Identification Of Unique Oxidosqualene Cyclase Genes And Biosynthesis Of Plant Triterpenes In Microorganisms

Triterpenes are widely found in plants and show various pharmaceutical functions,such as hemolysis,antibacterial,anti-inflammatory,anti-tumor,anti-cancer,anti-virus,and cholesterol-lowering activities.Tetracyclic(e.g.euphol and tirucallol)and pentacyclic(e.g.lupeol)skeletons represent two types of the structural scaffolds for the triterpenes.Oxidosqualene cyclase(OSC)cyclizes the precursor 2,3-oxidosqualene to form various kinds of triterpenes and steroids.Identification of the unique OSC genes involved in the pathways to different triterpenes would not only help to understand the molecular mechanism underlying the specificity for the triterpene skeleton formation,but also provide necessary gene components for engineering their biosynthesis in surrogate hosts.To date,it is still not clear how the unique triterpene euphol is formed and the OSC gene responsible for its biosynthesis has not been isolated from any of the plant species.Because that the plant species Euphorbia tirucalli L.produces a high amount of euphol in its latex,a transcriptome data set was constructed from different E.tirucalli tissues,including its latex sample.Through an extensive bioinformatics analysis of the transcriptome,a complete list of the genes involved in the biosynthesis of euphol were discovered in the database,especially the OSC gene for euphol synthesis was identified by this study.On the other hand,to establish a powerful pipeline for engineering the biosynthesis of plant triterpenes in microorganisms,we assessed the triterpene biosynthetic pathway in Escherichia coli or yeast hosts using the lupeol pathway as a maker system.Finally,we were successful in creating a robot yeast factory for producing lupeol in a relatively high titer.The results obtained from this study were summarized in the following three parts:(1)Comparative transcriptome analysis and expression analysis of the OSC encoding genes from E.tirucalli:Phytochemical analysis of different E.tirucalli tissues revealed a contrasting tissue-specificity for the biosynthesis of triterpenoids and sterols: euphol and tirucallol mainly accumulated in the latex,whereas ?-sitosterol did not accumulate in the latex.A comprehensive transcriptome of E.tirucalli derived from its root,stem,leaf and latex was constructed using the Illumina HiSeq X Ten sequencing platform and a total of 22.87 G nt of raw data was obtained.91,619 unigenes were generated by de novo assembly with 51.08% being successfully annotated against the non-redundant(NR)protein database.A total of 2922 unigenes associated with the secondary metabolite pathways were relatively highly expressed in the latex.The up-regulation of the transcriptional levels of most genes in the upstream steps of triterpenoid backbone biosynthesis(except the MEP pathway)led to the large accumulation of euphol in the latex.The differentially expressed genes(DEGs)discovered in the different E.tirucalli tissues revealed the regulation mechanism for the tissue-specificity in the biosynthesis of triterpenoids and sterols.Among them,seven of the putative OSC encoding genes were notably identified.EtOSC5 and EtOSC6 were significantly up-regulated in the latex,which might be the candidate genes for euphol and/or tirucallol biosynthesis.(2)Functional analysis of E.tirucalli EtOSC5 and EtOSC6:The putative OSC encoding genes EtOSC5 and EtOSC6 were introduced into the lanosterol-deficient Saccharomyces cerevisiae strain GIL77 for heterologous expression and functional verification.Both EtOSC5 and EtOSC6 were multifunctional triterpene synthases.EtOSC5 cyclized 2,3-oxidosqualene to yield euphol and tirucallol,at the yields of 0.81 and 0.41 mg/L,respectively.EtOSC6 cyclized 2,3-oxidosqualene to produce ?-amyrin and taraxasterol,with the yields being 0.53 and 1.74 mg/L,respectively.Site-directed mutagenesis revealed the sites of E123,F261 and I534 on the amino acid sequence of EtOSC5 had significant effects on the production of euphol,whereas showed less influences on the formation of tirucallol.After mutating the LADQ(475-478)sites of EtOSC5 to FSTA(475-478),the new product generated by the mutant might be a isomer of euphol,tirucallol,and lanosterol.Similarly,the consecutive NL(415-416)sites on the amino acid sequence of EtOSC6 played important roles in the proton rearrangement of germanicyl cation and biosynthesis of taraxasterol.After mutating the NL(415-416)sites of EtOSC6 to FS(415-416),the product generated by the mutant became ?-amyrin.(3)Construction of a highly triterpene-producing platform using the lupeol pathway as a marker system:The candidates of three lupeol pathway genes(SQS,SE and LUP)were respectively selected from different organisms and codon-optimized for their expressions in E.coli or yeasts.After comparing their in vivo performance,the squalene synthase from Thermosynechococcus elongatus BP-1(tSQS),squalene epoxidase from Rattus norvegicus(rSE)and lupeol synthase from Olea europaea(OeLUP)were confirmed to have higher activities compared with their respective counterparts investigated here.As a result,tSQS,rSE and OeLUP were firstly introduced into E.coli to reconstitute the lupeol synthesis pathway,however,only a trace amount of lupeol could be detected.Then these three genes were recruited to reconstitute the lupeol pathway in two S.cerevisiae strains WAT11 and EPY300.The engineered strain EPY300(namely strain ECTROe)co-expressing these three genes was found to process the best lupeol-producing ability with the lupeol yield being 225.7 mg/L at 30°C for 72 h,which was 27-fold higher than that produced by previously reported engineered yeast strain NK2-LUP.In this study,two new OSCs were identified from E.tirucalli.Both of them are multifunctional triterpene synthases.EtOSC5 cyclized 2,3-oxidosqualene to yield euphol and tirucallol,respectively.EtOSC6 cyclized 2,3-oxidosqualene to produce ?-amyrin and taraxasterol,respectively.The identification of two new OSCs will contribute to the polymorphism of oxidosqualene cyclases in plants and and provide new theoretical basis for the analysis of the catalytic mechanism of OSCs.The E.coli and S.cerevisiae are two well-established ‘Microbial factories',which have been widely used for engineering the production of plant chemicals.In this study,lupeol has been biological synthesized in E.coli for the first time.Secondly,the lupeol pathway was reconstituted by recruiting the better pathway candidate genes,which were identified from the screening experiments.Finally,a highly lupeol-producing yeast strain ECTROe was engineered by this study,which will provide a new reference for the triterpene biosynthesis.