Heterologous Biosynthesis Of Ginsenoside Compound K And Oleanolic Acid In Engineered Yarrowia Lipolytica

Ginsenoside compound K is a tetra-triterpenoid dammarane-type ginsenoside in ginseng which presents antitumor and antioxidation effects.And protopanoxadiol,the precursor of ginsenoside CK,also presents antitumor,antidepressant and antioxidation effects.Oleanolic acid as precursor of pentacyclic triterpenoids also presents anti-inflammatory,antibacterial,anti-ulcer,anti-oxidation and hypoglycemic functions.However,the production capacity and quality are greatly restricted by the traditional plant extraction and production methods of these natural products limited by the shortage of raw materials,pesticide residues and environmental pollution.First of all,the pathway of CK synthesis was constructed in Yarrowia lipolytica ATCC 201249,and protopanoxadiol synthesis strain was obtained through coexpress cytochrome p450 monooxygenase PPDS and NADPH-P450 reductase ATR1.Level of single gene and multiple genes overexpression including MVA pathway and 2,3-oxidized squalene synthesis pathway genes were taken.After introducing the key glycosyltransferase(UGT1)of ginsenoside CK production to the highest protopanoxadiol production strain,shake flask production of ginsenoside CK achieved 21.7 mg/L,which was the highest yield reported.Secondly,we reconstructed oleanolic acid synthesis pathway by introducing mixed AS synthase,cytochrome P450 monooxygenase CYP716A12 and ATR1 into Yarrowia lipolytica ATCC 201249.Engineering strains was obtained by optimization of precursor metabolic pathway whose production oleanolic acid was 48.1mg/L.Then,engineering strain was obtained by introducing ?-amyrin synthesis gene b AS,CYP716A12 and ATR1 into Yarrowia lipolytica ATCC 201249.The positive rate of the three gene fragments was increased to around 55% from 1.5% by multi-fragment recombination integration of the zeta site.Finally,the optimal genes overexpression combination of MVA and downstream precursor pathway were integrated in zeta multi-copy site,and the production of the oleanolic acid in strain YL-OA7 was 92.1 mg/L.We systematically analyzed the changes of the intracellular metabolism of the engineering YL-CK0,YL-OA7 and the original strain ATCC 201249.The amino acid metabolism of glutamine,prosine,isoleucine,tryptophan was associated with the synthesis of triterpenoids,which confirms the competitive substrate acetyl-co A between the MVA pathway and the fatty acid synthesis is the synthetic pathway.When P450 and its reductase ATR1 were coexpressed,the relative content of hexadecenoic acid(C16:1)and palmitic acid(C16:0)increased,indicating that the P450 enzyme system in the fatty acid metabolism process could compete ATR1 to deliver the electron from NADPH.Thus,the activity of PPDS and CYP716A12 cannot be fully activated,resulting in incomplete conversion of the precursor DMD or amyrin.In the synthesis of triterpenes,the MVA pathway and the downstream precursor pathway genes resulted in the increase the accumulation of DMD or amyrin,while the accumulation of 2,3-oxidesqualene increased genes overexpression in lanosterol synthesis pathway,resulting in the synthesis of metabolites to lanosterol.Finally,we constructed and optimized the fusion protein of PPS-ATR1 and CPY716A12-ATR1.The effects of P450 enzyme and ATR1 transmembrane division and length of linker on the vitality of P450-ATR1 were studied.Through rational optimization,the conversion rate of catalyzed substrate by fusion protein was increased by 38%,and the conversion efficiency of the DMD increased from 71% to over 98%.The conversion rate of ?-amyrin increased from 82% to nearly 100%.Supplemented by building MVA pathway optimization process,the fusion protein PPDS-ATR1,CYP716A12-ATR1 can play a stable role with high catalytic activity and conversion efficiency.The production of ginsenosides reached 161.8 mg/L,and the yield of oleanolic acid was 540.7 mg/L in 5 L fermenter.