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In E. coli , the genes of the D-xylonate catabolic pathway are present in two distinct operons, yagEFG  and yjhIHG , which are part of two cryptic phages found in the genome of this bacterium. These operons are under the control of two transcription factors: XynR which acts as a repressor of yagEFG and yjhI which is an activator of yjhIHG . Although D-xylonate is known to induce these two operons, the true inducer has not been identified yet. Through the construction of biosensors based on xynR and yjhI , using syfp2 as reporter gene in combination with mutants of genes involved in D-xylonate catabolism, it has been demonstrated that the effector of these operons is 2-keto-3-deoxy-D-xylonate, which is formed by the dehydration of D-xylonate catalyzed by the dehydratases encoded by yagF and yjhG genes. Building on the finding that these two operons were also upregulated in E. coli challenged with high concentration of platform molecule 2,4-dihydroxybutyric acid, it is reported that both XynR- and YjhI-based biosensors were also responsive to the intermediate non-natural molecule 2-oxo-4-hydroxybutyric acid, with characteristic performances in terms of response threshold, sensitivity, cooperativity, and dynamic response comparable to those of the natural effector. Given that the bottlenecks in the production of 2,4-dihydroxybutyrate from C2 and C5/C6 carbon are D-threonate dehydratase and L-homoserine transaminase, respectively, which catalyze the formation of 2-oxo-4-hydroxybutyric acid from either D-threonate or homoserine, we showed that XynR and YjhI-based biosensors could be efficient tools for the screening and selection of more active enzymes producing this compound, thereby improving the production of 2,4-dihydroxybutyrate.

(L)-2,4-dihydroxybutyrate (DHB) is a versatile compound that can serve as a precursor for the synthesis of the methionine analog 2-hydroxy-4-(methylthio)butyrate and new advanced polymers. We previously implemented in Escherichia coli an artificial biosynthetic pathway for the aerobic production of DHB from glucose, which relies on the deamination of (L)-homoserine followed by the reduction of 2-oxo-4-hydroxybutyrate (OHB) and yields DHB by an enzyme-bearing NADH-dependent OHB reductase activity. Under aerobic conditions, using NADPH as a cofactor is more favorable for reduction processes. We report the construction of an NADPH-dependent OHB reductase and increased intracellular NADPH supply by metabolic engineering to improve DHB production. Key cofactor discriminating positions were identified in the previously engineered NADH-dependent OHB reductase ( E. coli malate dehydrogenase I12V:R81A:M85Q:D86S:G179D) and tested by mutational scanning. The two point mutations D34G:I35R were found to increase the specificity for NADPH by more than three orders of magnitude. Using the new OHB reductase enzyme, replacing the homoserine transaminase with the improved variant Ec.AlaC A142P:Y275D and increasing the NADPH supply by overexpressing the pntAB gene encoding the membrane-bound transhydrogenase yielded a strain that produced DHB from glucose at a yield of 0.25 mol DHB mol Glucose −1 in shake-flask experiments, which corresponds to a 50% increase compared to previous producer strains. Upon 24 h of batch cultivation of the most advanced DHB producer strain constructed in this work, a volumetric productivity of 0.83 mmol DHB L −1  h −1 was reached.

The fermentative production of the functional precursor 2,4-dihydroxybutyrate (DHB) enables sustainable synthesis of the methionine analogue hydroxy-4-(methylthio) butyrate, which is currently still produced from fossil fuels. In this work, we aimed to optimize the aerobic production of DHB from glucose through the synthetic malyl phosphate (MalP) pathway, which comprises the conversion of the natural TCA cycle intermediate malate into MalP and the subsequent reactions to yield malate semialdehyde (MalSA) and finally DHB. We first implemented the synthetic pathway in an engineered Escherichia coli strain previously reported to over-produce malate through the oxidative TCA cycle. However, DHB was only detected in trace amounts, while acetate and malate were secreted in high quantities. Subsequent construction of strains producing malate, but negligible amounts of acetate, revealed that an increased supply of malate alone is not sufficient for improved production of DHB. Instead, we discovered metabolic inefficiencies in the DHB pathway as we found that deleting the endogenous succinate semialdehyde dehydrogenase Sad, whose natural substrate is structurally similar to MalSA, strongly improved performance of the DHB pathway. Specifically, with the single knock-out of sad we could achieve a 3-fold increase in DHB production with a yield of 0.15 mol mol -1 compared to the wildtype host in shake flask experiments. With additional chromosomal expression of the mutant ppc K620S gene encoding the malate-insensitive phosphoenolpyruvate carboxylase under control of a weak constitutive promoter, we achieved a DHB yield of 0.22 mol mol -1 , which corresponds to 17% of the maximal yield under aerobic conditions.