Fructose-Driven Biosynthesis of 5-Aminolevulinic Acid in the Engineered Probiotic <i>Escherichia coli</i> Nissle 1917

5-Aminolevulinic acid (5-ALA) is a versatile precursor in tetrapyrrole biosynthesis, with applications in photodynamic therapy and agriculture. Although microbial production offers a sustainable alternative to chemical synthesis, most current studies rely on laboratory strains that lack clinical relevance. Here, we address the gap by engineering the probiotic strain Escherichia coli Nissle 1917 (EcN), a GRAS-certified host with therapeutic potential, for efficient 5-ALA biosynthesis. Chromosomal integration of T7RNA polymerase and overexpression of hemA from Sphingobium amiense (SahemA) enabled robust expression control. To increase precursor flux, pyruvate carboxylase (pyc) and citrate synthase (gltA) were coexpressed, redirecting carbon into the TCA cycle. Fructose consistently supported superior cell growth and 5-ALA production compared with glucose under all conditions. Notably, EcN exhibited more efficient fructose utilization and higher biosynthetic output than the commercial BL21(DE3) strain, reinforcing its suitability as a probiotic production chassis. A stepwise feeding strategy with fructose and glycine further increased 5-ALA up to 10.74 g/L in a flask and 9.44 g/L in an Ultra Yield flask within 36 h, the highest yield reported in EcN to date. Overall, fructose represents a novel and effective carbon source for establishing a modular and clinically relevant platform for sustainable biomanufacturing.