Engineering of ethylene glycol (EG)-trophic Escherichia coli enables fast growth on EG for closed-loop PET biodegradation

Abstract

In polyethylene terephthalate (PET) enzymatic hydrolysate, terephthalic acid (TPA) can be efficiently recovered via acid-based precipitation, leaving a TPA-free PET hydrolysate rich in ethylene glycol (EG) as a non-grain carbon source suitable for microbial bioconversion. In this study, Escherichia coli MG1655(DE3) was engineered to assimilate EG for growth by plasmid-mediated overexpression of alcohol oxidoreductase (FucO) and aldehyde dehydrogenase (AldA) in tandem under the control of promoter gyrA (P_gyrA), achieving the highest cell density (2.59 of OD₆₀₀) and 42.3 % EG consumption within 72 h. This EG-assimilation cassette was then chromosomally integrated using a CRISPR-associated transposase system, yielding the plasmid-free strain MGEG27 as EG-trophic E. coli for the first time, which consumed 79 % of EG within 72 h. Adaptive laboratory evolution (ALE) was then applied to enhance EG assimilation of E. coli MGEG27, producing E. coli ALE1-7 with OD₆₀₀ = 5.45 and 98.7 % EG consumption within 48 h. Finally, E. coli ALE1-7 was engineered to express PET hydrolases BHR4M, TFU6M, and FastPETase, enabling the simultaneous degradation of PET and EG-based growth. In a closed-loop PET degradation system, E. coli growth and PET hydrolysis occurred concurrently, with the production of 1.12 mg/L, 0.79 mg/L, and 0.32 mg/L of TPA, respectively. This study presents a plasmid-free microbial platform for closed-loop PET recycling and the sustainable upcycling at ambient temperature.

Keywords: Adaptive laboratory evolution; Closed-loop recycling; Ethylene glycol; Metabolic engineering; PET biodegradation.