The enormous complexity of metabolic pathways, their regulation and crosstalk create major obstacles for metabolic engineering, because small changes made to the system, by introducing a new metabolic pathway for example, can often have unpredictable consequences. Thus, effective production strains need to go through many rounds of time-consuming optimization.

For metabolic engineering to become most effective, ideally an autonomous operating metabolic module is introduced decoupled from the cell’s regulatory and metabolic networks. Self-assembling, easy-to-modify and interspecies transferable Bacterial microcompartments (BMCs) are promising scaffolds for the next generation of metabolic engineering addressing the current challenges of containing toxic intermediates, minimizing metabolic crosstalk and creating a favorable microenvironment within compartment.

We have developed a comprehensive tool kit to produce synthetic BMC shells in E. coli, load these in vivo with multiple heterologous cargo enzymes and measure the BMC shell molecular permeability. These enabling tools allowed us to construct a synthetic cellular organelle capable of producing pyruvate out of format and acetate. Application of BMC based synthetic cellular organelles enabling metabolic routes that wouldn’t be feasible without compartmentalization will be discussed. As an example, we will highlight New-to-Nature CO₂ fixation routes, promising to outperform Rubisco mediated CO₂ fixation.