Lignin modifying enzymes from bacteria: discovery and engineering

Lignin, the most abundant aromatic biopolymer on earth, represents a potential source of high value aryl compounds. Efficient conversion of lignin into bio-fuels and value-added products is also fundamental to sustainable bio-refining. Although efforts to establish lignin-depolymerizing biocatalyst have focused on fungal enzymes, recent advances in genomics and other high-throughput approaches are revealing the lignolytic potential of bacteria. We identified Rhodococcus jostii RHA1 as a lignin-degrading actinomycete and characterized DypB – a dye-decolorizing peroxidase – as the first bacterial lignolytic enzyme capable of oxidizing Mn²⁺. We engineered the heme binding pocket of DypB and produced a variant, DypB^N246A, with improved Mn²⁺-oxidizing activity (80- and 15-fold, respectively, higher k_cat and k_cat/K_m values). DypB^N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and produced 2,6-dimethoxybenzoquinone and syringaldehyde as major degradation products.  We also devised a high-throughput screening method to identify lignin-modifying enzymes from metagenomic libraries based on a co-culture screening method that utilizes a biosensor responsive to lignin-derived aromatic compounds. Using this approach we identified 24 metagenomic fosmid clones producing compounds such as vanillin, 1,4-dihydroxy-2,6-dimethoxybenzene, and syringaldehyde. Mutagenesis and sequence analyses of these clones revealed the presence of numerous genes encoding enzymes with lignolytic potential, such as multi-copper oxidases and aryl alcohol oxidases. The ongoing work aims to characterize bacterial lignin-modifying enzymes, develope tunable scaffolds from metagenomic clones and ultimately engineer improved biorefining strains capable of efficient lignin transformation.