Metabolic Engineering To Improve Cannabigerolic Acid Production In Pichia Pastoris And Gene Mining Of Cannabigerolic Acid Synthase

Cannabis sativa contains more than 500 compounds,in which cannabinoid is the main compound showing bioactivities.Cannabinoid was reported to exhibit a positive effect on some diseases,such as chronic pain,epilepsy,and amyotrophic lateral sclerosis.Cannabigerolic acid(CBGA)is the metabolic precursor of cannabinoid,thus the synthesis of cannabinoids by microbial cells indicates significances for its industrial application.Pichia pastoris is the methyl-trophic yeast that has some advantages,including strong promoter,high density fermentation,high efficiency of exogenous protein expression,and growth in cheap medium,which reveals the potential for produce of CBGA.In this study,the original strain Pichia pastoris GMva07 was modified and optimized via serious of metabolic engineering strategies,such as optimizing the copy number of key enzymes,removing the speed-limiting step,strengthening the supply of precursor acetyl-Co A,deleting the competitive pathway,and peroxisome compartment engineering,which resulted in the improvement of the production of CBGA.In addition,according to the reported information of CBGA synthases,the potential CBGA synthase sequences were further investigated in NCBI database,and two potential CBGA synthase sequences were found.The main results are as follows:(1)Modifications on peroxisome of Pichia pastoris.the isopentenol utilization(IUP)pathway of was constructed in the peroxisome,firstly.Then,the latest strain was modified via constructing the synthesis pathway of CBGA,and the copy number of key enzymes in this pathway was further increased.The resulting strain GMIup04 produced 1.23 mg/l CBGA,which is 56% of increase compared with the original stain GMva07.(2)Enhancing the synthesis of CBGA via metabolic engineering modifications on the cytoplasm of Pichia pastoris.Firstly,the fusion expression of ERG20 and Nph B was conducted to increase the copy number of key genes.Then,the rate-limiting enzyme HMGR was overexpressed to eliminate the feedback inhibition of MVA pathway.Furthermore,considering acetyl-Co A is the original substance compound of MVA pathway,exogenous citrate lyase(ACL)and Ec PDH were expressed to accumulate acetyl-Co A in cytoplasm.Moreover,the ethanol metabolic pathway was weakened to improve the production of CBGA.The resulting strain produced 19.04 ± 0.24 mg/l CBGA,which is 25-fold increase relative to the control strain GMva07.(3)Two new potential CBGA synthases Ptfst and St PT2 were obtained by mining the NCBI database.Meanwhile,Ptfst has been successfully expressed in E.coli.The catalytic products of Pftst were analyzed by LC-MS.The mass-to-charge ratio of Ptfst was similar to that of the by-product Nph B(WT)under ESI(-)mode.It means that Ptfst can catalyze OA and GPP to CBGA’s isomer 2-O-GOA(2-O-geranyl olivetolic acid).