Silaffins-Driven Genetic Engineering of Diatom Cell Walls: Insight into Biosilica Morphology and Nanomaterial Design

Abstract

Diatoms synthesize silica cell walls (frustules) with genetically encoded morphologies, ranging from nanopatterns to micropatterns, that far exceed current synthetic chemistry. Silaffins, a family of phosphoproteins undergoing complex post-translational modifications, have been isolated from frustules and shown to facilitate and regulate biosilica formation in vitro with long-chain polyamines. However, their particular role in frustule morphogenesis and functionality remains unclear. In this study, functions of two representative silaffins, TpSil1 and TpSil3, were investigated in the model organism Thalassiosira pseudonana using gene overexpression and CRISPR/Cas9-mediated knockout approaches. Due to high sequence homology, TpSil2 was concurrently disrupted in TpSil1 knockout strains, while the homozygous knockout of TpSil3 proved to be lethal. Quantitative morphological analysis revealed distinct yet complementary roles: TpSil3 governs both microscale overall size and mesoscale features, including macropore (fultoportula) density and mesopore (cribrum pore) pattern, whereas TpSil1/2 exclusively contribute to macropore morphogenesis and mesopore density. Overexpression of silaffins increased silica deposition, while knockouts exhibited reduced silicification but enhanced cell growth and photosynthetic efficiency. Furthermore, these genetic modifications significantly influenced the physicochemical and optical properties of bulk frustules, potentially enhancing the hemostatic, catalytic and photonic performances, thereby positioning them as versatile candidates for a wide range of biotechnological and industrial applications. Collectively, our findings elucidate the distinct roles of TpSil1/2 and TpSil3 in diatom physiology and frustule morphology, highlighting a promising pathway for engineering nanostructured silica materials with tailored properties through synthetic biology.