Evidence for Improved Encapsulated Pathway Behavior in a Bacterial Microcompartment through Shell Protein Engineering

Abstract

Bacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2-propanediol, likely by controlling diffusion of one or more metabolites. This study highlights the use of two strategies to engineer microcompartments to control metabolite transport: altering the existing shell protein pore via mutation of the pore-lining residues, and generating chimeras using shell proteins with the desired pores.

Bacterial microcompartments

“…This is consistent with studies that show changes in BMC function when residues surrounding the pore are mutated 32,33 . The double-stacking BMC-T d proteins contain a relatively large pore (12–14 Å) that can be open or closed depending on the conformation of the surrounding sidechains 28–31 .…”

“…There are two subtypes of BMC-T proteins: a single trimer form, (BMC-T s ) and a second type in which two trimers dimerize across their concave faces, referred to as ‘double ’ BMC-T d proteins (Figure 3A). A pore, typically formed at the central symmetry axis of hexamers and pseudohexamers, serves as a channel for metabolites to traverse the shell 27, 29, 33–35 .…”

“…Mutations of the residues that flank the pores of shell proteins from the PDU metabolosome 32, 33 and carboxysomes 145 showed that altering the size and charge of the pore to influence permeability does not impair shell formation. This provides the basis for the further rational design of channels that are specific to desired metabolites that are not native to natural BMCs; this is a key design requirement as the shell serves as the interface between the lumen of the BMC and its cellular environment.…”

“…For example, chimeric shells were obtained by replacing the major shell protein CcmK2 of β-carboxysomes with the CsoS1 protein of α-carboxysomes 145 , and EutM was successfully integrated into a PDU BMC 33 . Likewise, chimeric shells were produced by the simultaneous expression of the pdu and eut operons 149 , and a β-carboxysome shell protein (CcmO) has been incorporated in the PDU BMC 149 , highlighting the potential use of BMC shell proteins as generic structural building blocks (Figure 5).…”

An estimate is worth about a thousand experiments: using order-of-magnitude estimates to identify cellular engineering targets

“…Controlling expression of MCP genes enables increased culture density [46]. Further, controlling permeability of the protein shell to metabolites changes the concentration of substrates in the MCP [45, 47], enabling operation at substrate-saturating regimes. Finally, the loading of enzymes in the MCP can be modulated via the targeting sequence and expression levels [48], and the low fractional volume of enzymes in MCPs enables modulation of enzyme loading.…”