Silaffins as functional biomacromolecules in regulating frustule morphogenesis and biosilica properties

Diatoms synthesize silica cell walls (frustules) with genetically encoded nano- to micropatterned morphologies that surpass current synthetic chemistry. Silaffins, highly post-translationally modified peptides found in frustules, facilitate biosilica formation and regulate silica architectures in vitro, in coordination with long-chain polyamines. However, the precise roles of silaffins in diatom frustule morphogenesis remain unclear. This study investigates the morphological and functional impacts of TpSil1 and TpSil3 on diatom frustule in the model organism Thalassiosira pseudonana using gene overexpression and CRISPR/Cas9-mediated knockout approaches. The inability to generate biallelic TpSil3 knockout mutants suggests that TpSil3 may be essential, possibly leading to lethality upon complete knockout. In contrast, biallelic TpSil1 knockout mutants also disrupted TpSil2 due to high sequence homology. Morphological analysis revealed distinct roles for these proteins: TpSil3 regulates overall cell size and macropore (fultoportula) density, while TpSil1/2 primarily contributes to macropore morphogenesis; mesopore (cribrum pore) patterns, however, remained consistent across the mutants. Beyond morphology, genetic manipulation of silaffins significantly affected diatom physiology. Overexpression of silaffins increased cellular silicification, while knockouts reduced silica deposition but enhanced cell growth and photosynthetic efficiency. Moreover, these modifications altered the physicochemical and optical properties of bulk frustules, enhancing potential applications in hemostasis, catalysis and photonics. This study elucidates the role of silaffins in frustule morphogenesis, linking frustule-associated proteins to diatom physiology and frustule properties, and provides a framework for engineering nanostructured silica through synthetic biology.