Biocatalytic demethylation of lignin-derived compounds investigated by ancestral reconstruction and characterization of Rieske non-heme iron enzymes

Abstract

Lignin is considered one of the most abundant renewable raw materials to serve as analternative to petroleum in the production of chemicals, thereby contributing to theestablishment of a green and renewable economy. However, one of the main challengesfor its use as a feedstock is its heterogeneous structure, which poses considerabletechnical challenges to achieve high-purity products. To overcome this, a clever strategyis the use of engineered microorganisms capable of transforming complex mixtures oflignin-derived compounds into a specific high-value metabolite. One crucial step in thisprocess is the removal of methyl groups - abundantly present in lignin - by enzymesknown as aryl-O-demethylases. There are few enzymes known to have this activity andthey are basically divided into three classes: Rieske non-heme iron oxygenases, P450monooxygenases, and tetrahydrofolate -dependent enzymes. Our group has identified anddemonstrated the demethylation activity for vanillate and syringate compounds (othersare under investigation) of a Rieske non-heme iron oxygenase, annotated as VanA, fromthe phytopathogen Xanthomonas citri pv. citri 306 (Xac 306). This oxygenase dependson the presence of a ferredoxin for electron transfer (annotated as VanB). However, thelow solubility and stability of these enzymes have hindered enzymatic characterizationassays. Therefore, we believe that obtaining ancestral enzymes through the AncestralSequence Reconstruction (ASR) technique may provide more soluble and stable targets,suitable for enzymatic kinetic assays. Additionally, we intend to characterize these enzymesand their reconstructed ancestors using electron paramagnetic resonance (EPR), providingvaluable information about the electronic environment of the iron-sulfur cluster presentin these enzymes.