Study On The Regulation Of DNA Methylation On Ganoderic Acids Biosynthesis In Ganoderma Lucidum

Ganoderic acids (GAs), mainly produced by Ganoderma lucidum, are a group of highly oxygenated lanostane-derived triterpenoids with many important pharmacological activities including antitumor effect, inhibition of histamine and anticancer effect. In recent years, many studies have been devoted to investigating fermentative optimization of G. lucidum, the separation and pharmacological effects of GAs. There were also some reports about the research on the regulatory mechanism of GAs in the fermentation process. As a sort of secondary metabolite, biosynthesis mechanisms of GAs are extremely sophisticated and influenced by many factors. It is helpful for people to understand the regulation of secondary metabolism of fungi, and the study will has a wide range of application value. In recent years, the epigenetic regulation of secondary metabolism in the genetic process of fungi gradually attracted people’s attention. Among them, DNA methylation is the most studied modification method. In this study, 5-Azacytidine (5-AC), an inactivator of DNA methyltransferase, was applied to control DNA methylationlevel of G. lucidum cells. After detecting cell differentiation, expression of key enzymes in the biosynthesis and the level of total methylation of G. lucidum, the regulation mechanism of DNA methylation on GAs biosynthesis was investigated.In this work, G. lucidum cells were treated with different concentration of 5-AC (0, 0.1,0.4,0.8,1.0 and 2.0 mmol/L), which were cultivated by combining liquid shaking culture with static culture. Then, cells biomass, GAs production, sugar consumption and culture pH values of G. lucidum at different concentration of 5-AC were analyzed and compared. There is no abnormal growth of mycelium from G. lucidum treated by 5-AC in the liquid static culture. As 5-AC reached to the concentration of 1.0 mmol/L, the content of GAs increased rapidly and reached their maximum values 13.11 mg/g,1.67 folds higher than those in the control without 5-AC. It is interesting that treatment with 5-AC (1.0 mmol/L) resulted in reduction of spore production and concurrent increment of GAs accumulation in spores, which reached 103.2 mg/g,2.88 folds higher than that without 5-AC treatment.To investigate 5-AC influence on biosynthesis pathway of GAs, the expression level of four important biosynthetic genes, global regulator laeA gene and velvet gene were examined by real-time PCR. The expression level of hmgr, sqs, se and ls genes at the condition of 1.0 mmol/L 5-AC treatment showed 6.26,3.10,3.42 and 4.32 folds higher compared with that in the control group without 5-AC, respectively. The amount of GAs accumulation in the 5-AC treatment was concomitant with induction of those genes. The results suggested that higher GAs content was related to increased transcription of those genes by treatment with 5-AC in the liquid culture. Besides,5-AC also affected gene expression of laeA and velvet gene, which involved in the regulation of morphological development and secondary metabolism in fungi. Expression level of laeA showed 2.83 folds higher than that without 5-AC treatment, and expression of velvet fell by 15 percent.Considering the relation between demethylation of 5-AC and gene expression, the genomic DNA methylation level of G. lucidum cells and DNA methylation status of important gene promoter at different 5-AC concentration were detected by high performance liquid chromatograph and bisulfite sequencing PCR, respectively. We found that with the increase of concentration of 5-AC, the genomic DNA methylation level gradually reduced, but the methylation status of gene promoter had no significant difference. These data show that 5-AC did not change methylation status of gene promoter region in CpG to regulate gene expression.The effects of epigenetic regulation of secondary metabolism on cell growth and GAs synthesis metabolism were studied from the point of DNA methylation. Possible regulation mechanism of DNA methylation in GAs biosynthesis was also disscussed. This research not only is helpful to people’s understanding of GAs biosynthesis, but also has certain reference value in the studies on the epigenetic genetic regulating secondary metabolismin fungi.