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

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 (OD₆₀₀ = 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.