Construction Of The Taxadiene Biosynthetic Module And The Fitness Between The Module And Chassis

Biosynthsis of nature products is one of the main aims of synthetic biology. Terpenoids is an important part of nature products. It is widely used and has development prospect in the areas such as medicine and energy sources. In this article S. cerevisiae was taken as the chassis, and construct taxadiene artificial biosynthetic cell.Taxadiene is a kind of diterpenoids. Four molecules of isoprene pyrophosphate (IPP) generate geranylgeranyl diphosphate (GGPP) under the catalysis of the enzyme ERG20and GGPP synthetase, and then generate taxadiene under the cyclization of taxadiene synthetase. In this article, to funtuning the mevalonate pathway which is responsible for the synthesis of IPP, the N-terminal truncated HMG-CoA reductase gene(tHMGR), the mutant version UPC2.1from transcription factor UPC2were cloned, and the down-regulation device of the squalene synthase (ERG9) was constructed. Then, by introducing the synthetic heterology genes TS, GGPPSsa and CarG into chassis, I constructed the biosynthetic module of taxadiene and achieved the biosynthesis of taxadiene in S. cerevisiae. After that, I redesigned the taxadiene biosynthetic module by means of DNA Assembly, a new version of taxadiene biosynthetic module which was insert into a CEN plasmid was constructed.In this article, I fit the different taxadiene biosynthetic module with different chassis,20kind of taxadiene artificial biosynthetic cell were constructed, and I compared the taxadiene yield of these artificial cells in the fermentation with selective SD medium. The S. cerevisiae chassis used in this article were W303, YSG50, BY4742and the single knockout strains of BY4742:YPL062W、YIL074C YLR085C、YNL280C and YDR215C. The outcome of the fermentation showed that the taxadiene biosynthetic module constructed by DNA Assembler had higher taxadiene yield in every chassis than the other modules, and the assembled module in chassis YPL062W YIL074C、YNL280C and YDR215C had even high yield than the other chassis, the highest yield was13.52mg/LAt last, parts of the artificial cells were chosen to ferment in the medium of YPD. The data show that the production of the strains containing the module inserted into a single CEN plasmid was highly improved.The highest production of taxadie new as74.84mg/L.