Deep scanning lysine metabolism in Escherichia coli

Our limited ability to predict genotype–phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype–phenotype relationships at a single‐protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR‐EnAbled Trackable genome Engineering to attempt a parallel and high‐resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism in Escherichia coli . We designed over 16,000 mutations to perturb this pathway and mapped their contribution toward resistance to an amino acid analog. By doing so, we identified different routes that can alter pathway function and flux, uncovering mechanisms that would be difficult to rationally design. This approach sets a framework for forward investigation of complex multigenic phenotypes. Synopsis Genotype—phenotype mapping is used to allow a high‐resolution interrogation of different routes that alter lysine metabolism and confer resistance to an antimetabolite analog. This approach narrows the search space down to specific targets to guide engineering efforts. 19 genes affecting lysine metabolism are investigated for their contribution toward resistance to the antimetabolite AEC in E. coli , totaling 16,300 mutations in the pathway. Loss‐of‐function mutations in the transporter are found to be the dominant selection winner. CRISPR‐EnAbled Trackable genome Engineering (CREATE) allowed exploring beyond the dominant selection winner, identifying mutations in biosynthetic genes and regulatory proteins that confer resistance by overproduction of lysine. Graphical Abstract Genotype—phenotype mapping is used to allow a high‐resolution interrogation of different routes that alter lysine metabolism and confer resistance to an antimetabolite analog. This approach narrows the search space down to specific targets to guide engineering efforts.