English
Functional Analysis of OAH Gene in Perenniporia fraxinea Using CRISPR/Cas9 System
Yeon-Jae Choi and Hyeon-Su Ro*
Department of Bio&Medical Bigdata (BK21plus) and Research Institute of Life Sciences,
Gyeongsang National University, Jinju, Korea
*Email: rohyeon@gnu.ac.kr
Perenniporia fraxinea, one of the polyporales mushrooms, characteristically produces mycelia with higher tensile strength than those of other fungal species. In addition, P. fraxinea secretes strong organic acids that negatively affect host cell growth and promote host cell wall decomposition by enhancing the activity of lignin-degrading enzymes. Oxalic acid (OA) is a major organic acid produced via the activity of oxaloacetate acetylhydrolase (OAH), using oxaloacetate as a substrate. OA plays an important role in fungal mineral interactions by forming calcium oxalate crystals, which regulate intracellular calcium levels and facilitate interactions with environmental minerals. Furthermore, it contributes to ecological processes by altering soil chemistry and supporting carbon cycling. These functions enable fungi to effectively colonize and degrade recalcitrant substrates such as woody biomass. However, excessive accumulation of OA causes browning and weakening of the mycelium, resulting in decreased tensile strength. In this study, we aimed to analyze the function of the OAH gene using the CRISPR/Cas9 system and to investigate its effects on mycelial mat formation. PEG-mediated transformation was performed on a dikaryotic strain using pre-assembled RNP complexes to disrupt the OAH gene, along with homologous recombination fragments for hygromycin selection. As a result, we successfully generated 13 transformants, including 11 dikaryons and 2 monokaryons. The transformants showed reduced calcium phosphate utilization and lignin degradation ability, while forming slightly stronger mycelial mats. This study confirms the role of oxalic acid and suggests that genetically improved strains can be developed for the production of eco-friendly materials.
