Rapid, high-titer biosynthesis of melanin using the marine bacterium Vibrio natriegens

Abstract

Melanin is one of the most abundant natural biomolecules on Earth. These macromolecular biopolymers display several unique physical and chemical properties and have garnered interest as biomaterials for various commercial and industrial applications. To this end, extensive research has gone into refining methods for the synthesis and extraction of melanin from natural and recombinant sources. In this study, we developed and refined a procedure using a recombinant microbial system for the biosynthesis of melanin using the tyrosinase enzyme Tyr1 and tyrosine as a substrate. Using the emergent microbial chassis organisms Vibrio natriegens, we achieved maximal yields of 7.57 g/L, and one of the highest reported volumetric productivities of 473 mg L^-1 h^-1 with 100% conversion rates in an optimized, minimally defined medium. Additionally, we identified and investigated the use of a native copper responsive promoter in V. natriegens for stringent regulation of heterologous protein expression as a cost effective alternative to traditional IPTG-based induction. This research represents a promising advancement towards a green, rapid, and economical alternative for the biomanufacture of melanin.

Keywords: Vibrio natriegens; biomanufacturing; copper induction; melanin; melanin biosynthesis; tyrosinase; tyrosine.

FIGURE 1

Schematic portraying the enzymatic and spontaneous reactions for biosynthesis of eumelanin starting from L-tyrosine as a substrate and intermediates along the biosynthetic pathway. The conversion of DOPA to dopaquinone can be either enzymatic via a tyrosinase (i.e. Tyr1 or MelA) or can occur spontaneously in the presence of oxidants [O] such as molecular oxygen. All enzymatic steps along the pathway require molecular oxygen (O₂) for the initial oxidation reactions. DOPA–3,4-dihydroxyphenylalanine; DHICA–5,6-dihydroxyindole-2-carboxylic acid.

FIGURE 2

(A) Image of melanin producing E. coli BL21 (DE3) cells transformed with the IPTG-inducible pJV plasmid expressing the indicated enzymes. Cultures were induced with 1 mM IPTG, supplemented with 2 mg/ml tyrosine, and allowed to incubate for 24 h at 37 °C. The visible dark, black pigmentation is characteristic of melanin formation. (B) Visualization of melanin formation in cell-free culture supernatants from pJV-Tyr1 expressing E. coli BL21 (DE3) cells. Melanin formation was monitored spectrophotometrically by recording absorbance values at 492 nm. Time points above indicate time post-induction with IPTG and addition of tyrosine.

FIGURE 3

(A) Optical density readings (OD₆₀₀) of recombinantly expressed HpaBC in E. coli. Cultures were grown in M9 (blue traces) or LB (black traces) media and HpaBC was expressed constitutively from the pCB1D5 plasmid (dashed lines) or induced by 1 mM IPTG using the pJV plasmid (solid lines). Measurements were taken from control cultures not supplemented with tyrosine. (B) Absorbance readings at 492 nm (A₄₉₂) for the detection of melanin from recombinantly expressed HpaBC in E. coli as in Figure 2A. Cultures were supplemented with 2 mg/ml tyrosine as substrate. A₄₉₂ readings taken on cell-free culture supernatants after centrifugation. Measurements taken from three independent biological replicates. (C) HpaBC SDS-PAGE protein expression analysis of E. coli soluble cell extracts with the indicated plasmids in either M9 (left) or LB (right) media. For all figures, indicated time points are post-induction with IPTG (for pJV plasmids) and after addition of substrate trysosine. 20 µg total protein loaded in each lane.

FIGURE 4

Analysis of growth profile (A), melanin production (B), and protein expression (C) as in Figure 3, but for recombinantly expressed Tyr1 in E. coli with either the constitutive pCB1D5 or inducible pJV plamsids in M9 or LB medium as indicated.

FIGURE 5

(A) Growth curves of V. natriegens in VnM9v2 vs. LBv2 recorded on a Bioscreen (C). Traces represent averages of three biological replicates. (B) Measurement of V. natriegens cellular biomass in VnM9v2 vs. LBv2 media from 50 ml overnight shaking culture flasks. (C) Quantitation of melanin production of V. natriegens pJV-Tyr1 cells supplemented with 2 mg/ml tyrosine and 40 µM CuSO₄. Biological triplicates were used for all studies. Error bars represent standard deviation of the mean. *p < 0.05; ***p < 0.005.

FIGURE 6

V. natriegens cultures in VnM9v2 medium expressing either Tyr1 (black traces) or HpaBC (blue traces) in the pJV plasmid after induction with IPTG and supplementation with tyrosine and CuSO₄ (for Tyr1 only). (A) OD₆₀₀ culture density measurements taken from control cultures not supplemented with tyrosine. (B) Melanin production by measuring A₄₉₂. Measurements were taken from cell-free culture supernatants after centrifugation. Readings were taken on biological triplicates. (C) Protein expression profiles of HpaBC and Tyr1 after induction with 1 mM IPTG. Values above each lane represent hour post-induction.

FIGURE 7

Using disodium tyrosine as a substrate significantly increased melanin biosynthetic yields due to increased substrate solubility. (A) Images of overnight expression cultures of V. natriegens supplemented with the indicated concentrations of disodium tyrosine and incubated at either 37 °C (top) or 30 °C (bottom). Black pigmentation is indicative of melanin biosynthesis. (B) Quantification of melanin extracted from V. natriegens cultures supplemented with 8 g/L disodium tyrosine and incubated at 30 °C. Average total yields were 7.40 g/L. Representative of three biological replicates. Error bars represent standard deviations of the mean.

FIGURE 8

V. natriegens biosynthesis of melanin in 100 ml VnM9v3 medium using small-scale bioreactors. Reported yields and product conversion rates are listed below for each reactor.

FIGURE 9

(A) Bioscreen C growth curves of V. natriegens cultures in VnM9v2 medium supplemented with increasing concentrations of CuSO₄. (B) qRT-PCR analysis of tyr1 transcript induction using the indicated concentrations of IPTG or CuSO₄ for the pJV-Tyr1 or pCopA-Tyr1 plasmids, respectively. Transcript abundances were normalized to the housekeeping gene gapA. Error bars represent standard deviations of the mean. ***p < 0.005. (C) Copper induction and melanin biosynthesis with the copA promoter. V. natriegens cultures containing the indicated plasmids were supplemented with 100 µM CuSO₄ and/or 2 mg/ml disodium tyrosine (Na₂Tyr) as indicated and incubated overnight, or up to 48 h for the pCopA-Tyr1 strains. Values below each culture represent absorbance values at 492 nm from cell free culture supernatants for measurement of melanin formation.