Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis

Structural insight into the Clostridium difficile ethanolamine utilisation microcompartment.

Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen

Predicting the Loading of Virus-Like Particles with Fluorescent Proteins

The virus-like particle (VLP) of the Cowpea Chlorotic Mottle Virus (CCMV) has often been used to encapsulate foreign cargo. Here we show two different rational design approaches, covalent and noncovalent, for loading teal fluorescent proteins (TFP) into the VLP. The covalent loading approach allows us to gain control over capsid loading on a molecular level. The achieved loading control is used to accurately predict the loading of cargo into CCMV VLP. The effects of molecular confinement were compared for the differently loaded VLPs created with the covalent method. We see that the loading of more than 10 fluorescent proteins in the 18 nm internal cavity of the CCMV capsid gives rise to a maximum efficiency of homo-FRET between the loaded proteins, as measured by fluorescence anisotropy. This shows that already at low levels of VLP loading molecular crowding starts to play a role

Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ϕX174 in Escherichia coli

Abstract Background Recombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ϕX174 inside recombinant BMCs to enhance its expression and achieve higher yields during downstream purification. Results E was fused with various N-terminal BMC targeting tags (PduP-, PduD-, and EutC-tags, 18–20 amino acids) and co-expressed with appropriate BMC shell proteins that associate with the tags and are required to form BMCs. Only BMC targeted E fusions, but not non-tagged E, could be successfully cloned, suggesting that the BMC tags reduce the toxicity of E. A PduP-tagged E system appeared to achieve the highest expression of E. Co-expression of Pdu BMC shell proteins with PduP-E increased its expression by 20–50%. Affinity purification of PduP-E via Ni–NTA in the presence of Empigen BB detergent yielded 270 µg of PduP-E per L of induced culture. Removal of the PduP-tag via proteolysis resulted in a final yield of 200 µg of E per L of induced culture, a nearly order of magnitude (~sevenfold) improvement compared to prior reports. Conclusions These results demonstrate improved expression of ϕX174 lysis protein E via re-directed BMC systems and ultimately higher E purification yields. Similar strategies can be used to enhance expression of other toxic proteins in recombinant Escherichia coli systems