Improved biosynthesis of C4 derivatives by engineered thiolase

Area of Science:

• Synthetic biology
• Metabolic engineering
• Biotechnology

Background:

• Ethylene glycol (EG), derived from polyethylene terephthalate (PET) enzymatic degradation, is a potential feedstock for sustainable biomanufacturing.
• Current methods for producing C4 compounds like 1,4-butanediol (1,4-BDO) and 1,2,4-butanetriol (1,2,4-BTO) often rely on petrochemical sources.
• There is a need for efficient biological routes to convert C2 substrates into high-value C4 chemicals.

Purpose of the Study:

• To develop a novel metabolic pathway in Escherichia coli for the biosynthesis of C4 compounds from C2 substrates like glycolate and EG.
• To enhance the catalytic efficiency of key enzymes through directed evolution for improved substrate assimilation.
• To demonstrate the direct utilization of PET-derived EG for the production of valuable chemicals, contributing to plastic waste recycling.

Main Methods:

• Engineered Escherichia coli (E. coli) with a novel metabolic pathway.
• Employed directed evolution to optimize the β-ketoacyl thiolase B (CnBktB) enzyme from Cupriavidus necator, identifying the L89S mutant.
• Utilized a growth-coupled screening platform for enzyme evolution.
• Integrated an upstream module for converting EG to glycolate.
• Performed fermentation experiments using glycolate, glucose, and PET-derived EG as substrates.

Main Results:

• Achieved production titers of >200 mg/L for 1,4-BDO, 266 mg/L for 1,2,4-BTO, and 9.22 g/L for succinate using glycolate and glucose.
• The L89S mutant of CnBktB showed enhanced catalytic efficiency for glycolyl-CoA and acetyl-CoA assimilation.
• Direct utilization of PET-derived EG yielded 11.4 g/L succinate with 93% conversion efficiency.
• Demonstrated a scalable route for converting C2 precursors into high-value C4 compounds.

Conclusions:

• Successfully established a synthetic biology platform for producing 1,4-BDO, 1,2,4-BTO, and succinate from C2 substrates.
• The engineered E. coli strain and optimized enzymes provide a foundation for sustainable biomanufacturing.
• This work integrates plastic waste recycling with bioeconomy strategies, offering a promising approach to reduce environmental impact.