Directed Evolution of Silicatein Reveals Biomineralization Synergism between Protein Sequences.

Abstract

Biomineralization is a green synthesis route for a variety of metal nanoparticles. Silicatein is a biomineralization protein originally found in marine sponge Tethya aurantia that converts inorganic precursors to metal oxide nanoparticles. In this work, we investigate the popular catalytic triad hypothesis and implement directed evolution with the aim to improve the solubility and kinetics of silicatein to enable increased nanoparticle synthesis. Site-directed mutagenesis with catalytic triad residues did not abolish biomineralization activity, aligning with the results seen in one previous study. Recombinant production of silicatein and mutants in Escherichia coli following library generation and a survival screen yielded several mutant proteins with augmented biomineralization activity. Sequence analysis of these mutant proteins reveals multiple sequences within a single cell that contribute to enhanced biomineralization. Combined with the sequence analysis of silicateins from different marine sponges, these results suggest the protein is permissive to wide sequence variations and that multiple protein sequences act synergistically for enhanced biomineralization.