Biochemical Characterization of Recombinant Oxalate Decarboxylases of the White Rot Fungus Dichomitus squalens

Background: Oxalic acid is a common metabolite of wood-degrading basidiomycete fungi, and it has been proposed to possess several important roles in fungal physiology, including assistance in degradation of lignocellulose polymers. Because high concentrations of oxalic acid are toxic to fungi, they produce oxalate degrading enzymes, i.e. oxalate decarboxylases (ODC) and oxalate oxidases (OXO), to regulate the amount of oxalic acid in the vicinity of fungal hyphae. In addition, oxalate degrading enzymes are of interest for several biotechnological applications, such as in diagnostics to determine oxalic acid concentrations and in bioprocess industry to prevent the formation of oxalate deposits. Our previous studies have shown that the wood-rotting white rot fungus Dichomitus squalens actively secretes and degrades oxalic acid during growth both in liquid cultures and on natural spruce wood cultures. Methods: To study the biochemical properties of D. squalens ODCs, the five ODC encoding genes were expressed in Pichia pastoris under control of the methanol inducible alcohol oxidase I (AOXI) promoter. Results: The secretion of recombinant ODCs (rODCs) from P. pastoris cells was driven by both the Saccharomyces cerevisiae α-mating factor fused to mature ODCs and native leader peptides of ODC proteins, thus showing that the native signal sequences of D. squalens ODCs were functional. All the rODCs were produced as multimeric proteins, which had dissimilar biochemical characteristics. The rODC3 and rODC4 degraded oxalic acid at the widest pH and temperature range. Conclusion: All the rODCs were produced as active enzymes with rODC3 and rODC4 having the highest thermostability and showing activity over the broadest pH range, thus having potential as robust biocatalysts in various biotechnological processes.