Role Of Calcineurin-responsive Zinc Finger CRZ1 In Ganoderic Acid Biosynthesis By Ganoderma Lucidum

Higher medicial fungi full of diverse bioactive metabolites with complicated stucture like polysaccharides,sterols,terpenes and so on are valuable sources for modern pharmacological researches and development.Ganoderma lucidum is a famous medicinal basidiomycete that has been used as a traditional herb for thousands of years in China and East Asia.Ganoderic acid（GA）extracted from G.lucidum,displays immunomodulatory,carcinoma suppressing and cholesterol inhibiting activities.Previously,our lab revealed that addition of Ca²⁺in the G.lucidum mycelial fermentation could significantly stimulate the GA production and the calcium signaling pathway was involved in the GA biosynthesis,but the role of calcineurin-responsive zinc finger（CRZ1）in the biosynthetic regulation remains not verified.As we know,calcium ion is an important signal to regulate the cellular growth and development upon the change of external environment.In the fungal cells,CRZ1 is a vital calcium signaling transcription factor that was generally investigated to participate in ion homeostasis,cell wall synthesis and pathogenesis.Besides,the function of CRZ1 has been largely unknown in the higher fungi due to the immaturity of their genetic manipulation tools.In this dissertation,Blast searches was performed in the proteome of G.lucidum with the homologous sequences of CRZ1 and two highly conserved CRZ1 orthologs were found,i.e.,GL25464 and GL26097,which was named Gl CRZ1 and Gl CRZ2,respectively.With our newly developed CRISPR/Cas9gene editing platform for G.lucidum,we disrupted the gene of glcrz1 and glcrz2separately and the experimental data showed that without calcium addition,the glcrz1 knocked-out strain displayed similar growth and mycelia morphology as the wild type（WT）strain.However,high concentration of calcium ion（100 m M）could at some degree inhibit the glcrz1disrupted strain（Δglcrz1）.Under the stimulus of low concentaion of calcium ion（10 m M）,the GA production of the WT strain increased profoundly with the individual ganoderic acids GA-Mk,GA-T,GA-S and the total crude GA 2,2.4,5.9 and 2.6 fold of the control group（without calcium addition）,respectively.While forΔglcrz1,the addition of 10m M calcium,the GA production had no significant difference with the non-calcium treated group.The above results demonstrated that Gl CRZ1 was required in the calcium-induced GA biosynthesis as an essential component of the calcium signaling.Different from the disruption of glcrz1,the growth ofΔglcrz2 was attenuated and the mycelia were yellowish.What’s more,Δglcrz2 displayed hyper sensitivity to high concentration calcium and almost stopped growing on the plate with 100m M Ca²⁺.In the meantime,we found that the GA production ofΔglcrz2 declined severly and unlike the WT strain,the airal mycelium ofΔglcrz2 failed to form the uniformed membrance during the static fermentation.Additionally,calcium stimulus（10 m M）did not increase the GA production but decreased the GA production inΔglcrz2 strain.In short,Gl CRZ2 was a vital transcription factor to maintain the cellular ion homeostasis,mycelium morphology and GA synthesis.To summarize,this thesis studied the function of Gl CRZ1 and Gl CRZ2 in G.lucidum for the first time.The findings will help us to further understand the role of CRZ1 in the calcium signaling and secondary metabolism of other higher fungi,not to mention G.lucidum.