2,4-Dihydroxybutyric acid (DHB) is a promising C 4 platform compound for the synthesis of methionine analogues and biodegradable polymers. However, aerobic DHB production from glucose in Escherichia coli involves transient acetate overflow prior to product synthesis, which could be challenging for process scalability. Therefore, we engineered Escherichia coli K-12 MG1655 for optimized DHB production by replacing the phosphotransferase system mediated glucose uptake with the galactose permease GalP, coupled to ATP-dependent phosphorylation via endogenous glucokinase. In combination with targeted deletions of malate- and fumarate-consuming reactions, we obtained a strain with enhanced flux through the tricarboxylic acid (TCA) cycle and pentose phosphate pathway leading to improved NADPH availability and increased anaplerotic activity, as revealed by 13 C metabolic flux analyses. Deletion of the mdh gene encoding for the cytosolic malate dehydrogenase further promoted DHB formation. The resulting strain achieved DHB yields up to 0.20 mol mol -1 (2.43 g L -1 ), a 4-fold increase compared to the wildtype background (0.05 mol mol -1 , 0.60 g L -1 ), under aerobic conditions while suppressing acetate formation. Together, these results demonstrate that GalP-mediated glucose uptake and engineering of the TCA cycle provide a robust metabolic framework for efficient DHB biosynthesis and establish a foundation for further process and pathway development. • Replacement of PTS by GalP improves DHB production • TCA cycle engineering increases DHB yield • 13 C-MFA reveals altered central carbon flux distribution • Malate node controls flux toward DHB synthesis • Maximum DHB yield of 0.20 mol mol −1 achieved
Nguyen et al. (Wed,) studied this question.