A Study on the Ganoderic Acids Biosynthetic Pathway through Two gene disruption

A Study on the Ganoderic Acids Biosynthetic Pathway through Two gene disruption

Hyerang Eom 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

Ganoderic acids(GAs), secondary metabolites primarily produced by Ganoderma lingzhi, are triterpenoid from the precursor lanosterol through hydroxylation, acetylation, decarboxylation. While GAs have been reported to have various pharmacological activities such as anticancer, anti-inflammatory, and antibacterial, it’s yield limited in clinical application. In this study, to confirm the GAs biosynthesis pathway in G. lingzhi, cyp 5150L8 and cyp family 628 involved in the production of GAs were edited using the CRISPR/Cas9 system developed in previous study. The protoplast transformed with ribonucleoprotein(RNP) complexes and a deletion cassette containing a carboxin resistance gene with 300 bp homologous arms. After that, fruiting bodies cultivation revealed length of stipe is decreased 2.6-, 1.4-fold in cyp 5150L8, cyp 628 family deletion strain. Notably, the pileus size decreased 2-fold only in cyp 5150L8 deletion strain, which also showed a significant increase in the total number of fruiting bodies. HPLC analysis further demonstrated that the cyp 5150L8 deletion strain failed to produce the ganoderic acid derivatives, wherease the cyp 628 family deletion strain exhibited increased levels of ganoderic acid C2, C6, B, K, compared to the wild type. This study elucidates the GAs biosynthetic pathway, providing a genetic framework for metabolic engineering to produce specific high value GAs for pharmaceutical applications.