An Engineering Approach for Rewiring Microbial Metabolism

The introduction of synthetic pathways into microbes often requires substantial modifications of the host metabolism. Here, we present and discuss key experimental aspects required for modifying microbial central metabolism. We introduce the concept of dividing pathways into metabolic modules, the activity of which can be selected for and optimized in dedicated gene-deletion strains. We provide a comprehensive methodology for systematic pathway implementation in vivo, ranging from gene-deletion methods for the creation of selection strains to cloning strategies that allow fine-tuned expression of individual pathway enzymes in synthetic operons. We further describe pathway testing and validation via high-throughput growth experiments and ¹³C-labeling measurements. While we focus on Escherichia coli as bacterial host, the holistic approach we present could be easily adapted for the metabolic engineering of other microbes.