Tryptophan, a non-canonical melanin precursor: New L-tryptophan based melanin production by Rubrivivax benzoatilyticus JA2

Abstract

Melanins are chemically diverse ubiquitous pigments found across the life forms synthesized via different biochemical pathways mainly from L-tyrosine or acetyl CoA. Though few reports suggest the possibility of tryptophan-based melanin synthesis, however, such tryptophan-based melanin and its biosynthesis remained a biochemical riddle. Here we report tryptophan-based melanin production by bacterium, Rubrivivax benzoatilyticus JA2. Aerobic cultures of strain JA2 produced brown pigment when grown on L-tryptophan-containing media. Purified pigment showed typical physico-chemical properties of melanin. Further, extensive spectroscopic studies revealed that pigment is an amorphous, indole-type polymer with stable free radical centers. Further, hydrolysis of the brown pigment revealed the presence of indole moiety, confirming the indolic nature of the pigment. Demonstration of in vitro and in vivo pigment synthesis directly from L-tryptophan or hydroxytryptophan confirms tryptophan-based melanin synthesis in strain JA2. Interestingly, canonical melanin biosynthetic inhibitors did not affect the pigment synthesis indicating possible non-canonical tryptophan-based melanin biosynthesis in strain JA2. Further, the exometabolite profiling and precursor feeding studies suggests that L-tryptophan converted to hydroxytryptophan/hydroxyindoles and their subsequent polymerization lead to the formation of melanin. The current study sheds light on biosynthetic diversity of melanins and L-tryptophan can be a potential precursor for melanin synthesis in life forms.

Figure 1

Growth, tryptophan utilization and brown pigment production by Rubrivivax benzoatilyticus JA2 under aerobic conditions. Values are the mean ± standard deviation of two biological replicates.

Figure 2

Image showing the brown pigment in acidified culture supernatant obtained from tryptophan-amended aerobic culture of strain JA2 (A), Dry purified pigment (B), SEM micrograph showing aggregated granules of pigments (C) and dispersed individual spherical granules of pigment (D).

Figure 3

Spectroscopic characterization of purified brown pigment. (A) UV-Visible spectrum; (B) FTIR spectrum; (C) ESR spectrum; (D) X-Ray Diffraction spectrum.

Figure 4

Nuclear Magnetic Resonance spectra of purified brown pigment. (A) Solid-state CPMAS ¹³C and (B) ¹⁵N NMR spectra.

Figure 5

Brown pigment production by strain JA2 in the presence of various inhibitors and substrates. (A) Effect of canonical melanin specific biosynthetic pathway inhibitors on brown pigment production under tryptophan-amended aerobic conditions. (B) Brown pigment production by strain JA2 grown in the presence of tryptophan and 5-hydroxytryptophan. Values are the mean ± standard deviation of three biological replicates. **P- value <0.005; ***P-val <0.0005 compared to Trp supplemented cultures, calculated using unpaired t-test. Trp, tryptophan; OH-trp, hydroxytryptophan; Glyph, glyphosate: KA, kojic acid; Quer, quercetin; Na-Az, sodium azide.

Figure 6

In vitro formation of brown pigment in the presence of tryptophan and 5-hydroxytryptophan. (A) In vitro pigment formation. (B) Time-dependent in vitro pigment formation. Figure shows UV spectra of pigment obtained from in vivo and in vitro assays from Trp, Inserts shows in vitro pigment formation.

Figure 7

Schematic representation showing melanin synthesis from canonical precursors and non-canonical tryptophan-based melanin synthesis in strain JA2. Predicted trp-based melanin biosynthetic pathway in strain JA2. Metabolites in parenthesis are unknown hydroxyindoles involved in polymerization.