Impacts Of Calcineurin Signal Transduction On Production Of Antitumor Ganoderic Acids In Mycelia Fermentation Of Medicinal Mushroom Ganoderma Lucidum

Mushrooms, as higher fungi, are a natural powerful cell factory for producing variousunique biologically active compounds including terpenes, heterocyclics, polyketides, andsterols. Ganoderic acids （GAs） are one type of triterpene compounds produced byGanoderma lucidum （Ling-zhi in Chinese, Reishi in Japanese）, which is a very famoustraditional Chinese medicinal mushroom. As reported, GAs have various importantbiological activities such as antitumor, anti-invasion and anti-HIV. However, to meet theneeds for the large-scale （pre-）clinical study and commercialization of individual GAs, theyield of each individual GA should be further enhanced by modern biotechnologystrategies.Recently, signal transduction engineering of secondary metabolism is receiving greatinterest as a powerful tool towards efficient production of valuable secondary metabolites.Calcineurin signals are involved in secondary metabolite biosynthesis in someAscomycota species, but there have been no reports in Basidiomycota, which is anothermajor division （phyla） of higher fungi. Moreover, there is still lack of informationregarding whether and how calcineurin signals could be triggered to regulate triterpenebiosynthesis, not to mention the mechanism how Ca²⁺sensors and GA biosynthetic genesrespond to calcineurin signals in G. lucidum fermentation process, in spite of the potentialsignificance of such information for biotechnology application such as productionimprovement.In this thesis, static liquid culture of G. lucidum, which is an efficient process for hyper-production of GAs developed in our lab, was used. In order to gain an insight intothe molecular events why the two stage culture was superior to the shaking culture,comparative transcriptome analysis using suppression subtractive hybridization （SSH）technique was performed to identify preferentially expressed genes in the two-stageculture vs. the traditional shaking culture. After macroarray analysis of1920cDNAs fromSSH library,147unigenes which exhibited high expression in the two-stage culture wereidentified. Among them, calmodulin, Ca²⁺channel and store-operated Ca²⁺entry, whichare involved in the Ca²⁺signals, were found to upregulate in the two-stage culture. Theresults suggested that Ca²⁺signals might be important in regulating GA biosynthesis.Therefore, in the following work, the impact of Ca²⁺signaling and its regulatory roles onthe GA biosynthesis were investigated in detail in the two-stage culture system.At first, the addition of Ca²⁺, Na⁺or Mn²⁺at the beginning of the static liquidcultivation was proved to be a useful strategy to enhance GA production by G. lucidum.The optimal dosage of Ca²⁺, Na⁺or Mn²⁺was identified to be10mM,100mM and10mM,respectively, and their highest production of total crude GAs （detected byspectrophotometer） was1.58g/L,1.32g/L and0.96g/L for each, which resulted in4.03,3.38and2.45times higher than that of control, respectively. The maximum individual GA（detected by HPLC） accumulation of GA-T,-S and–Me was reached at10mM Ca²⁺whileGA-Mk was at100mM Na⁺. The highest production of GA-Mk,-T,-S, and Me was274.15±9.1mg/L,327.8±13.5mg/L,122.1±8.4mg/L and153.6±6.5mg/L, respectively,which led to3.54-,5.50-,3.67-and4.55-fold improvement for each compared to thecontrol. Those were the highest reported GA yields to date.Under high salt stress of Na⁺, Mn²⁺or Li+, cytosolic Ca²⁺was reported to burst andfurther induce Ca²⁺signals to regulate the biosynthesis of abscisic acid in Arabidopsis. Inour study, the fluxes of Ca²⁺, Na⁺or Mn²⁺in response to external Ca²⁺, Na⁺or Mn²⁺addition were assayed during the fermentation process of G. lucidum. When addingmedium Ca²⁺, intracellular Ca²⁺level was significantly raised and reached maximum on day4, while the concentration of extracellular Ca²⁺was dropped to minimum on day4,implying that medium Ca²⁺flew into cells at first in response to the higher Ca²⁺concentration stress, but from day4intracellular Ca²⁺gradually effused to medium. Whenadding medium Na⁺or Mn²⁺, similarly the increase of intracellular Na⁺or Mn²⁺and thesimultaneous drop of extracellular Na⁺or Mn²⁺were observed. Moreover, it wasinterestingly found that intracellular Na⁺or Mn²⁺led to the influx of Ca²⁺from medium tothe cells, suggesting that Ca²⁺signals might be triggered to regulate GA biosynthesis.To probe the mechanism underlying the enhanced GA production by calcium signals,calcineurin inhibitor cyclosporin A was applied to investigate the involvement ofcalcineurin signals in regulating GA biosynthesis. Spore number and contents of totalcrude and four individual GAs were enhanced with Ca²⁺, Na⁺or Mn²⁺induction, declinedby adding cyclosporine A and partially relieved by supplementing with Ca²⁺, Na⁺or Mn²⁺,indicating that Ca²⁺, Na⁺or Mn²⁺stimulation might regulate GA biosynthesis throughcalcineurin signaling pathway. Intracellular Ca²⁺was also detected in order to evaluate therelationship among intracellular Ca²⁺, calcineurin signals and GA biosynthesis. Fromintracellular Ca²⁺imaging experiments, strong green fluorescence, representing high levelof free intracellular Ca²⁺, was observed in the cells under Ca²⁺, Na⁺or Mn²⁺addition butthe fluorescence decreased notably when the cells were treated with cyclosporine A and itbecame comparatively stronger when supplementing Ca²⁺, Na⁺or Mn²⁺. The factssuggested that calcineurin signals might be involved in regulating the level of intracellularCa²⁺under Ca²⁺, Na⁺or Mn²⁺induction.To further investigate the molecular events underlying the enhanced GA productionby calcineurin signals, the transcriptional levels of genes of Ca²⁺exchangers, Ca²⁺sensorsand GA biosynthesis were all detected during the fermentation process. The geneexpression levels of Na⁺/Ca²⁺exchangers (ENA1and Ca²⁺-ATPase) and Mn²⁺/Ca²⁺exchanger （PMR1） were assayed to clarify the relationship between medium Na⁺or Mn²⁺and calcineurin signals. The expression levels of ena1and Ca²⁺-ATPase were raised by Na⁺stimulation and that of pmr1was also elevated by Mn²⁺induction while dropped byinhibitors addition, suggesting that these exchangers might help for ion homeostasis torespond to high salt stress and further probably activate signal pathway. Furtherquantitative gene transcription analysis revealed that the expression levels of genes of GAbiosynthesis and Ca²⁺sensors were up-regulated with Ca²⁺, Na⁺or Mn²⁺addition whiledown-regulated under the inhibitors addition. The expression patterns of these genes wereconsistent with the GA accumulation. The above results suggested that the enhanced GAsbiosynthesis by Ca²⁺, Na⁺or Mn²⁺stimulation may result from the higher expression levelof these genes.As higher transcriptional levels of GA biosynthetic genes were found important tohigher GA production, overexpression of such a biosynthetic gene was thus attempted inthe next step. But, the genetic modification of mushroom （i.e., G. lucidum） is widelyknown as a very difficult issue. We successfully established the gene transformationplatform for G. lucidum by using the Agrobacterium tumefaciens-mediated transformation（ATMT）. Interestingly, overexpression of3-hydroxy-3-methylglutaryl coenzyme Areductase （HMGR） by ATMT led to a2-fold increase in ganoderic acid content. It also ledto the accumulation of intermediates （squalene and lanosterol） and the upregulation ofdownstream genes such as farnesyl pyrophosphate synthase, squalene synthase, andlanosterol synthase. The results implied that transgenic basidiomycete G. lucidum was apromising system to achieve metabolic engineering of the ganoderic acid pathway.In summary, the work of this thesis identified over100upregulated genes in the twostage culture by SSH technique, which implied the importance of Ca²⁺signals inregulating GA biosynthesis. It was found that addition of Ca²⁺, Na⁺or Mn²⁺could enhancethe GAs production in static liquid culture of G. lucidum. Investigation by intracellularCa²⁺imaging and using a calcineurin inhibitor （i.e., cyclosporin A） revealed that additionof Ca²⁺, Na⁺or Mn²⁺led to the influx of Ca²⁺from medium to the cells and calcineurinsignals were thus triggered. Then, the mechanism of the effect of calcineurin signals on GA biosynthesis was revealed at molecular level. This work provides not only an efficientand simple induction strategy by Ca²⁺, Na⁺or Mn²⁺addition for the improved GAsproduction but also the regulation mechanism of Ca²⁺, Na⁺or Mn²⁺on the GAbiosynthesis through calcineurin signaling transduction. Overexpression of a calcineurinsignal-induced biosynthetic gene （hmgr） by using a self-developed genetic transformationsystem （i.e., ATMT） resulted in a conspicuous increase in total GA content. The strategyproposed here might be useful for the large scale efficient fermentation of anti-tumor GAsor other secondary metabolite production by mushrooms.