Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application

Abstract

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as “melanin ghost” structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens. Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis. IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.

An Overview on Industrial and Medical Applications of Bio-Pigments Synthesized by Marine Bacteria

“…in protecting mammalian cells from UV irradiation. Results revealed 90% survival rate of HeLa cells in melanized cell culture [ 128 ]. In another report, Bio lip balm made from crude pigment (extracted from S. bellus sp.…”

Melanins as Sustainable Resources for Advanced Biotechnological Applications

“…[ 171–173 ] Melanins display paramagnetic character due to free radicals in their structures (e.g., semiquinone free radicals) which absorb microwaves under magnetic fields yielding spectra characteristic of the radical species present. EPR/ESR spectroscopy is therefore a potent method of studying melanins, with reports of its use for fundamental biochemistry studies (e.g., melanin type, [ 169,174–177 ] effect of pH [ 178–180 ] ), characterising melanins from different species including bacteria ( Rubrivivax benzoatilyticus JA2, [ 121 ] Vibrio natriegens , [ 181 ] Streptomyces cyaneofuscatus [ 182 ] ), yeast ( Cryptococcus neoformans [ 167 ] ), mushrooms ( Inonotus hispidus [ 183 ] ), black soldier flies ( Hermetia illucens [ 184,185 ] ), squid ( Loligo opalescens [ 175 ] ), cuttlefish ( Sepia officinalis [ 186 ] ), and cephalopod ink sacs from the Jurassic era. [ 187 ] Ionizing irradiation changes the EPR/ESR signals of fungal melanins due to changes in the electronic structure of the melanins, which informed a fascinating study of melanized fungal cells ( Wangiella dermatitidis , Cryptococcus Neoformans and Cladosporium Sphaerospermum ) which displayed increased growth relative to nonmelanized cells after exposure to ionizing radiation.…”

“…[ 356–360 ] Static light scattering (SLS) observes the average scattering intensity of a solution/suspension over a period of time, whereas dynamic light scattering (DLS) observes fluctuations of the scattered light over very short periods of time, offering insights into molecular weights of polymers and particle sizes (typically nanometer scale and upward). Light scattering has been used to study synthetic melanins (e.g., polydopamine [ 361,362 ] ), naturally occurring melanins in bacteria ( Vibrio natriegens [ 181 ] ), yeast ( Cryptococcus neoformans [ 167 ] ), fungi ( Aspergillus oryzae [ 363 ] ), mushrooms ( Inonotus hispidus [ 183 ] ), cuttlefish ( Sepia officinalis [ 362 ] ), and can be used to study the health of eyes for patients with various conditions (e.g., for patients with pigmentary dispersion glaucoma [ 364 ] ).…”

“…TEM uses a beam of electrons to illuminate samples and creates images from measurements of electrons that pass through very thin specimens (and contrast in images caused by differences in electron densities within different regions of the samples). [ 386,482,483 ] TEM has been used to examine melanins and materials containing melanins from a variety of sources including: synthetic melanins (based on DHICA/DHI, [ 496 ] l ‐DOPA, [ 236 ] DHN [ 231 ] ); bacteria ( Pseudomonas maltophilia , [ 497 ] Pseudomonas stutzeri , [ 234 ] Vibrio natriegens [ 181 ] ), yeast (Cryptococcus neoformans [ 167 ] ), fungi ( Aspergillus fumigatus , [ 484 ] Gaeumannomyces graminis var. graminis [ 498 ] ), human bone osteosarcoma cell lines in vitro [ 446 ] and human tissues ex vivo (e.g., aortic valves, [ 493 ] bone, [ 344,494 ] cartilage [ 170,499 ] ); and TEM equipped with EDX/EDS has been used to quantify the elemental composition of Al, C, Ca, Cl, Cu, Fe, O, P, Si, and Zn in human melanosomes in the eye ex vivo.…”

Marine Bacteria Is the Cell Factory to Produce Bioactive Pigments: A Prospective Pigment Source in the Ocean

“…Bacterial Melanin is a product of biosynthesis of Ltyrosine via the enzyme tyrosinase oxidizing it into L-3, 4dihydroxyphenylalanine which is further metabolized into dopachrome and finally converted to melanin by oxidoreduction where the color usually ranges from black-brown in color (El-Naggar and El-Ewasy, 2017). Traditional melanin nanoparticle synthesis usually takes around 12 h, but Wang et al (2020) demonstrated the production of melanin nanoparticle synthesis in bacteria within 30 min by controlling the growth of cell-free conditions by changing the salinity concentration of the medium and incubation period. This demonstrates the exciting potential of bacteria as melanin pigment factories and melanin has a broad range of usages such as anti-oxidative, anti-cancer, anti-bacterial, anti-viral properties also displaying thermal resistance, radiation damage by absorption of wide range electromagnetic spectrum, and even chemical resistance (Narsing Rao et al, 2017).…”