2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.