Dual Antibiotic-Free Plasmid Systems Enable High-Efficiency l-Fucose Biosynthesis

Area of Science:

• Biotechnology
• Metabolic Engineering
• Microbial Production

Background:

• l-Fucose is a valuable monosaccharide with applications in pharmaceuticals, nutraceuticals, and cosmetics.
• Traditional microbial production methods rely on antibiotic-dependent systems, posing environmental and regulatory concerns.
• Achieving high-efficiency and stable microbial production of l-fucose remains a challenge.

Purpose of the Study:

• To develop a dual antibiotic-free plasmid strategy for high-efficiency l-fucose biosynthesis in engineered Escherichia coli.
• To enhance genetic stability and plasmid retention without antibiotic selection.
• To optimize metabolic pathways for increased l-fucose yield.

Main Methods:

• Engineered Escherichia coli BL21(DE3) with a dual antibiotic-free plasmid system.
• Integration of the hok/sok toxin-antitoxin system and cysC-based auxotrophic selection.
• Metabolic pathway optimization including promoter replacements, genomic integration of key enzymes, and blocking l-fucose degradation.

Main Results:

• Achieved robust l-fucose production in an antibiotic-free system.
• Produced 7.99 g/L of l-fucose in shake-flask fermentation.
• Reached a record titer of 61.91 g/L via fed-batch cultivation.

Conclusions:

• The combined toxin-antitoxin and auxotrophic selection strategy ensures genetic stability and high-level l-fucose production without antibiotics.
• This approach represents a significant advancement in sustainable and high-productivity microbial manufacturing of functional monosaccharides.
• The developed strain and methodology offer a promising platform for industrial-scale l-fucose biosynthesis.