Construction And Optimization Of Pichia Pastoris Strain For Cannabidiol Acid Synthesis

Cannabidiol has a positive regulatory effect on various metabolic,immune and physiological functions of the human body.Therefore,cannabis drugs can be used to treat viral diseases,and can also be used as raw materials for fiber and oil,which has great research value.At the same time,it can also be used as a raw material for fiber and oil,which has great research value.Cannabis compounds are isolated from hemp plants,but there are problems such as long cycle,large environmental impact,and impure products.Heterologous host production may be an effective alternative.Pichia pastoris has been widely used as a protein production plant.In recent years,Pichia pastoris has also been used as a host for metabolic engineering to conduct many studies to produce valuable products.In this paper,the potential ability to synthesize cannabinoids in Pichia pastoris is explored,which provides a research foundation for the de novo biosynthesis of cannabidiol acid and monoterpenoid synthesis.The specific research content and results are as follows:（1）Construction of Pichia pastoris strain for synthesis of cannabidiol acidOverexpression of isopentenyl transferase and cannabidiol acid synthase CBDAS,respectively,to achieve the co-expression of these two enzymes in Pichia pastoris.After gene source screening and gene copy number optimization,the GNC03 strain containing Nph B^G286S-Y288Afrom Streptomyces sp.and three copies of CBDAS gene was fermented and cultured with 1m M olivetolic acid（OA）as a substrate for 96 hours,can get 0.22 mg/L cannabigerolic acid（CBGA）..（2）Optimization of the biosynthetic pathway of cannabidiol acid side chainTransform the flux of the precursor side chain GPP,including the optimization of the mevalonate（MVA）pathway and the introduction of the heterologous pathway IUP.The optimization of the MVA pathway was to overexpress EfmvaE,EfmvaS*,ERG19,ERG8,ERG12,IDI1,ERG20^wwin the GNC03 strain,and the recombinant strain was cultured to obtain 0.97 mg/L CBGA,which was 4.4 times that of the control strain GNC03 without these genes.（3）Optimization of fermentation process for synthetic cannabidiol acid recombinant strainThe shake flask fermentation process was optimized for the concentration of substrate OA,the time of adding substrate OA,the induction concentration of methanol,the pH of the medium and the temperature.The pH of the medium and the induced concentration of methanol have a relatively large effect on the yield of CBGA,and the time of OA addition has almost no effect.When pH=5.0,the yield of CBGA reaches 1.73 mg/L,which is more than 1times higher than pH=6.0.When the induced concentration of methanol was 2%,the yield of CBGA reached 4.66 mg/L,which was 2.2 times higher than when the methanol was 1%.