D-glycerate is an attractive chemical for a wide variety of pharmaceutical, cosmetic, biodegradable polymers, and other applications. Now several studies have been reported about the synthesis of glycerate by different biotechnological and chemical routes from glycerol or other feedstock. Here, we present the construction of an Escherichia coli engineered strain to produce optically pure D-glycerate by oxidizing glycerol with an evolved variant of alditol oxidase (AldO) from Streptomyces coelicolor. This is achieved by starting from a previously reported variant mAldO and employing three rounds of directed evolution, as well as the combination of growth-coupled high throughput selection with colorimetric screening. The variant eAldO3-24 displays a higher substrate affinity toward glycerol with 5.23-fold than the wild-type AldO, and a 1.85-fold increase of catalytic efficiency (k_cat/K_M). Then we introduced an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 expression system in E. coli to overexpress the variant eAldO3-24, and deleted glucosylglycerate phosphorylase encoding gene ycjM to block the consumption of D-glycerate. Finally, the resulting strain TZ-170 produced 30.1 g/l D-glycerate at 70 h with a yield of 0.376 mol/mol in 5-l fed-batch fermentation.

Background: Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative. Results: This study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the −35 region and into the spacer region between the −10 and −35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 μM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 μM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants. Conclusions: The methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.