Decoupling cellular complexity: Comparative mass transport analysis of wild-type and mutant PhoE in nanoreactors

Abstract Precise control of substrate transport across compartment boundaries is critical for the design of enzymatic nanoreactors, yet selective and tunable molecular flux remains a major bottleneck in confined reaction systems. Outer membrane porins offer a promising solution, as their transport properties can be modulated through protein engineering. Among these, the moderately anion-selective porin PhoE from Escherichia coli is an attractive candidate due to its defined pore architecture. Several PhoE mutants have previously been described and analyzed, predominantly in whole-cell assays. However, such systems limit the quantitative resolution of substrate transport, as specific flux cannot easily be disentangled from expression levels, membrane insertion efficiency, and variations in the amount of functional protein. To overcome these limitations, we employed enzyme-loaded nanoreactors as a minimal and well-defined platform to directly quantify PhoE-mediated substrate flux independent of cellular complexity. Wild-type PhoE was compared to mutants exhibiting distinct transport behaviors in bacterial cells, with F111C showing approximately 50% lower transport. Additionally, we designed and characterized the novel deletion mutant PhoEΔ102–107, which exhibited 2.4-fold higher transport than wild-type PhoE. The bottom-up synthetic biology system enabled a direct comparison of specific transport rates, demonstrating the potential of these variants for transport tuning in engineered enzymatic nanoreactors. Complementary molecular dynamics simulations provided structural insight into the molecular basis of the observed transport behavior of the PhoE mutants.