Metabolic engineering of a plasmid-free, non-auxotrophic Escherichia coli for efficient glycolate production

Glycolate, an α-hydroxycarboxylic acid, is widely used in industries such as bioplastics, food, and pharmaceuticals. However, current microbial production methods are limited by the use of plasmids and chemical inducers, hindering their industrial scalability. In this study, a stable and efficient Escherichia coli platform was developed for glycolate production. The glycolate biosynthetic pathway was reconstructed through the identification of a highly efficient glyoxylate reductase (GhrA) from Acetobacter aceti. Carbon flux toward glycolate synthesis was optimized through strategies including enhancing precursor supply, blocking competing pathways, and fine-tuning gene copy numbers. Cofactor engineering was employed by engineering GhrA cofactor preference from NADPH to NADH. Additionally, a non-auxotrophic strain (eliminating exogenous nutrient requirements) for glycolate production was engineered by implementing a growth-stage-dependent molecular switch to dynamically regulate the expression of isocitrate dehydrogenase. Through fermentation optimization, the engineered strain E. coli GA26 achieved a glycolate titer of 81.5 g/L, a yield of 0.49 g/g glucose, and a productivity of 1.9 g/L/h in a 5-L bioreactor, representing the highest reported glycolate titer from glucose to date. These results pave the way for sustainable and cost-effective industrial glycolate production.