/ the ability of H. titanicae to synthesize pyomelanin from 4-HPA and the property of the pigment to reduce Fe 3+ , raise the question of the survival of the bacterium at 4,000 m depth by maintaining a Fe 3+ /Fe 2+ ratio. A few remarks are worth noting about the oxidation of HGA. Besides the biological implications of metalcatalyzed oxidations, true autoxidation of biomolecules does not occur in biological systems, instead, this autoxidation is the result of transition metals bound to these biomolecules 53 . By analyzing the Pyo CHEM structure, surprisingly the 13 C solid-state NMR spectrum revealed an unexplained loss (~ 40%) of carboxylic moiety during the alkaline Mn 2+ -autoxidation of HGA without observable by-products of this degradation and hence contributes to the low pyomelanin yield. To date, the in vitro polymerization of HGA by a laccase has never been studied before. Here, the rMt laccase had been found to efficiently catalyze the HGA polymerization in terms of yield, and still confirmed the involvement of these oxidases in biological environments. It should be noted that pyomelanin-forming bacteria generally grow at pH 6-7, while the autoxidation is optimal at pH 8-9, one more element in favor of the laccase(s) action in living cells. The rMt enzyme supplied as a rich and concentrated extract is largely available and one of the cheapest in the market. Partial purification by ultrafiltration of the rMt extract would be an additional stage unnecessary. Indeed Aljawish et al. 54 showed that, if the brown color decreases after UF (⁓90%), it eliminates only 2.5-fold of total proteins and the specific activity of the UF-enzyme increased by only 2.1-fold. Other laccases had also been assayed in parallel. At their optimal parameters, we evaluated that the Trametes versicolor enzyme furnished ~ twofold less pyomelanin than rMt, while the purified recombinant Pycnoporus cinnabarinus laccase gave a quite similar yield, i.e. 1.1-1.2 g pyomelanin per g of substrate, at pH 5 in an acetate buffer.
The molecular weight of pyomelanin rarely reported was first evaluated by GPC/SEC at 3,000 Da for the pigment of the bacterium Alcaligenes eutrophus, and at 1,700 Da for autoxidized HGA, however, using unconventional PEG/PEO standards 29 . Turick et al. 14 estimated the size of Shewanella algae BrY pyomelanin ranging from 12 to 14 kDa, however by high-speed sedimentation and with proteins for calibration. In this work and as a suitable method in an alkaline eluent, the optimized processes led to close M w of 5,400 and 5,700 Da for Pyo ENZ and Pyo BACT , respectively (Table 2), a size much higher than those of laccase-synthesized polymers of catechol (M w 1,268 Da), resorcinol (1,489 Da), and hydroquinone (1,157 Da) 27 .
Biological pyomelanin is a C ar -C ar assembly polymer that contains two decarboxylation-issued products. Because alkaline-H 2 O 2 hydrolyses and pyrolysis experiments failed, the chemical structure of the three pyomelanin was determined by 13 C solid-state NMR, and partly confirmed by FTIR analyses. Like the hydroquinone polymerization 27 , C ar -C ar bindings between the rings predominated in Pyo ENZ , Pyo BACT, and Pyo CHEM , a finding deduced from the absence of C ar -O-C ar (ether linkages) resonance in the NMR spectra of these polymers. The reactions that govern the polymerization of HGA by the rMt laccase were proposed in Fig. 4A and showed two main suggested assembly modes, C 4 -C 6 (α-bindings) and C 3 -C 6 (β-bindings), giving preference to the C 3 -C 6 mode because of less subject to steric effects. Based on the NMR data, it was not possible to differentiate between the eight possibilities (Fig. 4A). The mechanisms of polymerization through radical reactions have also been proposed in Fig. 4B, considering the high reactivity of the primary phenoxy radicals in favor of aryl . In these structures, gentisyl alcohol (major) and gentisaldehyde (minor) issued from the decarboxylation mechanism (laccase process, bacteria) are supposed to be incorporated into the polymer in the same manner as HGA radicals at locations of the chain that could not be determined at this time. www.nature.com/scientificreports/ radicals and still showing C 3 -C 6 and C 4 -C 6 linkages as the most probable structures for pyomelanin. In any case, analytical techniques are still not able to deliver the exact structure and the location of the minor HGA derivatives in the polymer, this is the most problematic for all melanin and especially pyomelanin. Nonetheless, there is still to understand the mechanism of polymerization of HGA, particularly the relationships with the laccase structure. In this work, we notably reported a biological decarboxylation from BQA and caused by the action of the rMt laccase or suggested bacterial oxidase(s) (Fig. 1). Such a mechanism led to the formation of gentisyl alcohol and gentisaldehyde representing about 11-13% of the total components, and which polymerized together with BQA. Until today, HGA decarboxylation was attributed to an abiotic reaction from BQA at acidic pH (near [4][5], forming gentisaldehyde as the major product along with minor gentisyl alcohol 34 . In this work, Pyo BACT and Pyo ENZ contained both products, but in reverse order of level, gentisyl alcohol (major) and gentisaldehyde (weak compound). No decarboxylation was observed during the abiotic and alkaline synthesis of Pyo CHEM .
The pyomelanin Pyo ENZ for multiple applications. In addition to a DPPH-antioxidant activity equivalent to ascorbic acid, a high thermostability over time, a non-degradability in cells, Pyo ENZ efficiently scavenges ROS from irradiated human keratinocytes much better than the concurrent Mel SYNTH (Table 3). Comparatively and with a similar technique, 400 µg/mL of L-Dopa melanin isolated from Pseudomonas maltophilia has been reported to almost scavenge ROS totally from UVA-induced fibroblast cells 55 . Human eumelanin and pheomelanin photogenerate ROS meanwhile they photoconsume O 2 and are protective against skin cancer 56 . Nevertheless, they photochemically generate melanin degradation products that are responsible for sunlight-induced melanoma formation by inducing cyclobutane-pyridine dimers (CPDs) from DNA 57 . In contrast, strong irradiation of Pyo ENZ in solution did not generate degradation products, UV-visible spectroscopy, GPC/SEC, and RP-HPLC being reliable techniques to determine that Pyo ENZ is also a photostable polymer. At first glance, the two decarboxylation products in Pyo ENZ and Pyo BACT structure did not seem to influence the Fe 3+ -reducing activity (Fig. 3). In any case, ferric-reducing activity may be considered as a marker of the redox cycling nature of pyomelanin, a property that might be used to conduct electricity like an electronic-ionic hybrid conductor. It was advanced that only a few femtograms per cell were assumed to be enough amount for electron-transfer in bacterial systems 3,14,16 . Consequently, Pyo ENZ could be exploited as a hyperthermostable and Fe 3+ -reducing agent, and for bioelectronic applications better than melanin 58 . As an evident cosmetic ingredient and consistently with recent reports on microbial pyomelanin from Yarrowia lipolytica 48 and Pseudoalteromonas lipolytica BTCZ28 46 , all tested at a 100 µg/mL, Pyo ENZ was found non-cytotoxic and non-phototoxic on keratinocyte cells, until 500 µg/mL.

Conclusions
Pyomelanin issued from the three processes has different properties, giving a large priority to Pyo ENZ that can now be produced in interesting yield and at low cost. The pigment efficiently scavenges ROS, exhibits high DPPH-antioxidant activity, is non-degradable, photostable, non-toxic, and can be stocked indefinitely without any precaution. As a representative pigment of microbial pyomelanin, Pyo ENZ becomes an available standard for laboratories, might be used for applications that require extreme conditions, as an electron-transfer agent, why not for energy storage, and exploited for skin protection, assuming it cannot penetrate the blood skin vessels. Process for the production of pyomelanin (Pyo ENZ ) by the rMt laccase. The first part of the procedure consisted of an adapted HGA synthesis 25 . The second part is the polymerization step by the rMt laccase. The starting compound 2,5-dimethoxyphenylacetic acid (2,5-DMPA) 5 g was solubilized in 40 mL of 48% HBr and refluxed gently for 4.5 h in a 100 mL-bicol flask provided with a refrigerant maintained at 10 °C. The resulting deeply red solution was evaporated to dryness in vacuo, the residue (3.80 g, 99.7% yield, 99.8% purity) identified as HGA-lactone following its UV spectrum (λ max 232, 289 nm, bands slightly lower than that of HGA), elution in RP-HPLC (retention time 4.2 min), and GC-MS analyses of the TMS-derived compound (rt 16.3 min), similarly to the standard. In the second step of the procedure and typically, 1.0 g of HGA-lactone was dissolved by agitation in 130 mL hot milliQ-H 2 O (70 °C), stayed 3-5 min and few drops of NaOH 2 N added until pH 9.3 (pHmeter) to hydrolyze the lactone into HGA (in HPLC-DAD, rt 2.7 min, λ max 290 nm), complete ring-opening was ensured by analysis of a 5 µL sampling diluted 10 × in MeOH. Immediately after, 35 mL of Na-phosphate buffer 0.3 M pH 6.8 were added, the concentration of HGA and buffer at this stage was 40 mM and 65 mM, respectively. Once the temperature of the solution has reached 30-40 °C, 3-4 mL of concentrated laccase rMt were added (2250-3000 U in total), the enzyme activity was 750 U/mL (SD < 5%) as determined by the syringaldazine assay (see Fig. 2S, Additional information). Then the mixture was agitated at 130 rpm in dark at 30 °C for 48 h. The formed brown-black pigment was further precipitated by adding 34 mL of HCl 37% (2 N final concentration), agitated for 2 min, and stayed for 24 h, at ambient temperature in dark. The precipitated pyomelanin was centrifuged, washed with milliQ-H 2 O and ethanol, dried, and weighed as previously for Pyo BACT and Pyo CHEM (Fig. 1,  step 4). The yield of the process was determined as a mean of three independent preparations from 2,5-DMPA. For optimal pyomelanin yield, laccase activity and HGA concentration to be used were determined in 4 mL glass vials tightly closed and containing 500 µL of Na-phosphate 100 mM buffer pH 6. To determine the optimal (HGA)/(Mn 2+ ) ratio, concentrations of HGA (1-300 mM) and MnCl 2 (0.5-20 mM) were assayed in the same manner, the black-brown solutions diluted 50 × in NaOH 0.1 N and absorbance read at 400 nm (A 400 nm ). Cytotoxicity. The viability of cells exposed to melanin was expressed as the concentration-dependent reduction of the vital dye Neutral Red (NR) uptake in intracellular lysosomes. Assays were carried out with the three prepared pyomelanin and the two melanin standards ( www.nature.com/scientificreports/ complete medium containing the melanin (8 concentrations, 0-500 µg/mL), and cells were incubated again for 24 h. After removing the medium, cells were washed, placed into the NR medium (50 µg/mL NR in the complete medium), and incubated for 3 h (37 °C, 5% CO 2 ). The medium was removed, cells were washed three times with 0.2 mL of HBSS (Hank's Balanced Salt Solution, from DUTSCHER) to eliminate the excess dye, and 50 µL per well of a distaining solution (50% ethanol, 1% acetic acid, 49% milliQ-H 2 O) was added. The plates were shaken for 15 min at room temperature in the dark. The membrane damage degree, i.e. the increase of released NR, was determined by the A 540 nm in an Infinite M200 Pro (TECAN, Swiss) reader. The results obtained for wells treated with the pigment were compared to those of untreated (100% viability) and converted to percentage values. Cell viability was calculated as Viability (%) = [A 540 (test well) -A 540 (blank)] / [A 540 (negative control) -A 540 (blank)]. The concentration of the pigment causing a 50% release of NR as compared to the control culture (IC 50 , in µg/mL) was calculated by non-linear regression analysis using the Phototox v2.0 software (ZEBET, Germany).

Homogentisic acid and gentisyl alcohol syntheses.
Phototoxicity. The in vitro and normalized 3T3 NRU assay (OECD number 432) was used. Balb/c 3Τ3 mouse fibroblasts (3Τ3-L1, ATCC CL-173, from US type Culture Collection) were grown in DΜΕΜ supplemented with L-glutamine 4 mM and 10% of inactivated calf serum, seeded into two 96-well plates (0.1 mL per well) at 1.10 5 cells/mL concentration, and incubated (37 °C, 5% CΟ 2 ) for 24 h until semi-confluent. The medium was decanted and replaced by 100 µL of HBSS (see before) containing the appropriate pigment concentrations (8 concentrations, 0-500 µg/mL), then cells were incubated (37 °C, 5% CO 2 ) in the dark for 60 min. From the two plates prepared for each series of pigment concentrations and the controls, one was selected, generally at random, for the determination of cytotoxicity without irradiation (− Irr), and the other for the determination of phototoxicity with irradiation (+ Irr). For each set of experiments, a negative control (in HBSS) and positive control (chlorpromazine final concentrations from 1 to 100 µg/mL (− Irr) and 0.01 to 1 µg/mL (+ Irr), diluted in ethanol) were performed. The percentages of cell viability were calculated as previously (cytotoxicity). Irradiation was performed with a solar simulator Suntest CPS + (ATLAS MATERIAL TESTING TECHNOLOGY BV, Lochem, Netherlands) device equipped with a xenon arc lamp (1100 W), a glass filter restricting transmission of light below 290 nm, and a near IR-blocking filter. The irradiance was 750 W/m 2 corresponding to 4.5 J/cm 2 for one-min irradiation (0.41 J/cm 2 of UVA and 4.06 J/cm 2 of visible light). The Photo-Irritation-Factor (PIF) defined by the ration IC 50 (− Irr) / IC 50 (+ Irr) was expressed to finalize the results. Based on validation studies (OECD 432 guideline), a test substance exhibiting a PIF < 2 predicts no phototoxicity, 2 < PIF < 5 a probable, and PIF > 5 a phototoxicity.

Photostability of Pyo ENZ in solution.
It was evaluated on 4 mL glass-closed tubes containing 3 mL each of Pyo ENZ solution at 0.05, 0.1, and 0.5 mg/mL NaOH 0.05 N. The tubes were placed horizontally and irradiated in the Suntest CPS + solar simulator, respecting the ICH Q1B guidelines (European Medicines Agency). A strong irradiance by the xenon lamp was maintained with light energy of 550 W/m 2 during 1 h (i.e. 200 J/cm UVAvisible irradiation). Changes in the polymer structure were monitored by UV-visible spectroscopy from 200 to 700 nm and GPC/SEC (see Fig. 6S), comparatively to non-irradiated samples.
Metabolites identification, pyomelanin monitoring. Phenolic compounds along the three processes were identified by RP-HPLC-DAD and GC-MS according to our previous works [33][34][35] . To control the pigment formation during the bacterial culture and for optimization of the processes, the black-brown solution was diluted 20 and 50 × in NaOH 0.1 N (qsp 1 mL), respectively, and absorbance (A 400 nm ) read against the same alkaline reference 2 .