Dual Antibiotic-FreePlasmid Systems Enable High-Efficiency l‑Fucose Biosynthesis

l-Fucose, a functional monosaccharide with significant commercial potential in the pharmaceutical, nutraceutical, and cosmetic industries, faces challenges in microbial production due to antibiotic-dependent plasmid maintenance systems. This study presents a dual antibiotic-free plasmid strategy in engineered <i>Escherichia coli</i> BL21(DE3) to achieve high-efficiency l-fucose biosynthesis. By integration of the <i>hok/sok</i> toxin-antitoxin system and a <i>cysC</i>-based auxotrophic selection into two plasmids, genetic stability and plasmid retention were ensured without antibiotics. Metabolic pathway optimization involved enhancing GDP-l-fucose supply via promoter replacements, genomic integration of key enzymes (α1,2-fucosyltransferase and α-l-fucosidase), and blocking l-fucose degradation. The engineered strain demonstrated robust performance, producing 7.99 g/L of l-fucose in shake-flask fermentation and 61.91 g/L via fed-batch cultivationboth antibiotic-free. This titer represents the highest reported l-fucose yield to date, highlighting the effectiveness of combining toxin–antitoxin and auxotrophic systems for sustainable, high-productivity microbial manufacturing.