2.6 The pathway of the synthesis of triterpenes
The triterpenes have been reported of great importance in G.lucidum because of their significant roles in immune regulation and other biological activities［4- 7］.In plants，there are two pathways to synthesize terpenoids：the Mevalonate(MVA)pathway and MEP/DOXP pathway.It has been suggested that the MEP/DOXP pathway don't exist in fungi［8］.We checked the genes of G.lucidum in the“terpenoid backbone biosynthesis(map00900)”pathway，we found that the genes mainly distributed in the MVA pathway，almost no genes existed on the MEP/DOXP pathway.The same results were found in other basidiomycetes and ascomycetes.These verified that terpenoid backbone biosynthesis only could be through the MVA pathway in fungi at the genome level.
By integrating MVA pathway in KEGG and plant triterpenoid saponins biosynthesis from literatures，we summarized the potential triterpenoids biosynthesis pathway in G.lucidum(Figure 3).The pathway contained 14 steps catalyzed by different enzymes.The first 11 steps are the common steps for terpenoid skeleton biosynthesis and the last 3 steps may specific for different triterpenes and different species.We identified and summarized the enzymes in the ganoderic acids(GAs)biosynthesis in table 5.In this study，6 putative UDP-glycosyltransferases(UGTs)genes were found(Table 5).
Table 5 The putative genes involved in triterpene biosynthesis
Continued table
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Figure 3 Putative ganoderic acid biosynthesis pathway in G.lucidum
Enzymes involved in this pathway are：AACT：acetyl-CoA acetyltransferase，［EC：2.3.1.9］，K00626；HMGS：3-hydroxy-3-methylglutaryl-CoA synthase，［EC：2.3.3.10］，K01641；HMGR：3-hydroxy-3-methylglutaryl-CoA reductase，［EC：1.1.1.34］，K00021；MVK：mevalonate kinase，［EC：2.7.1.36］，K00869；MPK：phosphomevalonate kinase，［EC：2.7.4.2］，K00938；MVD：pyrophosphomevalonate decarboxylase，［EC：4.1.1.33］，K01597；IDI：isopentenyl-diphosphate isomerase，［EC：5.3.3.2］，K01823；GPPs：geranyl diphosphate synthase，［EC：2.5.1.1］，K00787，K00804；FPPs：farnesyl diphosphate synthase，［EC：2.5.1.10］，K00787 K00804；SQS：squalene synthase，［EC：2.5.1.21］，K00801；SE：squalene monooxygenase，［EC：1.14.99.7］，K00511；OSC：2，3-oxidosqualene-lanosterol cyclase，［EC：5.4.99.7］，K01852；P450：cytochrome P450，［EC：1.14.—.—］；UGTs：uridin diphosphate glycosyltransferases，［EC：2.4.1.—］.Ingredients are in blue box.Limited enzymes are in red oval，key enzymes are in green oval while the other enzymes are in pink oval.Solid arrows and broken arrows represent single and putative multiple enzymatic steps respectively.
Interestingly，we observed a fusion gene in triterpenes biosynthesis pathway in 12 Basidiomycete fungi except for L.bicolor.The N-terminal of the protein was similar to the enzyme K01760(KEGG ID，cystathionine beta-lyase，metC)，while the C-terminal of it was similar to another enzyme K00869(mevalonate kinase，MVK)which is one enzyme of triterpenes biosynthesis(Figure 4A).These proteins were referred as metC-MVKs in the following.In all other species except basidiomycetes，no such a protein matched the two enzymes at the same time.By multiple sequence alignment of the fusion genes in basidiomycetes，the average length of metC-MVKs was～886 aa and about half of it matched with K01760 and half matched with K00869.The metC-MVK gene was the only homologous protein with K00869 in our analyzed basidiomycetes，so they should be involved in terpenoid backbone biosynthesis functioning as K00869 in other species.We also noticed that this metC-MVK gene was the only one gene which best hit K01760.In addition，seven of the 12 Basidiomycota fungi had a conserved insertion sequence with 16 amino acids in the middle of the MVK regions of the metCMVK gene(Figure 4B).
Figure 4 The features of metC-MVK in basidiomycetes
(A)The metC-MVK matched two enzymes at the same time and some of them have an addition sequence in the middle of K00869(red line).(B)The added conservative sequence in 7 of 13 Basidiomycota fungi.