Genetically Programmed Regioselective Immobilization of Enzymes in Biosilica Microparticles

Abstract

Diatoms are single‐celled microalgae that produce a large variety of hierarchically porous, silica‐based microparticles as cell wall material. The presence of genetically encoded silica nanopatterns endows the biosilica with favorable properties for a wide range of applications including catalysis, chemical sensing, photonics, and drug delivery. Enhancing the performance of diatom biosilica requires i) a better understanding of the structure–property relationship in this material, and ii) methods that enable the manipulation of the biosilica structure and properties in a targeted manner. Here, genetic engineering of the diatom Thalassiosira pseudonana is employed to immobilize enzymes (glucose oxidase and horseradish peroxidase) into structurally distinct regions of the biosilica, which are termed valves and girdle bands. Remarkably, glucose oxidase in girdle bands exhibits >3‐fold higher catalytic activities compared to its location in valves. It is demonstrated through enzyme accessibility studies, protein engineering, and genetic engineering of biosilica morphology that the divergent enzyme activities are caused by the differences in the inherent silica nanopatterns of valves and girdle bands. This work highlights the importance of silica nanoscale architecture for the activity of immobilized enzymes and provides unprecedented tools for the biotechnological production of silica microparticles with tailored catalytic activities and anisotropic functionalities.

An intimate view into the silica deposition vesicles of diatoms

“…In the plasmid expression of the ccSin1-GFP fusion gene is under control of the endogenous ccSin1 regulatory sequences. The biolistic transformation of C. cryptica and the selection of nourseothricin resistant transformants was performed as described previously [54].…”

Computational analysis of the effects of nitrogen source and sin1 knockout on biosilica morphology in the model diatom Thalassiosira pseudonana

“…Recently, a genetic engineering based method called live diatom silica incorporation (LiDSI) was established that allows for generating diatom strains, which contain desired fluorescent proteins, receptor proteins, or enzymes in the biosilica [31]. While biosilica functionalization using LiDSI is well developed and even enables regioselective incorporation of proteins [32], the genetic engineering of biosilica structure is still in its infancy. Only quite recently, the first genetically induced morphological mutants were generated in diatoms through knockdown and knockout of specific genes [15,17].…”

Shedding light on biosilica morphogenesis by comparative analysis of the silica-associated proteomes from three diatom species

“…pseudonana and C. cryptica, because transformation experiments using the protocols for both species (Poulsen et al, 2006;Kumari et al, 2020) did not succeed in obtaining transgenic clones when applied to T. oceanica. Schemes showing the position of the GFP tag for each protein are shown in Fig.…”