Multiple-round Mutagenesis Of Antrodia Cinnamomea For High-level Production Of Triterpene

Antrodia cinnamomea is also called antrodia,red camphor etc.,grows on trunks of Cinnamomum kanehirai that is unique in the high mountain areas of Taiwan,China.Wild A.cinnamomea produces polysaccharides,terpenoids,ubiquinone derivatives and other biologically active substances.It is one of the most expensive wild medicinal fungi.At present,A.cinnamomea fruiting bodies is mainly cultivated artificially through the basswood cultivation method,which takes long time and gives low yield,and cannot meet market demand.Liquid fermentation is a kind of efficient culture method for A.cinnamomea because of its short cultivation time.However,the low-yield bioactive substances of A.cinnamomea limited the industrial application of liquid fermentation technology of A.cinnamomea.In this study,previously isolated A.cinnamomea wild-type strain AC16101was used as the starting strain for mutagenesis by ethyl methyl sulfone（EMS）and atmospheric and/or room temperature plasma（ARTP）.A total of 244mutants were isolated.Among them,mutant E3-64 produced the highest yield of triterpene with 175±17.3 mg/g,which was 88%higher than that of wild-type strain AC16101.The culture conditions for high-level production of triterpene by the mutant strain E3-64 was further optimized.The optimal medium components turned out to be glucose 85 g/L,yeast extract 10 g/L,Mg SO₄·7H₂O0.5 g/L,KH₂PO₄1 g/L,p H 5.0.The optimal cultivation conditions were found to be culture temperature 28℃,inoculum amount 1%,culture time 28 days.The content of triterpene in the optimized mutant strain E3-64 was 225±28 mg/g,which was 178%higher than that of wild-type strain AC16101.Liquid chromatography-mass spectrometry analysis showed that compared with the wild-type strain,the yield of ergostane triterpene Antcin A and Antcamphin A in the mycelium of mutant strain E3-64 were increased by 94%and 178%respectively,and the contents of Antcin B and Antcin G were decreased by 107%and 15%respectively.Fluorescent quantitative PCR analysis showed that the transcription levels of the genes encoding the key enzymes in the ergostane triterpene synthesis pathway lanosterol-14 alpha-demethylase,mevalonate pyrophosphate decarboxylase,oxidosqualene cyclase,squalene epoxidase and squalene synthase in the mutant strain E3-64 increased by 83.9%,206.1%,182.5%,59.7%,and 184.1%,respectively,in comparison with those of the wild-type strain.Furthermore,the genome of mutant strain E3-64 was re-sequenced.A comparative analysis indicated that the mutant E3-64 existed 1293 single nucleotide variants（SNVs）,of which,429 were located in the coding sequence（CDS）regions,470 located in the intergenic regions and 394 located in untranslated regions（UTRs）.A total of 692 nucleotide insertions/deletions（In Dels）in the mutant strain E3-64 were found.Among them,95 nucleotide insertions and 89 nucleotide deletions were localized in the CDS regions.There were 257 nucleotide insertions and 251 nucleotide deletions occurred in intergenic regions.It is worth noting that GME2365 has a nucleotide mutation in the CDS region,which is mainly involved in the biosynthesis of ubiquinone and other terpenoid quinones,and GME9224 has a nucleotide mutation in the CDS region,which participates in deoxygenation during the biosynthesis of the terpenoid backbone.KEGG functional annotation analysis shows that the proteins encoded by the mutant genes are mainly involved in lipid metabolism,amino acid metabolism,and carbohydrate metabolism,etc.