Production Of Individual Ganoderic Acids And Identification Of Differentially Expressed Genes In Liquid Static And Shaking Cultures Of Ganoderma Lucidum

Ganoderma lucidum (Leyss.exFr), as a famouse tranditional Chinese medicinal mushroom, has been used as a traditional medication for the prevention and treatment of various human diseases for several thousand years. Ganoderic acids (GAs) produced by this fungi possess a variety of bioactivities such as anticancer, anti-HIV virus and modulation of immune system. Recent research has also shown that different individual GAs have different activities. Due to the important pharmacological functions of GAs, the interest in G. lucidum has increased around the world in recent years, and many researchers have focused on studying fermentation conditions to accelerate mycelial growth and optimize the total GAs production. However, commercial application of mycelia fermentation is still limited due to the low production of GAs. Until now there is no information on individual GAs formation during the fementation process and the molecular mechanism for the regulation of GAs biosynthesis is unknown, although those knowledge is significant for prodution of individual GAs and solving the low yield of the desired compounds in G. lucidum.Our previous work has shown that the two-stage culture process by combining liquid shaking culture with static culture is an efficient strategy to enhance the production of total ganoderic acids. As different GA have different bioactivities, it is critical to understand the kinetics of individual GA production during fermentation, but no information is yet available. To understand the regulation of GA biosynthesis, investigation of the accumulation of intermediate (lanosterol) and by-product (ergosterol) and of the expression of three important biosynthetic genes,3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), squalene synthase (SQS) and lanosterol synthase (LS), was also conducted in liquid shaking and static fermentations of G. lucidum. The results showed that the content of individual GAs increased rapidly in the liquid static culture and reached their maximum values 6-25 folds higher than those in the conventional liquid shaking culture. For lanosterol, one of important intermediates of GA biosynthetic pathway, its content in liquid static culture was lower than that in shaking culture, which suggested that the metabolism of lanosterol might be faster in liquid static culture. The genes transcript accumulation level was also examined by real-time PCR and quantitative RT-PCR in both culture systems. SQS, LS and HMGR transcript in liquid static cultures showed 4.3,2.1 and 1.9 folds higher compared with that in shaking culture, respectively. The amount of GA accumulation in both cultivation modes was concomitant with induction of SOS. The results suggested that higher GA content in liquid static culture was related to increased transcription of those genes especailly SQS.Until now the detailed biosynthetic steps from lanosterol to GA are unclear, the identification of genes involved in GA biosynthesis and regulation is necessary to understand the biosynthetic pathway and further develop new strategies for creation of transgenic strain that can more efficiently accumulate GA. To gain an insight into the molecular mechanism of GA biosynthesis regulation, a suppression subtractive hybridization (SSH) approach was taken to investigate the difference in gene expression between cells in liquid static and shaking cultures. Transcriptional profiling was performed using macroarray with two DIG-labeled probes. We obtained 601 clones from the forward SSH library. After clone sequencing and analysis,147 differentially expressed uniESTs were identified. Real-time PCR was performed to confirm the results, the expression levels of ten randomly selected uniESTs were consistent with both macroarray and real-time PCR. Bioinformatics searches revealed that among these sequences 13 had significant similarity to hypothetical proteins,46 showed low similarity to known genes and might represent novel genes, and 88 possessed significant similarity to known proteins involved in cell metabolism, protein biosynthesis, cell organization, signal transduction and transcription regulation. The results provided useful information on candidate genes related to GA biosynthesis and important cules to the molecular process underlying the cell differentiation and regulation of GA synthesis.From SSH library, we identified G-protein beta subunit (Gβ) and mitogen activated protein kinase (MAPK) uniESTs, which encode important proteins of signal transduction pathways in eukaryotic organisms, and it is known that those related signalling pathway regulate cell developmental processes and secondary metatolites biosynthesis in filamentous fungi. Considering the important role of those proteins, we cloned full length cDNAs of Gβand MAPK in G. lucidum by rapid-amplification of cDNA ends PCR(RACE-PCR) method. The results showed that Gβencode a protein with 313 amino aicds and shares high homology with Lentinula edodes (96%). The full length cDNAs (1660bp) of MAPK was also cloned, with 5'-UTR (78bp) and 3'-UTR(468bp). A comparison of deduced amino acid sequence revealed that this MAPK is highly homologous to Postia placenta Mad-698-R MAPK gene.The valuable information obtained here provides a good basis for further investigation of GA biosynthesis pathway and regulation of GA heterogeneity in G. lucidum. Moreover, this work is also helpful to study on the production and regulation of useful secondary metabolites by mycelia fermentation in other higher medicinal fungi.