Cloning And Functional Characterization Of Sesquiterpene Synthases GsSTPS1 And GsSTPS2 From Ganoderma Sinense

Lingzhi is a traditional chinese medicine,which has anti-tumor and immunomodulatory activities.At present,the research on the active components of Lingzhi mainly focuses on triterpenes and polysaccharides,but few studies on sesquiterpenes.The two basic sources of Lingzhi,Ganoderma sinense and Ganoderma lucidum,have completed the sequencing and data analysis of genome and transcriptome.Based on the genomic and transcriptome data of Ganoderma sinense,the screening,cloning and characterization of sesquiterpene synthase from Ganoderma sinense were carried out,and the sesquiterpene synthases and sesquiterpene compounds in Ganoderma sinense were analyzed.The main results are as follows:（1）By comparing the genomic data and transcriptional data of Ganoderma sinense,it was found that the c DNA sequence of Gs STPS1 was completely identical except that the c DNA sequence of Gs STPS2 was 42 bp less at the 5’end.The open reading frame（ORF）of Gs STPS1was 1029 bp,encoding a 342-amino acid protein.The ORF of Gs STPS2 was 1071 bp,encoding356-amino acid protein.Gs STPS1 and Gs STPS2 had D（D/E/N）XX（D/E）and NSE/DTE motifs,which are conservative motifs of sesquiterpenoid synthases and responsible for the binding of Mg²⁺.（2）In order to study the difference between Gs STPS1 and Gs STPS2,we cloned them and transformed them into E.coli for heterologous expression.It was found that there was no target protein expression when the Gs STPS1 and Gs STPS2 genes were ligated into p ET28a vector and transferred into BL21（DE3）.The heterologous expression could be finished by ligating them to p ET32a and transferring them into Rosetta（DE3）.The results fully showed that the heterologous expression of genes in E.coli is closely related to the vectors and strains.The soluble expression of Gs STPS1 and Gs STPS2 was different in E.coli Rosetta（DE3）,and the soluble expression of Gs STPS2 was significantly higher than that of Gs STPS1.By optimizing the cell density OD₆₀₀,induction temperature,time and IPTG concentration,the soluble expression of Gs STPS1 was increased from 9.87%to 13.12%,and the soluble expression of Gs STPS2 was increased from 17.62%to 28.07%.The best induction culture conditions for Gs STPS1 were as follows:the initial bacterial solution OD₆₀₀ value was 0.8,the induction temperature was 18℃,the induction time was 16h,and the final concentration of IPTG was 0.7 m M.The best induction culture conditions for Gs STPS2 were as follows:the initial bacterial solution OD₆₀₀ value was 0.8,the induction temperature was 18℃,the induction time was 12 h,and the final concentration of IPTG was 1 m M.（3）The results of HS-SPME-GC-MS analysis of the enzyme products showed that the enzyme products of Gs STPS1 and Gs STPS2 were different,and the enzyme activity of Gs STPS2 was much higher than that of Gs STPS1.Gs STPS2 could produce terpenoids（α-cubebene,trans-β-copaene,β-copaene,cadina-3,5-diene,cis-muurola-4（15）,5-diene,germacrene D,α-muurolene,γ-cadinene andδ-cadinene）and sesquiterpenoid oxygen-containing derivatives（gleenol,epicubenol andτ-muurolol）,while Gs STPS1 could only produce terpenoids（β-bourbenene,β-copaene,cis-muurola-4（15）,5-diene,γ-muurolene and germacrene D）.HS-SPME-GC-MS analysis of E.coli cultures results showed that Gs STPS1 had no catalytic activity in E.coli,Gs STPS2 catalyzed production of sesquiterpenoid oxygen-containing derivatives gleenol,epicubenol andτ-muurolol in E.coli.（4）The results of HS-SPME-GC-MS analysis of Saccharomyces cerevisiae cultures showed that Gs STPS1 did not catalyze the production of new sesquiterpene compounds in Saccharomyces cerevisiae,but doubled the production of sesquiterpene compoundα-nerolidol in Saccharomyces cerevisiae.Gs STPS2 catalyzed the production of sesquiterpenoid cubenene and sesquiterpenoid oxygen-containing derivatives（gleenol,epicubenol andτ-muurolol）in Saccharomyces cerevisiae.Gs STPS1 and Gs STPS2 in vitro enzyme products,E.coli in vitro products and Saccharomyces cerevisiae in vitro products had certain differences in type and quantity,indicating that the reaction microenvironment could affect the enzyme activity and product specificity of sesquiterpene synthases.（5）The results of subcellular localization of Gs STPS1 and Gs STPS2 were different.The main difference was that the green fluorescent protein co-expressed by Gs STPS2-m GFP5 was not localized in the nucleus,while the green fluorescent protein co-expressed by Gs STPS1-m GFP5 could be localized in the nucleus.The localization of the protein can reflect the function of the protein to a certain extent.The difference in the location of Gs STPS1 and Gs STPS2indicated that they might perform different functions in Ganoderma sinense.