Construction Of Engineered Saccharomyces Cerevisiae For High Yield Of Taxadiene And Kolavenol

Diterpenoids are a type of natural products with unique biological activity,and are widely used in food,pharmaceutical,and chemical industries.Taking the anticancer drug paclitaxel as an example,its important biosynthetic precursor,taxadiene,is generated from geranylgeranyl diphosphate(GGPP)by taxadiene synthase(TS).At present,the taxadiene production in Saccharomyces cerevisiae is much lower than that within prokaryotic chassis.This study aimed to discover the bottleneck of taxadiene synthesis in yeast(from the aspects of the subcellular distribution of TS and GGPP as well as the expression issue of TS),and improve the taxadiene titer through rational and irrational methods.And the similar research strategy is also used to achieve the biosynthesis of another diterpenoid compound,kolavenol.Previous studies have shown that GGPP synthase(GGPPS)located in cytosol,while t60TS(the TS truncated at the 60 th amino acid of its N-terminus)is located in the mitochondria of the existing taxadiene synthesis strain,suggesting the subcellular distribution of GPPS with t60 TS is not coordinated.In order to eliminate this problem,three strategies was adopted as(1)N-terminal truncation of TS,(2)t60TS fussed with GGPPS,and(3)GGPPS fused with mitochondrial targeting signal peptide.These strategies attempts to express TS and GGPPS simultaneously in the cytoplasm or mitochondria.As the results,the truncation at the 84 th amino acid at the N-terminus of the TS(t84TS)and the GGPPS-t60 TS fusion increased the taxadiene production by 26%and 115%,respectively.Further increasing the copy number of GGPPS-t60 TS by changing the expressing plasmid into a multi-copy one increased the taxadiene production to 24.9 mg/L which was 3.4 times of that in the original starting strain.In the meanwhile,western blot assay found that t60 TS was modified and cleaved when expressed in yeast.Three lysine sites,K760,K836,and K849,were selected at the junction of TS and the substrate.Site-directed mutation.It was found that the point mutation K760 R can increase the yield of taxadiene by 36% compared with the unmutated sequence,which proves that K760 is likely to be modified to cause low TS enzyme activity In addition,plasma mutagenesis(ARTP)was applied to conduct mutagenesis of the initial taxadiene producing strain.We gained a strain with 25%increase on taxadiene production of the initial strain.This strain would generate a better chassis for taxadiene synthesis to express the optimal TS sequence obtained from our rational design.At the same time,through combined screening three different sources of kolavenol synthase(CPS)in S.cerevisiae,the kolavenol biosynthesis was firstly achieved in yeast.And the effect of forward and reverse fusion of CPS and GGPPS on kolavenol titer was also tested by combining three different sources of KPS with two sources of GGPPS.The result demonstrated that the At CPS:H263Y-Sa GGPPS fusion strain have the kolavenol titer of 22.1 mg/L among all the tested combinations.This study explores the bottleneck factors that limit taxadiene in S.cerevisiae,as well as improves the synthesis level of taxadiene through rational and irrational design.It also realizes the de novo synthesis of kolavenol in yeast for the first time.It provides a promissing reference to produce other diterpenoids in S.cerevisiae.