Cofactor engineering for improved production of 2,4-dihydroxybutyric acid via the synthetic homoserine pathway

Nadine Ihle ¹ Ceren ALKIM ² Laura Grüßner ¹ Stefanie Tuyet Anh Nguyen ¹ Thomas Walther ¹ Cláudio José Remédios Frazão ^1*

• ¹ Technical University Dresden, Dresden, Germany
• ² Institut Biotechnologique de Toulouse (INSA), Toulouse, France

L)-2,4-dihydroxybutyrate (DHB) is a versatile compound which can serve as a precursor for the synthesis of methionine analogue 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) yielding DHB by an enzyme bearing NADH-dependent OHB reductase activity. As under aerobic conditions the utilization of NADPH as cofactor is more favorable for reduction processes, we here report the construction of a NADPH-dependent OHB reductase and the increase of intracellular NADPH supply by metabolic engineering for improving 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 over 3-orders of magnitude. Using the new OHB-reductase enzyme, replacing the homoserine transaminase by 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 which produced DHB from glucose at a yield of 0.25 molDHB molGlucose -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 mmolDHB L -1 h -1 was reached.