A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum

Abstract

Brown rot fungi are primary decomposers of wood and litter in northern forests. Relative to other microbes, these fungi have evolved distinct mechanisms that rapidly depolymerize and metabolize cellulose and hemicellulose without digesting the more recalcitrant lignin. Its efficient degradative system has therefore attracted considerable attention for the development of sustainable biomass conversion technologies. However, there has been a significant lack of genetic tools in brown rot species by which to manipulate genes for both mechanistic studies and engineering applications. To advance brown rot genetic studies, we provided a gene-reporting system that can facilitate genetic manipulations in a model fungus Gloeophyllum trabeum. We first optimized a transformation procedure in G. trabeum, and then transformed the fungus into a constitutive laccase producer with a well-studied white rot laccases gene (from Trametes versicolor). With this, we built a gene reporting system based on laccase gene’s expression and its rapid assay using an 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) indicator dye. The laccase reporter system was validated robust enough to allow us to test the effects of donor DNA’s formats, protoplast viability, and gene regulatory elements on transformation efficiencies. Going forward, we anticipate the toolset provided in this work would expedite phenotyping studies and genetic engineering of brown rot species.

Citation

Li, Weiran; Ayers, Charles; Huang, Weiping; Schilling, Jonathan S.; Cullen, Daniel; Zhang, Jiwei. 2023. A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum . Microbiology Spectrum. 11(1): 39. https://doi.org/10.1128/spectrum.04246-22.