Identification Of Key Genes In Antrodia Cinnamomea Triterpenoids Synthesis Pathway And Their Response To Environmental Factors

Antrodia cinnamomea,a precious edible and medicinal fungus in Taiwan,has anti-inflammatory,anti-tumor,and liver-protecting effects,attributed to abundant active compounds,such as triterpenoids.However,these active compounds have been in short supply for a long time due to the slow growth of A.cinnamomea fruit body.For artificial synthesis of A.cinnamomea triterpenoids（ACTs）,the relevant enzymes in the triterpenoid synthesis pathway and their regulatory mechanisms should be revealed,which is very meaningful for research and development of ACTs by fermentation or synthetic biology.Based on the optimization of solid and liquid fermentation in our previous study,the key enzyme genes in the triterpenoid synthesis pathway of A.cinnamomea were identified and expressed in E.coli for verifying the activity of the recombinant enzymes.Moreover,the response characteristics of key enzyme genes to different environmental factors in liquid fermentation were studied.The main results are as follows:1.Transcriptome sequencing analysis of A.cinnamomea mycelia form solid and liquid-state fermentation was performed.The results showed that the quality of sequenced bases and base distribution were good,and the sample correlation was high.The total number of differential expression genes（DEGs）was 1092.Compared to solid-state fermentation,there were 510 up-regulated DEGs and down-regulated 582 DEGs in liquid-state fermentation.In GO annotation analysis,DEGs were mainly involved in ion binding process,and catabolism process.In the KEGG annotation analysis,DEGs were mainly involved in the metabolic processes of starch and sucrose,drug metabolism,and CYP450 exogenous biotin metabolism.Combining the transcriptome sequencing results with the triterpenoid synthesis pathway,we obtained the ORF sequence of DEGs in the triterpenoid synthesis pathway under the two fermentation conditions.Two key enzyme genes,lanosterol synthase（LSS）and isopentenyl pyrophosphate isomerase（IDI）,were chosen for the subsequent study.2.The CDS sequences of the abovementioned genes（named AcLSS and AcIDI）were cloned in E.coli and analyzed by bioinformatics.AcLSS gene is 2205 bp and encodes 734amino acids.The protein RAcLSS is about 80.74 k Da and the isoelectric point is 6.13.The protein is an unstable hydrophilic protein without transmembrane region and signal peptide.AcIDI gene（810 bp）encodes 269 amino acids,and the protein is about 29.59k Da（p I 5.70）.The protein RAcIDI is also an unstable hydrophilic protein without transmembrane region and signal peptide.3.The recombinant plasmids p ET-AcLSS and p ET-AcIDI were transformed into the expression strain BL21（DE3）to construct the recombinant strains.After induction by 0.4m M of IPTG,the recombinant proteins were expressed successfully with correct sizes.The target protein is expressed in the supernatant.In vitro enzymatic reaction confirmed that recombinant RAcLSS catalyze the transformation from 2,3-oxidized squalene to lanosterol,indicating RAcLSS has the enzymatic activity of lanosterol synthase.4.Triterpenoid synthesis,biomass,and expression levels of AcLSS and AcIDI genes were analyzed under different fermentation conditions.When bran was used as carbon source,ACTs content and mycelial biomass were higher than those in the glucose and sucrose groups,but the expression levels of AcLSS&AcIDI genes did not change significantly.When ammonium sulfate was used as nitrogen source,ACTs content,biomass and the expression levels of AcLSS&AcIDI genes were lower than those of yeast extract and peptone groups significantly.Metal ions,such as Ca²⁺,Fe²⁺,Mn²⁺,and Mg²⁺,enhanced the biomass,but they reduced the triterpenoids content and inhibited the expression of AcLSS&AcIDI genes.With the respect of fermentation temperature,28℃was confirmed to be beneficial for the mycelial growth and ACTs synthesis.Moreover,different elicitors increased the mycelial biomass.Altough the co-solvent ethanol reduced ACTs content,the elicitors（methyl jasmonate,oleic acid,andα-terpineol）restored the triterpenoids synthesis.Oleic acid andα-terpineol significantly increased the expression level of AcLSS gene,but have no obvious effect on the expression level of AcIDI gene.