Heterologous Expression, Characterization, and Comparison of Laccases from the White Rot Causing Basidiomycete Cerrena Unicolor

Abstract

Lignin is the most abundant renewable source of aromatics on earth, and conversion of it to chemicals and fuels is needed to build an economically viable renewable biofuels industry. Biological routes to converting lignin to fuels and chemicals involve depolymerizing lignin using lignin-degrading enzymes that catalyze the breaking of ether and carbon-carbon bonds in the phenolic and non-phenolic subunits of lignin. Laccases are a crucial class of lignin-degrading enzymes and are copper-containing enzymes capable of oxidizing electron-rich organic substrates such as lignin using molecular oxygen as an electron acceptor. The genome of Cerrena unicolor was recently added to the JGI MycoCosm database and has eight laccases. Two of these laccases, designated Lc1 and Lc2, predicted to have the highest likelihood for successful expression in soluble, active form were selected for characterization. Lc1 and Lc2, which share 65% sequence identity, were heterologously expressed in Komagataella pastoris (formerly Pichia pastoris), allowing characterization and comparison of their purified forms. Lc1 and Lc2 had half-lives of 16 min and 185 min at 60°C, respectively, and, based on molecular dynamics simulations, the longer half-life of Lc2 was due to an increased number and persistence of salt bridges compared to Lc1. Using model lignin-like dimers and a nanostructure-initiator mass spectrometry assay to quantify catalysis of specific bond-breaking events, both Lc1 and Lc2 had their highest activity at pH 3 and in combination with syringaldehyde as a mediator, with Lc1 having a higher catalytic efficiency of β-O-4' ether and C-C bond breaking. This comparative study demonstrates the diversity, including thermostability differences, of laccases from the same fungus, and improves our understanding of laccase catalyzed breaking of bonds commonly found in lignin, which will facilitate the developing this important class of enzymes for applications in the conversion of lignin to valuable bioproducts.