Discovery of new metabolic pathways and enzymes for the valorization of lignin

LAURA E. NAVAS

Rosario

Congreso; V Encuentro y II Workshop de la Red de Tecnología Enzimática (Red TEz); 2023

Lignin is an aromatic biopolymer that constitutes up to 35% of lignocellulosic biomass. Due to its heterogeneous structure and recalcitrance, nowadays it is mostly underutilized in biorefineries usually being burned to provide heat energy for the extraction of carbohydrates. The catalytic fractionation is a process that not only separates biomass into lignin and carbohydrate fractions but also converts the lignin into aromatic monomers and oligomers. The monoaromatics can be used directly for industrial applications, such as polymer synthesis, or can be further converted by microbial cell factories to different useful chemicals in high yield. The sustainable production of chemicals using an engineered microorganism that transforms lignin monomers depends on elucidating and optimizing the requisite aromatic catabolic pathways. Here, we describe a microbial pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. To unravel the pathway we first performed transcriptomic analyses of Rhodococcus rhodochrous GD02, previously isolated for growth on acetovanillone, and revealed an eight-gene cluster up-regulated during growth on acetovanillone. Bioinformatic analyses predicted that the hydroxyphenylethanones (Hpe) pathway proceeds via enzymatic phosphorylation and carboxylation. We validated experimentally the proposed pathway producing each recombinant protein and performing enzymatic reactions. Furthermore, we determined substrate specificity performing kinetic studies. In addition, the biocatalytic potential of the Hpe pathway and enzymes was demonstrated by the ability of GD02 to grow on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting acetovanillone, vanillin and vanillate. By identifying a pathway responsible for the degradation of acetovanillone and characterizing its enzymes, this study facilitates the engineering of bacteria to biocatalytically upgrade lignin.