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

Abstract

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 (Pyo_CHEM yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (Pyo_BACT, 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (Pyo_ENZ, 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. C_ar–C_ar bindings predominated in the three polymers, C_ar–O–C_ar (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, Pyo_ENZ exhibited an M_w 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, Pyo_ENZ might also be useful for applications in extreme conditions and skin protection.

Introduction

Pyomelanin is a natural polymer of homogentisic acid (HGA, 2,5-dihydroxyphenylacetic acid) synthesized through the L-tyrosine pathway by bacteria, fungi, mammals, and plants, and belongs to the heterogeneous group of allomelanins¹. In living cells, the HGA 1,2-dioxygenase disruption or deletion leads tfigo pyomelanin accumulation² (Fig. 1S). The first property of the pigment is to protect microorganisms from UV light limiting free radicals and ROS generation³. The excitation of L-Dopa melanin by UV light produces cell-damaging ROS⁴, whereas the formation of ROS by light has never been reported with pyomelanin. Therefore, pyomelanin increases resistance to light, for instance in Legionella⁵. Its antioxidant role has also been demonstrated in Pseudomonas aeruginosa⁶, Burkholderia cenocepacia⁷, Aspergillus fumigatus², and many other strains. Pyomelanin also diminishes the oxidizing stress of the host microorganism, by its high tolerance to H₂O₂, as demonstrated in Ralstonia solanacearum⁸ and clinical isolates of Pseudomonas aeruginosa from infected patients⁹. Moreover, pyomelanin is an antibacterial and antifungal agent against microbial outside attacks, especially pathogenic¹⁰, reduces biofouling¹¹, chelates heavy metals^3,12, and contributes to microbial pathogenesis as it is associated with virulence in a broad range of pathogenic fungi and bacteria^9,13. Pyomelanin potentially reduces soluble FeIII in FeII^14–16, FeII being essential in many bacteria such as Legionella pneumophila, that ensure homeostasis by an appropriate Fe²⁺/Fe³⁺ ratio for their survival^17,18. Consequently, in vivo or in vitro, pyomelanin may serve as a terminal electron acceptor, electron shuttle, or conduit for electrons, a complementary iron acquisition to the siderophore role. Several ways for pyomelanin synthesis may be explored. In microorganisms, two distinct pathways led to HGA formation, through a 4-HPP dioxygenase (4-HPPD; EC 1.13.11.27; route 1), the most described from many bacteria mostly pathogen, and via a 4-HPA-1-hydroxylase (4-HPAH-1; EC 1.14.13.18; route 2) (Fig. 1S). To date, there are very little data on microbial pyomelanin production and yield reminds weak (max. 0.35 g/L¹⁹), comparatively to the eumelanin pigment produced at 28.8 g/L by a tyrosinase overexpression in a recombinant Streptomyces kathirae²⁰. The construction of a recombinant 4-HPPD enzyme is not an option because the substrate 4-HPP is expensive. Recently, as opposed to the plant enzyme-based assay, an optimized high-throughput screening assay using human 4-HPPD was constructed using the E. coli strain C43 (DE3) supplemented with L-Tyr in the culture medium, a useful tool to find new inhibitors against alkaptonuria disease²¹. However, no pyomelanin yield was reported. On the other hand, the 4-HPAH-1 enzyme responsible for HGA synthesis in Delftia acidovorans²² and Azoarcus evansii²³, had been partially characterized. In 2008, the sequence of the D. acidovorans enzyme had been given by several genomic approaches²⁴. The enzyme contains two components, hpaH which codes for a flavoprotein NAD(H)-dependent oxidase that transforms 4-HPA in a non-identified metabolite called Z, the second hpaC catalyzes the conversion of Z in HGA. hpaH and hpaC were cloned together, however, no HGA synthesis occurred from these constructions. To date, HGA is very expensive, at about $800/gram. Several methods of chemical synthesis have been developed but the majority not applicable for a large-scale process, the yield being comprised of 50–60% when indicated. Ultimately, the former procedure of DeForrest Abbott and Doyle-Smyth²⁵ through HGA-lactone as intermediate, remained the most convenient and proposed in two or three simple steps. For a long time, pyomelanin had been described as the result of the HGA autoxidation catalyzed by Mn²⁺ or Cu²⁺, from neutral to alkaline conditions²⁶. For 30 years, several authors hypothesized the action of oxidases, such as the multicopper-dependent laccases (EC 1.10.3.2) generally involved in the polymerization of dihydroxylated phenols²⁷. Laccases have also been suggested in that of HGA in Vibrio cholerae²⁸, Alcaligenes eutrophus (now Cupriavidus necator)²⁹, Metarhizium anisopliae³⁰, and their role definitively confirmed in Cryptococcus neoformans³¹.

At this time, no marketed pyomelanin exists, and a method to furnish acceptable quantities of pigment became necessary. We have therefore developed a large-scale and low-cost production process by reconsidering the chemical synthesis of HGA on the one hand, and the use of laccase on the other. From this optimized enzymatic process, the resulting pigment (Pyo_ENZ) and its chemical structure and properties had been examined. For comparison, bacterial pyomelanin (Pyo_BACT) from an induced Halomonas culture, the bacterial process, and that issued from the HGA autoxidation (Pyo_CHEM), the chemical process, was also extensively characterized. Furthermore, the mechanism of HGA polymerization was re-evaluated with a lot of care.

Results

Development of an enzymatic process (Pyo_ENZ)

To produce high quantities of pyomelanin, appropriate chemical synthesis of HGA has been combined with a polymerization step by a selected laccase in an on-line process (steps 1 to 4, Fig. 1). The first part of the adapted synthesis of DeForrest Abbott and Doyle-Smyth²⁵ yielded pure HGA-lactone (step 1, ~ 99% yield) as controlled by HPLC and GC-MS (see spectroscopic data in Methods). The alkaline hydrolysis of the lactone was followed by immediate buffering at pH 6.8 (step 2) and the addition of the rMt laccase extract (step 3) that reacted in two successive stages, the formation of BQA and the polymerization (step 3). In the final step 4, the polymer was precipitated, washed, and dried. The 2,5-DMPA as the starting molecule of the process could be prepared from 2,5-dimethoxyacetophenone²⁵, four times less expensive according to various suppliers. Finally, the optimal yield was reached with 40 mM HGA (final conc.) or HGA-lactone equivalent, 17–22 U (~ 23–30 µL) of rMt laccase extract per mL of reaction volume, at pH 6.8 for 24 h agitation (Fig. 2S and 3S). A temperature of 50 °C modified the polymerization rate but did not improve the yield at the end of the incubation time that must be 25 h (Fig. 4S). In these conditions, at 30 °C, the process yielded 1.25 ± 0.11 g of dried Pyo_ENZ per g of 2,5-DMPA (mean of 3 experiments).

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 type²⁵. 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 – CH₂CHO] at m/z 251), similarly to the NIST data bank and previous data³⁴. In step 2, the addition of sulfite (Na₂SO₃)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.

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^32–34. 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 (3,4-DHPA) in H. alkaliantartica, H. neptunia, H. sulfaedris (this work), and H. sp. HTB24³⁵, through routes 2b and 3, respectively (Fig. 1S). H. titanicae is a γ-proteobacterium isolated on the Titanic wreck, its genome now entirely available³⁶, 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 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 Mn²⁺ to enhance the catalysis²⁶, an (HGA)/(Mn²⁺) 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.

Table 2.

Summarized chemical and biological properties of pyomelanin and commercial melanin.

Production yield	PyoENZ	PyoCHEM	PyoBACT	MelSYNTH	MelSEPIA
	1.25 ± 0.11 g/g 2,5-DMPA	0.31 ± 0.03 g/g 2,5-DMPA	0.55 ± 0.09 g/L 0.24 ± 0.04 g/g 4-HPA	–	–
Polymer size
MW (g/mol)	5400	2300	5700	2000	NDf
Mn (g/mol)	360	350	470	250	–
Mp (g/mol)	3600	2500	4600	1600	–
Ð (MW/Mn)	15.3	6.64	11.9	7.95	–
Monomer composition	HGA, BQA, GA, GALDa	HGA, BQAa	HGA, BQA, GA, GALDa	DHI, DHICAb	DHI, DHICAb
Elemental analysis	(%)	(%)	(%)	(%)	(%)
C	49.15 ± 0.08	54.61 ± 0.11	40.58 ± 0.03	49.69 ± 0.10	34.10 ± 0.03
H	3.40 ± 0.02	2.59 ± 0.10	3.15 ± 0.03	2.83 ± 0.05	3.23 ± 0.02
N	2.75 ± 0.02	0	3.65 ± 0.05	6.34 ± 0.03	6.31 ± 0.08
Od	44.70	42.80	52.62	41.14	56.36
C/O	1.10	1.27	0.77	1.20	0.6
C/N	18	–	11	8	5.4
Formulac	C221H140N11O151	C105H59O61	C193H179N15O187	C83H57N9O51	–
Calc. mass (g/mol)	5362	2295	5697	1995	–
Solubility
NaOH 0.05 N	≤ 10 mg/mL	≤ 10 mg/mL	≤ 10 mg/mL	≤ 10 mg/mL	≤ 10 mg/mL
DMSO	≤ 0.5 mg/mL	≤ 0.5 mg/mL	≤ 0.5 mg/mL	≤ 0.5 mg/mL	≤ 0.5 mg/mL
Other solvents	Insoluble	Insoluble	Insoluble	Insoluble	Insoluble
DPPH-antioxidant activity (EC50 µg/mL)e	27.5	20.23	133.7	25.9	> 300
ROS scavenging activity (IC50 µg/mL)	82.2 ± 5.5	ND	ND	284.1 ± 12.3	> 500
UV spectrum range	200–700 nm	200–700 nm	200–700 nm	200–700 nm	200–700 nm
Fe3+-reducing activity
% Related to MelSYNTH	96	95	54	100	34
In ng Fe2+/h/µge	5.30	5.24	2.98	5.52	1.94
Stability to T°C	≤ 80 °C, 72 h	≤ 80 °C, 72 h	≤ 80 °C, 72 h	ND	ND
Cytotoxicity (c ≤ 500 µg/mL)	No	No	No	No	No
Phototoxicity (PIF)	No (< 2)	No (< 2)	No (< 2)	No (< 2)	No (< 2)

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.

^aIdentified by ¹³C solid-state NMR (Fig. 2 and Table 1).

^bTo confirm the global structure of Mel_SYNTH and Mel_SEPIA, assays on their degradation by alkaline-H₂O₂ were conducted similarly to those on human melanin⁴¹. 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.

^cElemental 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_xH_yN_z, each index was deduced by (x, y, z) = %atom (data from elemental analysis) x M_w/M_atom.

^dThe index w (for oxygen O_w) was deduced from 100% – C – H – N.

^eStandard deviations were < 5%. Composition (C, H, N) of Mel_SYNTH was similar to those described⁶³, that of Mel_SEPIA close to the reported values⁴² (see sample Com).

^fMolecular 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.

Structural data of the three pyomelanin

Better than ¹H-NMR, solid-state ¹³C-NMR analyses of polymers can provide not only structural features through the resonances of the monomers but also the types of bindings. Pyomelanin issued from the three developed processes, Pyo_ENZ, Pyo_BACT, and Pyo_CHEM were analyzed by solid-state ¹³C CP-MAS at their optimal signal resolution in conjunction with FTIR experiments.

Solid-state ¹³C 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 C₅) of the ring bearing the -OH group, with shoulders at 143.4 for the three pyomelanin (suggested C₂), 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 (-CH₂-) of the acetic acid moiety. From the three ¹³C 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 (-CH₂-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 ¹³C 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 (Fig. 2, data framed) indicates a correct -CH₂-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.

Figure 2.

¹³C Cross-Polarization Magic Angle Spinning (CP-MAS) solid-state NMR spectra of the three 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 ¹H and ¹³C nuclei, respectively, by using a 4 mm cross-polarization 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 ¹³C spectra, the acquisition time was 28.7 ms. The ¹H 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 ¹³C 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.

Table 1.

Summary of the ¹³C CP-MS solid-state NMR chemical shifts and their corresponding assignation for the three pyomelanin pigments, and comparison with standards.

Compound	Protonated carbons			Unprotonated carbons		References
	δ (ppm)	Assignation		δ (ppm)	Assignation
PyoENZ	17.2	–CH3 (lipid residue)		122 sh	Car1– (suggested)	–
	23.1	–CH2–R (lipid residue		143.4 sh	Car–OH
	34.3	–CH2– (acetic group)		149.3
	52.5 br	–CH2–OH (gentisyl alcohol)		172.0	–COOH (carboxylic C)
	67.9 br	Saccharide moiety (> C–OH)
	119.0	–CarH=Car
	191.0 w	–CHO (aldehyde group on Car1)
PyoBACT	17.4	–CH3 (lipid residue)		124 br	Car1– (suggested)	–
	23.9	–CH2–R (lipid residue		143.4 sh	Car–OH
	34.8	–CH2– (acetic group)		149.2
	52.5 br	–CH2–OH (gentisyl alcohol)		152 sh	Undetermined
	118.1	–CarH = Car		173.0	–COOH (carboxylic C)
	190.5 w	–CHO (aldehyde group on Car1)
PyoCHEM	33.0	–CH2– (acetic group)		143.4 sh	Car–OH	–
	110sh	Undetermined		149.4
	118.1	–CarH=Car		173.4	–COOH (carboxylic C)
Standards
HGA	33.9	–CH2– (acetic group)		122.3	–Car1–	Data similar to the literature64
	116.5	Car4–H	#	146.7	Car2–OH
	117.0	Car3–H		148.3	Car5–OH
	120.9	Car6–H		180.9	–COOH (carboxylic C)
Gentisyl alcohol	55.1	–CH2–OH		125.9	Car1–CH2OH	Data quite similar to those of Molbase site (www.molbase.com)
	115.9	–CarH=Car(Car3,4,6–H)*		147.1	Car2–OH
				154.4	Car5–OH
Gentisaldehyde	119.2	–CarH=Car(Car3,4,6–H)*		125.9	Car1 (–CHO attachment)	Data quite similar to those of Molbase site (www.molbase.com)
	200.6	–CHO (aldehyde group on Car1)		149.0	Car5–OH
				155.6	Car2–OH

Car, aromatic carbon; sh, shoulder; br, broad; w, weak.

*Unresolved; # well resolved.

FTIR analyses

Pyo_BACT and Pyo_ENZ exhibited very similar FTIR and ¹³C-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^29,37; (iv) 1623 and 1656 cm⁻¹ absorptions that were described as typical for aromatic C=C conjugated with C=O groups (quinones), with a stronger response in Pyo_CHEM; (v) 1384 and 1385 cm⁻¹ (both minor) would be assigned to the O–H bond of the hydroxyl groups attached to the ring; and (vi) strong bands at 1197 (Pyo_ENZ) and 1222 cm⁻¹ (Pyo_CHEM) of the phenolic-OH links.

Presence of N in Pyo_ENZ and Pyo_BACT

Especially, a reaction of substitution on the C₄ position of the BQA ring by primary and secondary amines had been reported³⁸, 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 ¹³C NMR, mainly at 3000–3500 cm^-1 (N-H stretching vibrations of aromatic 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 C₄ 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 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 ¹³C 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-H₂O₂ oxidation assays

This treatment has also been tried on the three pyomelanin and the commercial melanin⁴¹ (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 DHI-derived 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 chrysogenum⁴³ 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.). Exceptionally, all the pigments are soluble in DMSO at a concentration that does not exceed 0.5 mg/mL after 24 h agitation. Solubility in H₂O was not improved after 2 days at 80 °C. As solid form and/or solubilized in alkaline solutions, Pyo_ENZ, Pyo_BACT, and Pyo_CHEM were stable until 80 °C for 3 days (max. tested) with no degradation products detected by RP-HPLC, and GC–MS analyses, near size modification by GPC/SEC. Molecular weights (M_w) of the three HGA-pigments were successfully determined and have been found at 5,400 Da (dispersity Ɖ 15.3) for Pyo_ENZ, 5,700 Da (Ɖ 11.9) for Pyo_BACT, and a less high M_w at 2,300 Da (Ɖ 6.64) and 2,000 Da for Pyo_CHEM and Mel_SYNTH (Fig. 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 products⁴⁴. 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 IC₅₀ of 82.2 ± 5.6 µg/mL, while IC₅₀ 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.

Table 3.

Effect of Pyo_ENZ on scavenging ROS generated by UVA irradiation on keratinocyte cells.

	Concentration (µg/mL)	Fluorescence intensity	ROS release (%)	IC50 (µg/mL)
PyoENZ	50	331.6 ± 3.3	65.92 ± 2.64	82.2 ± 5.6
	100	244.3 ± 7.7	35.05 ± 1.71
	250	187.0 ± 6.6	14.86 ± 2.16
	500	164.0 ± 4.0	6.70 ± 2.28
MelSYNTH	50	412.6 ± 2.8	94.51 ± 2.61	284.1 ± 12.3
	100	373.3 ± 11.0	80.67 ± 4.90
	250	326.0 ± 12.2	63.97 ± 5.13
	500	231.6 ± 16.4	30.70 ± 6.67
MelSEPIA	50	414.6 ± 5.3	95.17 ± 1.70	> 500
	100	393.3 ± 4.9	87.70 ± 3.57
	250	334.6 ± 19.9	66.92 ± 5.87
	500	301.3 ± 5.3	55.19 ± 2.86
Non-irradiated control		145.0 ± 2.4	0	–
Irradiated control		428.3 ± 5.7	100	–

The ROS release was detected by fluorometric measurement and inhibition of the DCFH-DA reagent. Pyo_ENZ antiradical activity was compared to that of the melanin standards. Determinations resulted from triplicate assays and IC₅₀ values were calculated by the Phototox v2.0 software (ZEBET, Germany). Experimentally, keratinocyte cells (see phototoxicity protocol for preparation) were UVA-irradiated in presence of grads concentration (50–500 μg/mL) of Pyo_ENZ and standard melanin (Mel_SYNTH, Mel_SEPIA), or not (control). Cells were decanted, washed twice by a 25 mM PBS buffer at pH 7.4, then loaded with 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) to a final concentration of 20 μM, and incubated in dark at 37 °C for 30 min. ROS were then measured by fluorescence intensity of dichlorofluorescein (DCF) at an excitation wavelength of 499 nm and an emission wavelength of 521 nm, in an Infinite M200 Pro fluorescence reader (TECAN, Swiss) equipped with a 1-cm quartz cell. Irradiation was carried out with the Suntest CPS + solar simulator (xenon arc lamp 1100 W, with filters to restrict light transmission below 290 nm and near IR), at a dose of UVA-visible light of 138 kJ/m², the irradiance was at 765 W/m² and irradiation time 3 min. The temperature of the samples was maintained at 4 °C using a water-cooling system linked to the irradiation chamber. Inhibition of DCFH-DA fluorescence (Fluo) was expressed as a percentage as compared to the irradiated and non-irradiated control (without pyomelanin or melanin):

ROS release (%) = 100 × (Fluo_test − Fluo_{-irr control})/(Fluo_{+irr control} − Fluo_{-irr control}). IC₅₀ (μg/mL) values were calculated by the Phototox v2.0 software.

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 (EC₅₀ 27.5 µg/mL) and Mel_SYNTH (EC₅₀ 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 pro-oxidant when the glutathione pool is depleted⁴⁷, a feature that must also be controlled in the case of pyomelanin. Barely better than Pyo_ENZ, Pyo_CHEM EC₅₀ was 20.0 µg/mL, while EC₅₀ 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 (EC₅₀ 230 μg/mL⁴⁸), and far from eumelanin from Sepia officinalis (Mel_SEPIA, > 300 µg/mL, this work) and the synthetic butylated hydroxytoluene (BHT, EC₅₀ 722 μg/mL⁴⁹). Although the trihydroxylated benzoic ester, propyl gallate (EC₅₀ 4.2 µg/mL) (Fig. 7S-B), was one of the leading dietary antioxidants, it induced DNA damages⁵⁰; BHT was also found cytotoxic⁵¹, 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 Fe³⁺-reducing activity among the five polymers tested (Fig. 3). From these data, the equivalent Fe³⁺-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 Fe³⁺-reducing activity was evaluated at 1.73 µM per hour related to 50 µg of pigment, i.e. 5.30 ng Fe²⁺/h/µg.

Figure 3.

Fe³⁺ reduction by pyomelanin issued from the three processes (Pyo_ENZ, Pyo_BACT, Pyo_CHEM), and comparison to the commercial melanin (Mel_SEPIA, Mel_SYNTH). HGA and ascorbic acid were used as positive controls. The Fe³⁺-reducing activity was ada from other methods^17,18,62. Briefly, 400 μM ferrozine and 120 μM anhydrous FeCl₃ (final conc.) were extemporaneously mixed in a Tris-HCl 25 mM buffer pH 7.5 (solution A). Then, 5 μL (50 µg) of the melanin stock solutions, each at 10 mg/mL in NaOH 0.05 N, was added in 1 mL of A in closed glass tubes. Blanks were prepared identically without melanin. Mixtures were incubated for 12 h at room temperature and the developed color was measured at 562 nm. As positive controls, 10 µL (44 µg) of ascorbic acid 25 mM solution, and 20 µL (50 µg) of HGA 15 mM, which have defined reduction activity, were also mixed with 1 mL A, incubated, and measured identically. To quantify the activity, a standard curve was generated with known concentrations of ferrous sulfate complexed with ferrozine (solution A), a stock solution of FeSO₄,7H₂O 3 mM in milliQ-H₂O used. After correction, the concentration of Fe²⁺ varied from 0.92 to 13.8 µg, i.e., 6 to 90 µM, and a typical standard equation was: A_{562 nm} = 0.026 x [Fe²⁺ (µM)] + 0.0093. All values were expressed as a mean of three independent experiments, SD < 5% not given to lighten the figure. These experiments suggest that the reducing capacity of HGA is lost after polymerization, and ascorbic acid remains the stronger reducing agent which is however degraded on time.

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-Kindler²⁵. 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 culture⁵², second 0.173 g/L culture of the Shewanella algae BrY strain supplemented by 2 g of L-Tyr/L¹⁴, and third 0.35 g/L by random mutagenesis of Pseudomonas putida¹⁹. 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, the ability of H. titanicae to synthesize pyomelanin from 4-HPA and the property of the pigment to reduce Fe³⁺, raise the question of the survival of the bacterium at 4,000 m depth by maintaining a Fe³⁺/Fe²⁺ ratio.

A few remarks are worth noting about the oxidation of HGA. Besides the biological implications of metal-catalyzed oxidations, true autoxidation of biomolecules does not occur in biological systems, instead, this autoxidation is the result of transition metals bound to these biomolecules⁵³. By analyzing the Pyo_CHEM structure, surprisingly the ¹³C solid-state NMR spectrum revealed an unexplained loss (~ 40%) of carboxylic moiety during the alkaline Mn²⁺-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 standards²⁹. 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-H₂O₂ hydrolyses and pyrolysis experiments failed, the chemical structure of the three pyomelanin was determined by ¹³C solid-state NMR, and partly confirmed by FTIR analyses. Like the hydroquinone polymerization²⁷, 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, C₄-C₆ (α-bindings) and C₃-C₆ (β-bindings), giving preference to the C₃-C₆ 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 radicals and still showing C₃-C₆ and C₄-C₆ 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 alcohol³⁴. 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.

Figure 4.

The proposed mechanism of HGA polymerization by the laccase or in abiotic conditions, giving the most probable structure in (A) and the most active radical reactions detailed in (B). These two figures show the configurations of the C_ar-C_ar links present in the three pyomelanin structures, whereas C_ar-O-C_ar (ether) links are absent as demonstrated by ¹³C solid-state NMR (see text). 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.

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 cells⁵⁵. Human eumelanin and pheomelanin photogenerate ROS meanwhile they photoconsume O₂ and are protective against skin cancer⁵⁶. Nevertheless, they photochemically generate melanin degradation products that are responsible for sunlight-induced melanoma formation by inducing cyclobutane-pyridine dimers (CPDs) from DNA⁵⁷. 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 Fe³⁺-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 Fe³⁺-reducing agent, and for bioelectronic applications better than melanin⁵⁸. As an evident cosmetic ingredient and consistently with recent reports on microbial pyomelanin from Yarrowia lipolytica⁴⁸ and Pseudoalteromonas lipolytica BTCZ28⁴⁶, 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.

Methods

Chemicals and enzymes

Solvents of mass spectrometry grade were supplied by BIOSOLVE (Dieuze, France), media for human keratinocytes, and mouse fibroblasts cultures from DUTSCHER (Brumath, France), main chemicals including standard melanin from SIGMA. Natural melanin (Mel_SEPIA, reference M2649) consists of purified eumelanin from the ink of Sepia officinalis. Synthetic melanin (Mel_SYNTH, M8631) is an L-Dopa melanin obtained from L-Tyr in presence of H₂O₂. HGA and HGA-lactone were used as standards for HPLC and the GC-mass data bank. The Aspergillus sp. laccase (SAE0050) consisted of a highly concentrated and brown miscible-water solution (density 1.15 g/mL, stored at 4 °C) obtained by submerged fermentation of the recombinant Myceliophtora thermophile laccase expressed in Aspergillus oryzae. This enzyme originally furnished by Novozym under reference 51,003, was re-named here ‘laccase rMt’. Purified laccases from Pycnoporus cinnabarinus (recombinant, a gift of The Fungal Biodiversity and Biotechnology Laboratory, Marseille⁵⁹) and Trametes versicolor (SIGMA, 38429) were also used.

Halomonas strain selected and growth conditions

The strain Halomonas titanicae was provided from the DSMZ collection (Germany), isolated and taxonomically characterized in 2010⁴³, and compared in our laboratory among a large collection of Halomonas spp.³² The strain was grown in a shaker (130 rpm) at 30 °C in a basal medium containing (in g/L), yeast extract 1.0, NaCl 20, KH₂PO₄, K₂HPO₄ 0.6, NH₄Cl 1.0, MgCl₂·6 H₂O 10, CaCl₂·2 H₂O 0.1. The pH was adjusted to 7.0 with a 4 N NaOH solution. Aliquots (25 and 500 mL) were dispensed into flasks and sterilized by autoclaving at 120 °C for 20 min. Ca- and Mg-chloride stock solutions were sterilized separately, and the accurate volumes were added to the medium. L-Tyr and 4-hydroxyphenylacetic acid (4-HPA) stock solutions (250 mM in milliQ-H₂O, neutralized, heated moderately, sterilized by 0.2 µm pore size filtration) were added before inoculation at 5 mM final concentration. The strain was pre-cultivated twice for 2 days each, in 25 mL of the same saline basal medium containing either L-Tyr or 4-HPA. The preculture served as inoculum at 10% (v/v) for the culture in 500 mL volume that was agitated at 150 rpm, until the A_{400 nm} no longer changed. To overproduce pyomelanin, 500 mL cultures supplemented by repeated addition of 4-HPA amounts were carried out using the experimental design AZURAD software (a company of Marseille). The 8 experiments resulted from the defined parameters, the response Yi (mass of pyomelanin per Liter of culture), and the 6 entry parameters, X₁ for the 4-HPA concentrations added (2, 5, and 10 mM), and X₂ for the time of supplementation (at 0, 2 or 3 days of growth). An experimental domain of cubic form was chosen and a second-degree polynomial model applied⁶⁰.

Process for the production of pyomelanin (Pyo_ENZ) by the rMt laccase

The first part of the procedure consisted of an adapted HGA synthesis²⁵. 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-H₂O (70 °C), stayed 3–5 min and few drops of NaOH 2 N added until pH 9.3 (pH-meter) 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-H₂O 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 6.8 (final 50 mM), 50 to 500 µL (5–50 mM) of HGA 100 mM stock solution in milliQ-H₂O, 5–40 µL laccase rMt extract (3.75–30 U), and milliQ-H₂O (qsp), in one mL total volume. Assays were incubated at 30 °C or 50 °C, at incubation time varying from 1 to 30 h, under 120 rpm agitation in dark. The enzyme activity was stopped by placing the tubes in a boiling water bath for 10 min, and pyomelanin content was evaluated by spectrophotometry (see below pyomelanin monitoring). Each point value resulted from triplicate tubes and a mean ± SD.

Bacterial (Pyo_BACT) and chemical (Pyo_CHEM) pyomelanin preparation

Bacterial cultures at the stationary phase were centrifuged (8,500 g, 30 min), pyomelanin (Pyo_BACT) in the supernatant was precipitated by the addition of 2 N HCl (final conc.), and the solution left to rest for 24 h, at room temperature in the dark. After centrifugation (8,500 g, 20 min), the brown-black pellet was washed successively with milliQ-H₂O (3×) and ethanol (1×), centrifuged, dried at 70 °C for 2 days, weighed, and powdered as fine particles before storage in glass vials at room temperature. Chemical pyomelanin (Pyo_CHEM) was prepared from 2,5-DMPA (1 g, 5.1 mmol) that was demethylated by HBr (reflux) and giving HGA-lactone by rotative evaporation. The lactone was then dissolved in hot H₂O identically to Pyo_ENZ preparation (see previously), and the aqueous HGA (0.84 g, 98.1% yield, evaluated by HPLC with pure HGA as calibrant) formed by alkaline treatment at pH 9.3. Autoxidation of HGA was then continued in presence of 5 mM MnCl₂, 4H₂O, the solution agitated in dark for 3 days with a barrel at 30 °C, the pigment precipitated with 6 N HCl (final conc.), left to sediment for 12 h, and centrifuged (8,500 g, 20 min). The light-brown colored supernatant indicated the presence of many oxidized compounds that could not precipitate by increasing the acid until 10 N. After washing and drying, the pelleted Pyo_CHEM was weighed and stored as for the Pyo_BACT and Pyo_ENZ pigments. To determine the optimal (HGA)/(Mn²⁺) ratio, concentrations of HGA (1–300 mM) and MnCl₂ (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}).

Homogentisic acid and gentisyl alcohol syntheses

Alkaline hydrolysis of 1.0 g of HGA-lactone in hot 130 mL H₂O was immediately acidified until pH 5 with drops of HCl 37%, followed by the addition of 6.5 mL of a saturated NaCl solution (5% v/v final conc.). Then HGA was extracted 3 × in a funnel with AcOEt, the whole organic phase washed 1 × with milliQ-H₂O, clarified with solid Na₂SO₄, filtered, and evaporated to dryness. To eliminate residual BQA, the dried HGA was dissolved in 0.1% HCOOH and applied on a glass column (20 mL Luer-lock tip syringe mounted with a vacuum flask and a pump) containing 10 cm³ of Lichroprep RP₁₈ (from SIGMA) previously conditioned with MeOH and acidified H₂O. After washing by 2 vol. of acidified H₂O, HGA was eluted by a mixture of MeOH-acidified H₂O (1:9, v/v), evaporated to dryness, and resulted in a 99.9% purity light grey HGA (yields ~ 70 wt%), as determined by RP-HPLC and GC-MS analyses, indicating that recrystallization was not necessary. As standard for NMR analyses, pure gentisyl alcohol was synthesized from gentisaldehyde by NaBH₄-reduction in tetrahydrofuran (yield 49%)⁶¹, purity confirmed by GC-MS and ¹H-NMR in d₆-DMSO.

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 (Table 2), all prepared at 10 mg/mL in NaOH 0.05 N (stock solution). Human epidermal keratinocytes neonatal cells were maintained in a complete keratinocyte serum-free medium (Panserin 412, from DUTSCHER) supplemented with bovine pituitary extract (30 µg/mL), recombinant epidermic growth factor (rEGF, 0.2 ng/mL), and an antibiotic cocktail of 10 U/mL penicillin-100 µg/mL streptomycin. Precultures were seeded into 96-well plates (0.2 mL per well) at 1.10⁵ cells/mL concentration. After incubation at 37 °C (5% CΟ₂) for 24 h until semi-confluent, the medium was decanted, replaced by 200 µL of 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% CO₂). 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-H₂O) 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 (%) = [A₅₄₀ (test well) — A₅₄₀ (blank)] / [A₅₄₀ (negative control) — A₅₄₀ (blank)]. The concentration of the pigment causing a 50% release of NR as compared to the control culture (IC₅₀, in µg/mL) was calculated by non-linear regression analysis using the Phototox v2.0 software (ZEBET, Germany).

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 1.10⁵ cells/mL concentration, and incubated (37 °C, 5% CΟ₂) 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% CO₂) 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/m² corresponding to 4.5 J/cm² for one-min irradiation (0.41 J/cm² of UVA and 4.06 J/cm² of visible light). The Photo-Irritation-Factor (PIF) defined by the ration IC₅₀ (− Irr) / IC₅₀ (+ 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/m² during 1 h (i.e. 200 J/cm UVA-visible 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^33–35. 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 reference².

Abbreviations

DPPH

1,1-Diphenyl-2-picrylhydrazyl

FTIR

Fourier Transform InfraRed spectroscopy

GC–MS

Gas chromatography coupled to mass spectrometry

GPC/SEC

Gel permeation/size exclusion chromatography

L-Dopa

L-3,4-dihydroxyphenylalanine

OECD

Organisation for Economic Co-operation and Development

rMt

Recombinant laccase of Myceliophtora thermophile

PEG/PEO

Polyethylene glycol/polyethylene oxide

RP-HPLC-DAD

Reverse-phase HPLC coupled to a diode array detector

TMS

Trimethylsilyl

Author contributions

J.L. initiated this study, designed the experiments, analyzed the data, supervised the research, and reviewed the manuscript. F.L. designed and executed the experiments, collected and analyzed the data, wrote the manuscript, and prepared the figures. F.Z. executed the NMR spectra and corrected the concerned part of the text. A.A. studied with J.L. the H. titanicae genome (hpaH/C genes), proposed this strain as a good candidate for producing pyomelanin, and helped in the culture experiments. C. Di G. managed the cytotoxicity, phototoxicity and ROS experiments with F.L. M.R. synthesized gentisyl alcohol and HGA. P.P. participated in the strategy and entirely supervised the financial aspects of this work. All authors have read and approved the final manuscript.

Funding

FL works were supported in part by an ANRT-CIFRE (Association Nationale Recherche Technologie-Conventions Industrielles de Formation par la Recherche, France) fellowship.

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-021-87328-2.