TY - JOUR

T1 - Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization

AU - Zedler, Julie A.Z.

AU - Schirmacher, Alexandra M.

AU - Russo, David A.

AU - Hodgson, Lorna

AU - Gundersen, Emil

AU - Matthes, Annemarie

AU - Frank, Stefanie

AU - Verkade, Paul

AU - Jensen, Poul Erik

N1 - Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023

Y1 - 2023

N2 - 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 Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis 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.

AB - 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 Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis 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.

KW - cyanobacteria

KW - encapsulation peptide

KW - nanofilament

KW - PduA

KW - protein scaffold

KW - Synechococcus elongatus UTEX 2973

KW - Synechocystis sp. PCC 6803

U2 - 10.1021/acsnano.3c08600

DO - 10.1021/acsnano.3c08600

M3 - Journal article

C2 - 38065569

AN - SCOPUS:85180116039

VL - 17

SP - 25279

EP - 25290

JO - A C S Nano

JF - A C S Nano

SN - 1936-0851

IS - 24

ER -