Identification And Analysis Of Cytochrome P450 Genes Involved In Ganoderic Acid Biosynthesis In Ganoderma Lingzhi

Ganoderic acid(GA),a kind of highly oxygenated lanostane-type triterpenoid from Ganoderma lingzhi,possesses antitumor and other significant pharmacological activities.Until now,the genes involved in conversion from lanosterol to GAs in the biosynthetic pathway remains opaque,which limits the understanding of its regulation mechanism and metabolic engineering of G.lingzhi for overproduction of GAs.Previous studies indicated cytochrome P450 s may be responsible for modification of lanosterol skeleton in the GA biosynthesis.Among them,cyp5150l8 was found to catalyze a three-step oxidation of lanosterol at C-26 to synthesize 3-hydroxy-lanosta-8,24-dien-26 oic acid(HLDOA).However,the other related P450 genes is not identified and characterized.In this study,up-regulated genes in liquid static culture condition vs shaking culture condition of G.lingzhi were identified.Results showed that 3321 genes was up-regulated in liquid static culture condition.The P450 genes belonged to the CYP512 and CYP5144 families that can effectively modify steroid were screened.On the basis of previous report,10 CYP512 and CYP5144 genes co-expressed with lanosterol synthase(lss)which is a structure gene involved in GA biosynthesis pathway were screened out.Real-time quantitative PCR was used for identification the expression profiles of 10 selected genes under liquid static/shaking culture conditions.The results indicated that the transcription levels of cyp512a3,cyp512a11,cyp5144n1,cyp512u4 and cyp512v2 in liquid static culture were 7.2-,3.7-,3.4-,89.8-,63.4-fold higher than those in shaking culture,respectively.The correlation between the kinetic transcription levels of the 5 P450 genes with the dynamic accumulation of GA-T under both culture modes was detected.The results demonstrate that the transcription levels of cyp512a3 and cyp512v2 were positively correlated to the accumulation of GA-T,correlation coefficient was 0.954 and 0.992(p<0.05),respectively.Furthermore,the correlation analysis between the transcription levels of the 5 P450 genes with the accumulation of GA-T were performed under the liquid shaking,liquid static,liquid static with calcium ion addition and liquid static with nitrogen-limiting culture conditions.It was confirmed that the transcription level of cyp512v2 was positively correlated to the accumulation of GA-T,correlation coefficient and p value was 0.992 and 0.008,respectively.In orders to characterize the function of cyp512v2 in GA biosynthesis,we suppress the transcription level of cyp512v2 by using antisense RNA technology in G.lingzhi.Nine putative transformants was selected by carboxin resistance.The integration of cyp512v2 gene in the carboxin-resistance strains was confirmed with PCR.In addition,qRT-PCR analysis was conducted to compare the cyp512v2 gene expression levels in the 9 transformants and WT.the results showed that the cyp512v2 gene was suppressed in the 9 transformants by about 0.52,0.75,0.76,0.27,0.03,0.36,0.23 and 0.49 folds lower than that of the WT.Three cyp512v2 gene silenced strains with the WT strains were chosen to study the content of GA-T in liquid shaking culture condition.The results showed that the content of GA-T in the 3 cyp512v2 silenced strains were reduced 13%-69% compared to that of the WT strain.Taken together,cyp512v2 gene may involve in the biosynthesis of GA-T in G.lingzhi.In summary,we obtained cyp512v2 that may be involved in the biosynthesis of GA.Further research such as yeast heterologous expression and the deletion/replacement of the cyp512v2 gene is necessary to clarify the role of cyp512v2 in GA biosynthetic pathway in G.lingzhi.The work should be helpful for understanding the biosynthesis and metabolic regulation of GA.