We have developed a fluorescence-based metabolite sensor enabling in vivo detection of various aldehydes of biotechnological interest in Escherichia coli. YqhC is a transcriptional regulator that is known to be involved in the upregulation of the yqhD-dgkA operon in the presence of aldehydes. We took advantage of this property by constructing a bi-modular biosensor, in which a sensing module constitutively expresses yqhC while a reporter module drives the expression of the syfp2 reporter gene that is put under control of the yqhD promoter. The sensitivity of the sensor has been optimized by engineering the 5′-UTRs of both the sensing and the reporter modules resulting in a 70-fold gain of fluorescence in response to the model compound glycolaldehyde at 5 mM. The optimized sensor further responded to other aldehydes when supplemented to the cultivation medium at concentrations of 1–10 mM. We furthermore showed that this metabolite sensor was functional in vivo as it responded to the presence of glycoladehyde that is specifically produced upon induction of a synthetic xylulose-1-phosphate pathway expressed in E. coli. This bi-modular sensor can therefore be employed as an exquisite tool for FACS-based ultra-high-throughput screening of aldehyde (over) producing enzymes.

A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s−1), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (kcat/Km) between natural and synthetic OHB substrates (84 and 31 s−1 mM−1, respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis.