Biology subjects

Kerfeld, C.

Publications and source records attributed to Kerfeld, C..

3 recordsLinked to original sources

bioRxiv2026

Multi-lab, Multi-enzyme Study Demonstrates the Versatility of Bacterial Microcompartment Shells as a Modular Platform for Confined Biocatalysis

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d34f8dorg.highwire.dtl.DTLVardef@10bba9corg.highwire.dtl.DTLVardef@10868b7org.highwire.dtl.DTLVardef@112cb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

A chaotrope-based approach for rapid in vitro assembly and loading of bacterial microcompartment shells

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol, and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here we describe the development of a new, efficient, and rapid method for in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly, and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

synthetic biology↗

bioRxiv2023

The phycobilisome linker protein ApcG interacts with photosystem II and regulates energy transfer to photosystem I in Synechocystis sp. PCC 6803

Photosynthetic organisms harvest light using pigment-protein super-complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these super-complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show that the newly discovered PBS linker protein ApcG interacts specifically with photosystem II through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG exhibit reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with photosystem II is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with thylakoid membrane suggesting a regulatory role mediated by phosphorylation of ApcG. Low temperature fluorescence measurements showed increased photosystem I fluorescence in apcG deletion and complementation strains. The photosystem I fluorescence was the highest in the phospho-mimicking complementation strain while pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and implies that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

plant biology↗