Exploring enzymatic diversity in the environment: Discovery and characterization of enzymes from lignolytic soil bacteria

In order to explore new chemical strategies for lignin degradation, we have initiated studies aimed at discovery and characterization of oxidative and accessory enzymes in lignin-reactive soil bacteria that exhibit particularly rich activity towards the depolymerization and utilization of biomass-derived carbon sources. Our interest lies in studying the biochemical logic underlying the transformation of complex substrates by living organisms with the overall goal of elucidating new molecular strategies for lignin degradation. Towards this goal, we began by creating a pipeline for rapid discovery and functional identification of new enzymes from unsequenced bacteria under lignin-reactive growth conditions, an approach that can be applied to any culturable microbe of interest. To do this, we assembled a de novo genome for Amycolatopsis sp. 75iv2 and used this genome sequence to identify genes potentially involved in the degradation system of lignocellulose as well as their downstream degradation products. With a genome sequence now available for proteomics analysis, we analyzed the secretome of A. sp. 75iv2 grown in the presence of lignocellulose by extracellular peroxidase activity of the culture; separation of the secretome by SDS-PAGE and heme-staining revealed the presence of heme-containing proteins which we identified as a catalase-peroxidase and a catalase by LC-MS/MS. Biochemical characterization of the catalase-peroxidase revealed an inability to oxidize non-phenolic ring motifs but showed that phenolic moieties, which encompass ~20% sites in lignin, could be oxidized.Further analysis of the genome sequence allowed for identification of an interesting and relatively new family of enzymes, the dye decolorizing peroxidases (DyPs). One phylogenetically unique member of this family, DyP2, was heterologously expressed for biochemical and structural characterization. Comparison of the enzymatic activity of this protein indicated that it displayed not only high peroxidase activity against a suite of low- and high-potential DyP substrates, similar to that of fungal DyPs and LiPs, but also an unusually active manganese peroxidase activity akin to that of versatile peroxidases. Furthermore, studies showed DyP2 to have a Mn-dependent oxidase activity that expands its substrate scope to include the oxidation of substrates with non-phenolic aromatic rings. We also solved a crystal structure of DyP2 at 2.25A resolution which revealed the presence of a Mn binding pocket 15 Å from the heme active site. Finally to begin studying the physiological response of A. sp. 75iv2 to lignin, we developed a set of minimal media conditions that demonstrated A. sp. 75iv2 to be indeed competent to utilize lignin as a sole carbon source. Interestingly, the presence of lignin induced a differential growth response compared to growth on sugar controls. Analysis of the A. sp. 75iv2 secretome further showed the existence of an induction of extracellular peroxidase activity and changes in the secreted heme protein profile. RNA sequencing and proteomics studies were then utilized to profile the global response of A. sp. 75iv2 to lignin and identify candidate proteins and pathways involved in its metabolism. These studies revealed that the dominant response involved a strong activation of Fe assimilation pathways as well as significant upregulation in the expression of universal stress response regulators. Both up- and down-regulation of aromatic degradation pathways were also observed. In order to continue to explore this response, we developed a synthesis for 13C-labeled synthetic lignin as a tool for structural studies of lignin by NMR as well as a peroxidase fractionation method as an approach to begin identifying upregulated peroxidases in the A. sp. 75iv2 secretome