Saccharomyces Cerevisiae Construction For Heterologous Synthesis Of Levopimaric Acid

Levopimaric acid,an important diterpene resin acid in conifers can help defense against insects and pathogens.Levopimaric acid is also a significant industrial intermediate,and it's Diels-Alder reaction products are widely used for coatings,printing inks,plasticizers and adhesives.Some levopimaric acid derivatives show anti-inflammatry,cardiovascular and antioxidant high value-added activities.In this study,heterologous pathway of Levopimaric acid biosynthesis was introduced into Saccharomyces cerevisiae,and with optimizing,the production of Levopimaric acid increased.Firstly,expression vector with levopimaradiene synthase was constructed and transformed into strain WTI-BE,and levopimaradiene was detected.Then modifications of LPS protein were taken,including truncating N-terminal 40 aa,60aa,79 aa and site-directed mutants.The results showed that strain with T79?LPS produce highest levopimaradiene(6.9mg/L),and was 23 times as much as LPS(0.3mg/L).Protein fusion between T79?LPS and Bts1 was performed,and strain WTI-BE-(Bts1-linker4-T79?LPS)produced 10.2mg/L levopimaradiene,which was the highest.A yeast chassis WTE-9 was constructed for preferable levopimaradiene biosynthesis.Overexpressing Bts1 and T79?LPS in WTE-9 made the yield of levopimaradiene reached 49.2mg/L.Then,the CYP720B1 and CPR suitability experiments were carried out.The result comfirmed AtCPR1 from Arabidopsis thaliana and TcCPR from Taxus cuspidate can help CYP720B1 catalyze levopimaradiene to form Levopimaric acid,and TcCPR performed better suitability.Finally,CYP720B1 and TcCPR were overexpressed in WTE-9-Bts1-T79?LPS multiple to construct strain WTE-9-Bts1-T79?LPS multiple-CYP720B1 multiple-TcCPR multiple and the yield of levopimaric acid was 45.2mg/L.Through the shake flask feeding experiment and baffle shake flask experiment,the production of levopimaric acid increased to 109.5mg/L.