Highly efficient biosynthesis of 2,4-dihydroxybutyric acid by a methanol assimilation pathway in engineered Escherichia coli

Abstract

2,4-Dihydroxybutyric acid (2,4-DHB) is an important fine chemical, which can be used as a precursor in the production of important chemicals. Methanol, as an attractive C1 compound, is an ideal nonfood sugar feedstock for biomanufacturing. In this study, we reported for the first time the biosynthesis of 2,4-DHB in engineered Escherichia coli from a mixture of methanol and glucose. In this process, methanol dehydrogenase was used to convert methanol into formaldehyde, and the key intermediate 2-keto-4-hydroxybutyric acid was synthesized through the condensation of formaldehyde and pyruvate derived from glycolysis by pyruvate-dependent aldolase, which was subsequently transformed into the target product 2,4-DHB by an enzymatic hydrogenation reaction. The new pathway demonstrated the feasibility of producing 2,4-DHB from methanol and glucose in E. coli. For improving the production of 2,4-DHB step by step, dehydrogenases and aldo–keto reductases with higher activity towards 2,4-DHB were screened, and the pyruvate degradation pathway genes were knocked out to enhance the flux from pyruvate towards 2,4-DHB biosynthesis, resulting in the strain DHB9 with about 10-fold improvement compared to the initial strain DHB4. Additionally, the methanol dehydrogenase MDH_Cn with higher enzymatic activity was obtained by screening. The mutant MDH_Cn (G48F) with improved activity in vitro was obtained by rational analysis of MDH_Cn, and the results showed that the enzymatic activity of MDH_Cn (G48F) increased 3-fold in vitro compared with that of the wild type. Finally, on the basis of the above work, the best engineered strain DHB13 was constructed by knocking out the gene (frmRAB) responsible for the conversion of formaldehyde to formic acid, and the target product 2,4-DHB was accumulated up to 14.6 g L⁻¹ in a 5 L bioreactor. To date, the highest concentration of 2,4-DHB has been achieved in this work, which is promising to make the bioprocess feasible from an economic perspective. Moreover, one glucose molecule and two methanol molecules can be bio-transformed into two 2,4-DHB molecules with 100% conversion of carbon atoms in theory, showing the high carbon atom economy of the novel synthetic metabolic pathway.