Improved biosynthesis of C4 derivatives by engineered thiolase

Ethylene glycol (EG), a major product of the enzymatic degradation of polyethylene terephthalate (PET), provides a promising feedstock for sustainable biomanufacturing. Herein, we developed a novel metabolic pathway using Escherichia coli(E. coli) as a host for the biosynthesis of four-carbon compounds such as 1,4-butanediol (1,4-BDO), 1,2,4-butanetriol (1,2,4-BTO), and succinate, from two-carbon substrates such as glycolate and EG. This represents an efficient strategy of using C2 precursors for these high-value chemicals. Through directed evolution of the β-ketoacyl thiolase B of Cupriavidus necator (CnBktB), one of the rate-limiting enzymes in the pathway, via an established growth-coupled screening platform, we identified the L89S mutant, which exhibits significantly enhanced catalytic efficiency in assimilating glycolyl-CoA and acetyl-CoA. Using glycolate and glucose as substrates, the route achieves 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. Furthermore, integrating an upstream module for EG conversion to glycolate allows direct utilization of PET-derived EG, yielding 11.4 g/L succinate with 93 % conversion efficiency from EG. This work bridges the fields of synthetic biology and plastic waste recycling, demonstrating a sustainable and scalable route for converting PET-derived EG into valuable four-carbon compounds. The novel biosynthetic pathways developed in this study offer a foundation for advancing circular bioeconomy strategies and reducing the environmental impact of plastic waste.