Metabolic engineering of Escherichia coli for efficient production of d-phenylglycine

d -phenylglycine (D-PHG) is a valuable building block extensively used in the synthesis of β-lactam antibiotics and other high-value pharmaceuticals. However, its conventional chemical production relies on expensive starting materials and harsh reaction conditions, while existing biotechnological approaches remain constrained by low production efficiency. In the present work, we established a novel de novo biosynthetic pathway for D-PHG production from glucose in Escherichia coli by reconstructing a streamlined route from the native shikimate pathway intermediate phenylpyruvate. The pathway integrates 4-hydroxymandelate synthase, an FMN-dependent S -mandelate dehydrogenase that avoids H 2 O 2 formation, and a highly specific d -phenylglycine aminotransferase, enabling efficient D-PHG production. Systematic metabolic engineering strategies, including elimination of competing pathways, enhancement of precursor supply, optimization of amino group donor availability, and reinforcement of NADPH regeneration, increased the D-PHG titer to 6.98 g/L in shake-flask cultivation. Further scale-up in a 3-L fed-batch fermentation achieved 9.83 g/L, representing the highest reported titer for de novo D-PHG biosynthesis from glucose to date. This work establishes an efficient and sustainable biosynthetic route to D-PHG and provides a foundation for its large-scale industrial manufacturing.