Optimizing Heterologous Expression of Silicatein for Increased Biomineralization of Metal Oxide Nanoparticles

Biomineralization is a process by which biological systems produce and control the formation of inorganic materials with exquisite precision. Silicatein, a biomineralizing protein from sea sponges, represents a promising enzyme-based alternative to chemical synthesis of metal oxides such as nano cerium oxide by directly controlling the size and morphology of nanoparticles at ambient temperature and in aqueous environments. In addition to the direct implementation of the enzyme in the scale-up and production of ordered nanostructures manufactured sustainably, work to advance understanding of the mechanism and structure of silicatein will inspire more energy-efficient and eco-friendly synthesis methods in other industrial areas. However, the production of recombinant silicatein has presented challenges in solubility and intrinsic activity that severely limit its potential as an industrial catalyst. Currently, at least 90% of the enzyme produced is insoluble and virtually unrecoverable, and silicatein-mediated production of nanoceria requires 24 to 48 hours at the maximum achievable aqueous enzyme concentration of 4 μM. This work aimed to increase silicatein solubility by exploring different cosolutes for enhanced purification from insoluble fractions as well as genetic fusions for enhanced intrinsic solubility, and both prokaryotic and eukaryotic host organisms for production to address silicateins insolubility. Although purification optimization using cosolutes did not yield improved results, preliminary screens suggest an approximately 10-fold increase in soluble silicatein when fused to a trigger factor tag or enhanced green fluorescent protein. We are also the first to demonstrate that silicatein may be made recombinantly in the yeast Pichia pastoris. By demonstrating significantly improved solubility of silicatein, we have developed a platform for further work on enhancing the enzyme’s activity and scalability via protein engineering and directed evolution.Through such improvements, silicatein may prove a viable alternative to achieve industrial-scale synthesis of nanoparticles with precisely controlled size distributions in an energy-efficient and environmentally friendly way.