Lignin oxidation by bacterial dye-decolorizing peroxidase enzymes requires hydrogen peroxide as a co-substrate, an unstable and corrosive oxidant. We have identified a glycolate oxidase enzyme fromRhodococcus jostiiRHA1 that can couple effectively at pH 6.5 with DyP peroxidase enzymes fromAgrobacteriumsp. orComamonas testosteronito oxidise lignin substrates without addition of hydrogen peroxide.Rhodococcus jostiiRHA1 glycolate oxidase (RjGlOx) has activity for oxidation of a range of α-ketoaldehyde and α-hydroxyacid substrates, and is also active for oxidation of hydroxymethylfurfural (HMF) to furandicarboxylic acid. The combination of RjGlOx withAgrobacteriumsp. DyP orC. testosteroniDyP generated new and enhanced amounts of low molecular weight aromatic products from organosolv lignin substrates, and was able to generate high-value products from treatment of lignin residue from cellulosic biofuel production, and from a polymeric humin substrate.