Silica Nanostructures Produced Using Diatom Peptides with Designed Post‐Translational Modifications

Abstract

Diatoms produce intricately-patterned silica structures under ambient conditions, a process initiated by post-translationally modified silaffin peptides that nucleate silicic acid. Designing these peptides would enable the production of silica nanostructures with desired properties; however, the functional effects of modifications are poorly understood. Here, Escherichia coli is used to express and modify recombinant silaffin R5 peptide from the diatom Cylindrotheca fusiformis. A library of 38 enzymes is tested for R5 modifications in vitro, from which active methyltransferases, kinases, acetyltransferases, oxidases, and myristoyltransferases are identified from diatoms, humans, yeast, and bacteria. Modified R5 peptides are used for silica precipitation and the impacts on particle size, shape, porosity, and surface area are quantified. In vivo pathways are designed to co-express R5 and a modifying enzyme and the resulting peptide is used to nucleate silica nanostructures with controlled size (100-3500 nm), porosity (20-635 m 2 g -1 ), or embedded with melanin. We find phosphorylation reduces the need for inorganic phosphate during silica synthesis. The simultaneous methylation and phosphorylation of R5 leads to smaller particles requiring less inorganic phosphate.Inspired by diatoms, the use of post-translationally modified peptides will enable the control of silica morphology under ambient conditions, with potential applications in electronics and photonics.

Genetic Tuning of Iron Oxide Nanoparticle Size, Shape, and Surface Properties in Magnetospirillum magneticum

“…[ 97,98 ] The silica‐nucleating silaffin R5 peptide (19 amino acids) from Cylindrotheca fusiformis has been produced recombinantly in E. coli and used to create engineered silica nanostructures. [ 99 ] Natural silaffin polypeptides are highly decorated with post‐translational modifications, but it has been shown that the lysines in unmodified R5 peptides bind to the substrate tetramethyl orthosilicate (TMOS) and are sufficient to catalyze silica precipitation. [ 99,100 ] We hypothesized that the R5 peptide could form a silica shell around nanoparticles.…”

“…[ 99 ] Natural silaffin polypeptides are highly decorated with post‐translational modifications, but it has been shown that the lysines in unmodified R5 peptides bind to the substrate tetramethyl orthosilicate (TMOS) and are sufficient to catalyze silica precipitation. [ 99,100 ] We hypothesized that the R5 peptide could form a silica shell around nanoparticles.…”

“…Cells are natural architects to build composite materials that can be rationally designed by combining pathways from different sources, as we have done to produce silica shells around the metal nanoparticles by incorporating proteins from marine eukaryotic diatoms. [ 99 ] Understanding the bio‐manufacturing of such materials, and the possible products, is still nascent, where metabolic and cost models have focused on carbon‐based products. While slow growing, there has been work with fermentation of magnetotactic bacteria and material properties can be controlled with media, growth conditions, and purification.…”

“…Purified R5 peptide was produced from recombinant E. coli , following the protocol described previously. [ 99 ] Lyophilized peptide was reconstituted at 5 m m in autoclaved Milli‐Q water. Silica reactions were performed in 44 µL liquid volumes in 1.5 mL microcentrifuge tubes (USA Scientific #1415‐2500).…”

Synthetic biology as driver for the biologization of materials sciences

“… Protein class Description Reference Silicatein A bioorganic–inorganic hybrid material is produced by the incorporation of nanoparticles into sponge spicules [ 73 ] Bacterial biofilm protein (CsgA), silaffin A diatom frustule-like structure is built by harnessing a curli biofilm protein, a chitin-binding domain, and a silaffin peptide, which together serve as a scaffold for silica mineralization. The construction of an artificial photosynthesis system is achieved upon colonization of the material with living cells [ 74 ] Silaffin Recombinant silaffin peptides from diatoms, which nucleate the formation of silica nanostructures, are produced in E. coli with different post-translational modifications, obtaining nanostructures with different properties [ 75 ] A genetically engineered diatom produces silaffin peptides fused to proteins of interest, resulting in the immobilization of the protein in the diatom silica [ 75 ] A genetically engineered diatom produces a silica matrix displaying antibodies on its surface. This material is applied for the targeted delivery of chemotherapeutic drugs [ 76 ] A genetically engineered diatom produces a silica matrix that incorporates proteins of interest.…”