Characteristics Of Farnesene Synthase And Farnesene Synthesis In Saccharomyces Cerevisiae

Farnesene is a sesquiterpene compound which has a wide range of applications in plant defense and fuel production.Farnesene synthase（FS）is the key enzyme in the synthesis pathway of farnesene,which can catalyze farnesyl pyrophosphate（FPP）to form farnesene.Current research on farnesene synthase is insufficient,which hinders its application in synthetic biology.In this study,three terpene synthases from Citrus sinensis（FSCS）,Cajanus cajan（FSCC）and Glycine max（FSSO）were selected and expressed in Escherichia coli to study their enzymatic properties.Enzymatic properties were then used to guide farnesene production in recombinant Saccharomyces cerevisiae strains,which provided solid reference for the construction of microbial cell factories to realize the efficient production of farnesene.The main research results are as follows:（1）pET28a-fscs,pET28a-fscc and pET28a-fsso were constructed and transformed into E.coli BL21（DE3）to obtain recombinant strains E.coli BL21（DE3）/pET28a-fscs,E.coli BL21（DE3）/pET28a-fscc and E.coli BL21（DE3）/pET28a-fsso.The expression of recombinant protein was induced by adding 0.5 mmol·L^-1IPTG under 18℃.Three recombinant enzymes were functionally characterized through in vitro enzyme reaction and product detection.p TIG-TRX was used as an expression vector to enhance the soluble expression of three FSs.And the enzyme activity of FSCS,FSCC and FSSO increased by 3.4,4.0 and 3.2 times under the same induce condition,respectively.（2）Three FSs were purified by Ni²⁺affinity chromatography to study their enzymatic properties.The results indicated:When FPP was used as the substrate,all FSs could catalyze the formation ofα-farnesene.Farnesol was also detected in the crude enzyme,but its formation was not related to the recombinant enzyme,it was generated under the catalytic reaction of phosphorylase.Moreover,all FSs required Mg²⁺as cofactor,and FSSO required both Mg²⁺and K⁺as cofactors.Three FSs reached their highest enzyme activity and remained stable at 20 mmol·L^-1Mg²⁺and above.K⁺had a significant promotion effect on enzyme activity.FSCS and FSCC reached highest enzyme activity under 30 mmol·L^-1K⁺and above,which was 3.3 and 3.7 times higher than adding Mg²⁺alone.No enzyme activity was detected for three FSs when Mn²⁺was added alone,and Mn²⁺above 5 mmol·L^-1inhibited the enzyme activity based on the addition of Mg²⁺and K⁺.The optimal reaction temperatures of three FSs were:FSCS 20℃,FSCC 25℃,FSSO 25℃,and the optimal reaction p H of three FSs were:FSCS 7.0,FSCC 7.5,FSSO 7.5.（3）All three FSs and FS from Malus domestica（FSAP）were expressed in S.cerevisiae to construct four recombinant strains WYP4-Fscs,WYP4-Fscc,WYP4-Fsso and WYP4-Fsap.And the enzymatic properties were applied to recombinant strains to guide the synthesis of farnesene.The application of optimal metal ion conditions had the most positive effect on farnesene production.Farnesene yield of WYP4-Fscs increased by 30%to 43.76 mg·L^-1,farnesene yield of WYP4-Fscc increased by 40%to 44.99 mg·L^-1,farnesene yield of WYP4-Fsso increased by 18%to 141.76 mg·L^-1,and farnesene yield of WYP4-Fsap increased by 14%to 3.65 mg·L^-1.The optimal temperature and p H conditions of FSs would affect bacteria growth,but farnesene accumulation was improved in the late fermentation stage.The integration of optimal enzymatic properties conditions were then applied to recombinant strains.Compared with the original fermentation conditions,WYP4-Fscs farnesene yield increased by 43%to 48.54 mg·L^-1,WYP4-Fscc farnesene yield increased by39%to 46.39 mg·L^-1,WYP4-Fsso farnesene yield increased by 23%to 148.34 mg·L^-1,and WYP4-Fsap farnesene yield increased by 20%to 3.95 mg·L^-1.