Construction Of Efficient Expression System Of Sesquiterpene Synthase GsSTS26 And GsSTS27 From Ganoderma Sinense

Lingzhi is a fungus of the basidiomycetes subphylum Aphyllum.As a traditional and precious Chinese medicinal material,it has a medicinal history of more than 2,000 years.Its main active ingredients are terpenoids and polysaccharides,which have pharmacological effects such as antibacterial,antiviral,antitumor,anti HIV,antioxidant and cholesterol lowering.With the successive completion of the functional characterization of 21 sesquiterpene synthase genes from E.coli in our group,GsSTS26 and GsSTS27 can produce three identical sesquiterpene compounds in E.coli Rosetta(DE3).The main product,Gleenol,has the effects of killing termites,anti-helminth,and regulating plant growth.The two by-products Di-epi-1,10-cubenol and ?-Muurolol also have high application value in agriculture.It is worth noting that the three products are all proposed for the first time in Basidiomycetes,have good pharmacological activities,and have high potential application value.However,the content of the three products produced in the Rosetta(DE3)strain is low,which is difficult to meet the industrial production and market demand.Therefore,it is of great significance to increase the content of several sesquiterpenoids.In this experiment,three methods were used to construct recombinant expression plasmids: the same promoter initiates the expression of tandem genes,and different promoters independently initiate the expression of two genes and multiple copies of the target gene.Heterologous expression of the recombinant plasmid was completed in E.coli,and HSSPME-GC-MS was performed on the E.coli in vivo culture,with β-caryophyllene as the internal standard.The relative contents of sesquiterpene synthase GsSTS26 and GsSTS27 sesquiterpene synthases were calculated by the internal standard method.The main research results are as follows:1)Among the recombinant plasmids with the same promoter for tandem gene expression,the GsSTS26 and GsSTS27 recombinant plasmids have different expression results.The gene of interest was respectively associated with the rate-limiting enzyme IDI(named as E.coli IDI)gene from the MEP pathway of E.coli,and the IDI cloned from Ganoderma lucidum(named as Gl IDI)gene tandem.The results showed that the fusion expression of the two IDI genes could increase the expression of GsSTS26 protein,and the content of Gl IDI was much higher than that of the fusion protein of E.coli IDI.Interestingly,although GsSTS26 and GsSTS27 belong to the same branch of the evolutionary tree,the homology is 100%,and the gene structure is very similar,but the results of fusion expression with IDI are different.Gl IDI and E.coli IDI fused with GsSTS27 did not increase the target protein content,which may be due to the weak expression of IDI-GsSTS27.We performed HS-SPME-GC-MS detection on recombinant strain E.coli in vivo culture.The data results show that in the expression plasmid of the tandem gene promoted by the same promoter.Whether it is E.coli IDI or Gl IDI,the relative content of sesquiterpene products is significantly increased when it is connected in series with GsSTS26,and Gl IDI has a stronger effect than E.coli IDI.In pET32a-Gl IDIGsSTS26 E.coli culture,the content of ?-Muurolol was increased the most,about 25.2-fold;the content of Gleenol was increased by about 22.5-fold;and the content of Di-epi-1,10-cubenol was increased by about 22-fold.In pET32a-E.coli IDI-GsSTS26 cultures,the contents of Gleenol,?-Muurolol and Di-epi-1,10-cubenol were increased about 16.6-fold,16.2-fold and14.44-fold,respectively.The recombinant plasmid with the gene GsSTS26 close to the promoter does not increase the product content as much as the gene is far from the promoter,about 6-8 times.Unlike GsSTS26,E.coli cultures of all GsSTS27 recombinant strains were analyzed,and the data showed that none of the recombinant strains had increased product content,and due to the extremely low content of ?-Muurolol itself,the chromatograms of its recombinant strains did not automatically integrate.The peaks were integrated,and the GC-MS results were also consistent with the protein expression results.2)In order to further enhance the expression of the target gene,a recombinant plasmid with different promoters independently promoting the expression of the two genes was constructed,and the Lac promoter was added between the tandem fragments of the two genes,so that the Lac promoter and the T7 promoter on pET32 a independently promoted the two genes expression.To explore whether the Lac promoter can enhance the expression of IDI-GsSTS26 and IDI-GsSTS27 fusion genes.The results showed that after adding the Lac promoter,in the E.coli strain of the GsSTS26 recombinant strain,the amount of the target protein did not increase,but was lower than that of the original T7 promoter.The difference from before is that the content of the target protein of the pET32a-Gl IDI-Lac-GsSTS27 recombinant plasmid after adding the Lac promoter is significantly increased.It indicated that the Lac promoter could enhance the expression of IDI-GsSTS27 fusion gene to a certain extent.The recombinant plasmids were used to express proteins heterologously in E.coli,and the in vivo cultures of E.coli were detected by HS-SPME-GC-MS.According to the results,the relative content of the gene GsSTS26 product was lower than that of the same promoter.The contents of Gleenol,Diepi-1,10-cubenol and ?-Muurolol in the pET32a-Gl IDI-Lac-GsSTS26 recombinant strain were increased about 15-fold,11.5-fold and 13-fold,respectively.The content of Gleenol in pET32 aE.coli IDI-Lac-GsSTS26 recombinant strain was increased about 12.8 times,and the content of Di-epi-1,10-cubenol and ?-Muurolol was increased about 12-fold and 11.2-fold.Interestingly,in the GsSTS27 recombinant strain,the content of sesquiterpene product was increased.In the pET32a-Gl IDI-Lac-GsSTS27 recombinant strain,the relative contents of the three products were increased by 3.6-fold,2.89-fold and 4.72-fold,respectively.The effect of pET32a-E.coli IDI-Lac-GsSTS27 was second,which were increased by 2.66-fold,2.04-fold and 2.65-fold,respectively.3)The two-copy recombinant expression plasmids of the genes GsSTS26 and GsSTS27 were constructed by isocaudal enzymatic method.Sep I and Nhe I were selected as the homologous enzymes of GsSTS26,and Sep I and Avr II were selected as the homologous enzymes of GsSTS27,and the gene two-copy vector was constructed by the steps of enzyme digestion and ligation.The experimental results showed that the multi-copy strategy of the target gene did not increase the protein content of the gene.The product content in the twocopy pET32a-GsSTS26-GsSTS26 recombinant strain hardly changed,but the product content in the two-copy pET32a-GsSTS27-GsSTS27 recombinant strain decreased,which may have a feedback inhibition effect.This experiment concluded that Gl IDI is the optimal fusion expression gene of GsSTS26 gene,and pET32a-Gl IDI-GsSTS26 is the optimal expression system.The gene GsSTS27 needs to be independently promoted by different promoters to be effective.Two copies of the target gene cannot increase the expression of GsSTS26 and GsSTS27.In this experiment,highefficiency expression systems of sesquiterpene synthases GsSTS26 and GsSTS27 were successfully constructed.The results of this study will lay a foundation and provide a basis for in-depth research on terpenoid biosynthesis in basidiomycetes.