Analysis Of Triterpenoid And Polysaccharide High Yield Mechanism In Ganoderma Lucidum Based On Omics Technology

Ganoderma lucidum is a well-known edible and medicinal mushroom in China and also draw more attention in other parts of the world.Triterpenoid and polysaccharide are recognized as the major bioactive molecules of G.lucidum.However,the high costs and the low production of biologically active substances severely limits its wide application in various fields.The unclear biosynthesis pathways for active substances are the main reason for the phenomenon.Therefore,perfecting the triterpenoid and polysaccharide synthesis pathway and mining key regulatory genes have become the urgent problems to be solved for greatly improving the fermentation level of triterpenoid and polysaccharide,reducing production costs,and realizing its wide application.In this work,G.lucidum CGMCC5.26 is used to establish a new culture method for improving triterpenoid and polysaccharide production,and the biosynthesis mechanisms of triterpenoid and polysaccharide are revealed by omics technologies.The main results were summarized as follows:(1)The liquid superficial-static culture(LSSC)process was used to enhance the production of triterpenoid and polysaccharide.After optimizing,the best condition to produce triterpenoid in LSSC was initial medium height 0.8 mm,inoculum size 7.0 g/L,adding medium to 1.6 mm in 48 h and fermentative duration 7 d.Under the optimal conditions,the triterpenoid production was rechead 32.95±0.51 mg/g which was 2.10-fold than that in submerged culture,and the total polysaccharide production was 169.19±5.00 mg/g.The mycelia grown on the air-liquid interface in LSSC formed aerial mycelia in the air and submerged mycelia in liquid.The growing vigour of aerial mycelia(white mycelia)might be significant to triterpenoid production of G.lucidum in LSSC.Further,the triterpenoid production scaled 65.91±0.84 mg/g,being 4.19-fold that in submerged culture,and the productivity was 83.32 mg/L/Day which was the maximum that reported.(2)The biosynthetic mechanism of triterpenoid was investigated by comparing metabolites and transcriptome dynamics during LSSC and submerged culture.LSSC was a better method to produce ganoderic acid(GA)because eleven GAs and eleven GA derivatives were identified from mycelia by UPLC-QTOF-MS,and the content of all GAs was higher in LSSC than in submerged culture.Transcriptome dynamics analysis revealed eight hydrophobin genes were upregulated in LSSC,and hydrophobin was beneficial for aerial mycelia formation then inproving GA production.CYP5150L8 was the key gene regulating lanosterol flux into GA biosynthesis.Other sixteen CYP450 genes were significantly higher expressed during the culture time in LSSC and could be potential candidate genes associated with the biosynthesis of different GAs.Ergosterol was accumulated during the submerged culture process in G.lucidum.ERG11 was a key gene that regulates the flow of lanosterol to ergosterol synthesis.In addition,six of the ten expressed genes in ergosterol biosynthetic pathway shown upregulated at some time points in submerged culture.(3)The glycosyltransferases(GTs)and glycoside hydrolases(GHs)involved in the polysaccharide biosynthetic pathway of G.lucidum were obtained.Comparative genomics revealed that the nucleotide sugar biosynthetic pathways and glycosyltransferases were conserved in fungi.UDP-glucose,UDP-galatose and GDP-mannose could be synthesed in all the 37 strains,except the UDP-galatose in Pichia kudriavzevii.Twenty-two of the fourty glycosyltransferases families existing in the 37 strains.Other nucleoside sugar biosynthetic pathways and GT families were conserved in the same Phylum or Order.The GH family proteins associated with fungal polysaccharides biosynthesis were poorly conserved.Among the 35 GH families found in the 37 strains,only 11 families are relatively conserved.As glycoside hydrolases participate in the modification of polysaccharide,the abundant glycoside hydrolases might be related to the diversity of polysaccharides.In addition,we found 80 glycosyltransferases and 211 glycoside hydrolases in G.lucidum,and deeply analyzed the potential functions of 15 glycosyltransferases with unknown functions.Some of homologous proteins in ?-1,3-glucan,?-1,6-glucan,?-1,3-glucan and galactomannans biosynthetic pathways were annotated in G.lucidum.(4)Glycoside hydrolases played an important role in polysaccharide production of G.lucidum.When glucose and xylose were used as carbon sources,the yield of G.lucidum polysaccharides was significantly different.The transcriptome kinetics and proteomics results showed glycosyltransferases,GL20535-R1 and GL24465-R1(?-1,3-glucan synthase),GL22007-R1(mannosyltransferase)genes were highly expressed under glucose.In addition,compared with glycosyltransferases,there are a large number of glycoside hydrolases upregulated.Among them,GL15467-R1(GH16),GL15468-R1(GH16)and GL26618-R1(GH79)proteins are anchored on the cell membrane by GPI,and there are 19 proteins located in the extracellular space,including GL24039-R1(GH1),GL20743-R1(GH3),GL22586-R1(GH3),GL24911-R1(GH3),GL29912-R1(GH3),GL30087-R1(GH5),GL23820-R1(GH16),GL25603-R1(GH17),GL25033-R1(GH18),GL29255-R1(GH18),GL22188-R1(GH18),GL23450-R1(GH27),GL21451-R1(GH55),GL24581-R1(GH55),GL26459-R1(GH79),GL29243-R1(GH79),GL23706-R1(GH79),GL21185-R1(GH128),GL27365-R1(GH152).