Constructing a Novel Biosynthetic Pathway for the Production of Glycolate from Glycerol in Escherichia coli.

Glycolate is an important α-hydroxy acid with a wide range of industrial applications. The current industrial production of glycolate mainly depends on chemical synthesis, but biochemical production from renewable resources using engineered microorganisms is increasingly viewed as an attractive alternative. Crude glycerol is an abundant byproduct of biodiesel production and a widely investigated potential sustainable feedstock. Here, we constructed a novel biosynthetic pathway for the production of glycolate from glycerol in Escherichia coli. The pathway starts from the oxidation of glycerol to d-glycerate by alditol oxidase, followed by sequential enzymatic dehydrogenation and decarboxylation as well as reduction reactions. We screened and characterized the catalytic activity of candidate enzymes, and a variant of alditol oxidase from Streptomyces coelicolor A3(2), 2-hydroxyglutarate-pyruvate transhydrogenase from Saccharomyces cerevisiae, α-ketoisovalerate decarboxylase from Lactococcus lactis, and aldehyde dehydrogenase from Escherichia coli were selected and assembled to create an artificial operon for the biosynthetic production of glycolate from glycerol. We also characterized the native strong constitutive promoter Plpp from E. coli and compared it with the PT7 promoter, which was employed to express the artificial operon on the plasmid pSC105-ADKA. To redirect glycerol flux toward glycolate synthesis, we deleted key genes of the native glycerol assimilation pathways and other branches of native E. coli metabolism, and we introduced a second plasmid expressing Dld3 to reduce the accumulation of the intermediate d-glycerate. Finally, the engineered strain TZ-108 harboring pSC105-ADKA and pACYC184-Plpp-Dld3 produced 0.64 g/L glycolate in shake flasks, which was increased to 4.74 g/L in fed-batch fermentation. This study provides an alternative pathway for glycolate synthesis and demonstrates the potential for producing other commodity chemicals by redesigning glycerol metabolism.