Identification of 2-keto-3-deoxy-D-xylonate and 2-oxo-4-hydroxybutyrate as natural and artificial effectors of the transcription factors XynR and YjhI, respectively, and application to the development of biosensors for the bioproduction of 2,4-dihydroxybutyric acid in E. coli

In E. coli , D-xylonate catabolism is carried out by two distinct operons, namely yagEFG and yjhIHG , present on two distinct cryptic phages in the genome of this bacterium. These two operons are controlled by the transcription factors XynR and YjhI, the former acting as a repressor of yagEFG while the latter acts as an activator of yjhIHG . Although D-xylonate is known to induce these two operons, the effective inducer remains unknown to date. Through the construction of biosensors based on XynR and YjhI using syfp2 as a reporter gene, we showed that the true natural effector of the two D-xylonate catabolic operons is 2-keto-3-deoxy-D-xylonate, which is formed by dehydration of D-xylonate catalyzed by the dehydratases encoded by yagF and yjhG . Building on the finding that these two operons were also upregulated in E. coli strains producing the non-natural platform molecule 2,4-dihydroxybutyric acid, we discovered that both XynR- and YjhI-based biosensors were responsive to the non-natural molecule 2-oxo-4-hydroxybutyric acid, harboring comparable characteristic performances in term of response threshold, sensitivity, cooperativity and dynamic response as the natural effector 2-keto-3-deoxy-D-xylonate. Given that the enzymatic steps involved in the production of this non-natural metabolite from C2 and C5/C6 carbons, catalyzed respectively by threonate dehydratase and homoserine transaminase, constitute bottlenecks in the synthetic pathways for the production of 2,4-dihydroxybutyric acid, we showed that the transcription factors XynR and YjhI can be repurposed as biosensors to select more active variants of these enzymes, thereby improving the production of this platform molecule from carbon sources.