Engineering Plasmid‐Free Escherichia coli via Synergistic Metabolic Tuning and Fermentation Optimization for High‐Titer Salidroside Biosynthesis

ABSTRACT Salidroside, a major bioactive component of Rhodiola rosea , exhibits diverse pharmacological activities and broad applications, but faces biosynthesis challenges. Herein, we developed a synergistic strategy to construct a high‐level plasmid‐free salidroside production strain W6U4. First, multi‐copy genomic integration of the phenylpyruvate decarboxylase mutant ARO10 D331C and glycosyltransferase UGT85A1 was performed to enhance precursor tyrosol synthesis and glycosylation efficiency. Subsequently, systematic metabolic engineering was applied to redirect metabolic flux: reinforcing the pentose phosphate and shikimate pathways and eliminating competing pathways, which boosted salidroside titer to 2.63 g/L in shake flasks. Finally, optimized fed‐batch fermentation in a 5‐L bioreactor with two‐stage temperature control (37°C for growth, 30°C for induction) and early log‐phase induction (OD 600 = 15) resulted in 33.68 g/L salidroside, the highest reported titer to date, with only 0.40 g/L residual tyrosol. This integrated strategy establishes an efficient, stable microbial platform for salidroside production, highlighting the synergistic effect in advancing industrial‐scale biosynthesis.