Biosensor-Based Multigene Pathway Optimization for Enhancing the Production of Glycolate

Area of Science:

• Metabolic Engineering and Synthetic Biology
• Biotechnology
• Industrial Microbiology

Background:

• Glycolate is a valuable industrial chemical with applications in cleaning, cosmetics, and medical materials.
• Previous glycolate production methods relied on expensive inducers, hindering large-scale industrial fermentation.
• Optimizing multigene pathways for constitutive biosynthesis requires efficient high-throughput screening.

Purpose of the Study:

• To develop a cost-effective and efficient method for optimizing glycolate biosynthesis pathways.
• To establish high-throughput screening techniques for rapid identification of superior glycolate-producing strains.
• To enable constitutive, large-scale production of glycolate without expensive inducers.

Main Methods:

• Established a glycolate-responsive biosensor for strain screening.
• Developed agar plate and 48-well deep-well plate high-throughput screening methods.
• Randomly assembled a library of 22 gradient-strength promoter-5'-untranslated region (UTR) complexes upstream of pathway genes.

Main Results:

• Screened 6 × 10⁵ transformants in one week to identify an optimal strain.
• Achieved a glycolate titer of 40.9 ± 3.7 g/liter in a 5-liter bioreactor.
• Identified high expression of YcdW and GltA as beneficial for glycolate production; AceA had no significant impact.

Conclusions:

• The developed biosensor and screening strategy enable efficient optimization of multigene pathways.
• This approach facilitates constitutive glycolate biosynthesis, avoiding expensive inducers like isopropyl-β-d-thiogalactopyranoside (IPTG).
• The findings provide a paradigm for optimizing other complex biosynthetic pathways in industrial biotechnology.