Studies On The Construction Of The P450 Reductase Gene Replacement Vector For The Optimization Of The Engineered Artemisinin Producing Yeast

Artemisinin is a sesquiterpene compound that was first isolated from Sweet Wormwood Herb(Artemisia annua L.).A lot of work have been done in our lab at artemisinin production in the metabolic engineering approach.And we have got an engineered yeast which produces amorpha-4,11-diene and artemisinin.Cytochrome P450 reductase(CPR) plays an important role in the biosynthetic metabolic pathway of artemisinin.It participates all of the 3 steps from amorpha-4,11-diene to artemisinic acid.It is considered that the catalytic efficency of CPR strongly affect the production level of the artemisinin.However,the CPR in WHT does not come from A.annua but from Helianthus tuberosus.The encoded DNA sequences of the two enzymes only have 75%identity.It is indefinite about the catalytic activity of the Helianthus tuberosus CPR compared with the CPR from A.annua.And the genetic backgroud correlate with the cpr gene in WHT is also unclear.A series of tests have been done on WHT to identify and clone the sequences correlated to Helianthus tuberosus CPR, including the promotor,terminator,and complete A.annua.L CPR gene in this article. And then,a step by step ligation have been carried out and a integrative plasmid have been constructed.This plasmid contains the 5'CPR flanking,promotor,A.annua.L CPR gene,KanMX4 gene and 3'CPR flanking.It could take along the A.annua.L CPR gene integrate into the WHT genomic DNA.And it is a found work for the WHT optimization.