Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation Generally, melanin is classified into five types-eumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelanin-based on the various chemical precursors used in their biosynthesis Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melanin-based materials, while the synthetic analogues of other melanins have received far less attention In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure-function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials

Unraveling the Structure and Function of Melanin through Synthesis.

Microbial melanin: Recent advances in biosynthesis, extraction, characterization, and applications.

The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applications - from speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review, we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolism - key physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.

Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis

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.

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

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m2/g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

The fast-growing nonmodel marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order to further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences, and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that, while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this nonmodel chassis.

Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens.

Formaldehyde-free sugarcane-derived aldehydes to co-crosslink silk fabrics with diols for improving wet resilience and minimizing pollutions from wet processing of silk

Erin E. Kelly

Papers

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

Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens.

Formaldehyde-free sugarcane-derived aldehydes to co-crosslink silk fabrics with diols for improving wet resilience and minimizing pollutions from wet processing of silk