Publications

Laccases are oxidative enzymes containing 4 conserved copper heteroatoms. Laccases catalyze cleavage of bonds in lignin using radical chemistry, yet their exact specificity for bonds (such as the β-O-4 or C-C) in lignin remains unknown and may vary with the diversity of laccases across fungi, plants and bacteria. Bond specificity may perhaps even vary for the same enzyme across different reaction conditions. Determining these differences has been difficult due to the fact that heterologous expression of soluble, active laccases has proven difficult. Here we describe the successful heterologous expression of functional laccases in two strains of Saccharomyces cerevisiae, including one we genetically modified with CRISPR. We phylogenically map the enzymes that we successfully expressed, compared to those that did not express. We also describe differences protein sequence differences and pH and temperature profiles and their ability to functionally express, leading to a potential future screening platform for directed evolution of laccases and other ligninolytic enzymes such as peroxidases.

We are working to generate fundamental understanding of enzymatic depolymerization of lignin and using this understanding to engineer mixtures of enzymes that catalyze the reactions necessary to efficiently depolymerize lignin into defined fragments. Over the years the enzymes involved in these processes have been difficult to study, because 1) the enzymes thought to be most important, fungal laccases and peroxidases, are very difficult to express in soluble, active form; 2) the full complement of required enzymes and whether or not they act synergistically is not known; 3) analysis of bond cleavage events is difficult due to the lack of analytical tools for measuring bond cleavage events in either polymeric lignin or model lignin-like compounds.