(588ci) Systematic Design of Auxotrophic Strains and Media Conditions to Probe Metabolic Functions in E. coli

Despite progress in automated gene annotation, many deficiencies and knowledge gaps remain, even for well-studied organisms. Of particular concern is the accuracy and detail of annotations for transporters of various organic substrates and products of metabolism and for enzymes that do not share sequence homology with well-characterized ones. Unfortunately, annotation errors present in earlier genome-scale metabolic (GSM) models propagate to newer models with few opportunities for later correction. Here, we introduce a systematic computational procedure that applies GSM models of Escherichia coli to design auxotrophic gene deletion strains that can grow in glucose but fail to grow on different carbon substrate(s) unless rescued with the addition of an ORF encoding a rescue function. Using the E. coliGSM model supplemented with regulatory rules that quantify grow/no grow outcomes on different organic substrates, we identified 252 distinct auxotrophic designs for which specific single functions can uniquely complement them. We envision that this collection of auxotrophic strains can be used to disambiguate the metabolic role of unannotated or poorly annotated genes.