Metabolic Engineering of <i>Escherichia coli</i> for the Improved Malonic Acid Production

Malonic acid (MA) is a high-value chemical with diverse applications in the fields of food, agriculture, medicine, and chemical synthesis. Despite the successful biosynthesis of MA has been performed in <i><i>Escherichia coli</i></i>, <i>Myceliophthora thermophila</i>, and <i>Saccharomyces cerevisiae</i>, the resulting MA titers remain insufficient for industrial-scale production. In this study, three distinct metabolic pathways were designed and constructed to increase MA production in <i><i>E. coli</i></i>. Among these, the fumaric acid pathway comprising four key enzymes including the aspartase (AspA), the decarboxylase (PanD), the β-alanine-pyruvate transaminase (Pa0132), and the succinic aldehyde dehydrogenase (YneI) was identified as the most effective for MA production. Additionally, the supplementation of fumaric acid was found to significantly improve MA production. To further enhance the MA production, metabolic engineering strategies were employed, including the deletion of the <i>ydfG</i> gene, responsible for encoding the malonic semialdehyde reductase, and the <i>ptsG</i> gene, which encodes a glucose transporter. Finally, through the optimization of fermentation conditions and feeding strategies, the engineered strain achieved an MA titer of 1.4 g/L in shake flask and 17.8 g/L in fed-batch fermentation. This study provides new insights into the industrial-scale production of MA utilizing the metabolically engineered <i><i>E. coli</i></i> cells.