Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization.

Abstract

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts, but so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium <i>Synechocystis</i> sp. PCC 6803 and the fast-growing cyanobacterial strain <i>Synechococcus elongatus</i> UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of <i>pduA*</i>, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in <i>S. elongatus</i> UTEX 2973 and shorter filamentous structures in <i>Synechocystis</i> sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly had only a minor impact on cellular metabolism. Finally, as a proof-of-concept for co-localization with the filaments, we targeted a fluorescent reporter protein, mCitrine, to PduA* by fusion with an encapsulation peptide that natively interacts with PduA. The establishment of nanofilaments in cyanobacterial cells is an important step toward cellular organization of heterologous pathways and the establishment of cyanobacteria as next-generation hosts.