Heterologous Biosynthesis Of Betulinic Acid By Engineered Yarrowia Lipolytica

Betulinic acid(BA)is a pentacyclic lupane type of triterpenoid,which has many pharmacological activities such as anti-tumor,anti-virus and anti-malaria,showing great promise in anti-melanoma and anti-HIV especially.Betulinic acid is usually acquired by extracting from plants,chemical synthesis and microbial catalysis,however,these methods cannot meet the demand for it.Recently,synthetic biology is a hotspot and constructing genetic engineering strains to produce betulinic acid could help solve the problem of low production of betulinic acid.Yarrowia lipolytica engineering strains for heterologous biosynthesis of betulinic acid were constructed,and key genes of the biosynthetic pathway were regulated to further improve betulinic acid production with fermentation conditions optimized.At first,Yl-opAtLUP1 was constructed,successfully converted 2,3-oxidosqualene to the precursor lupeol.Then Yl-BA1,with opAtLUP1,opMtCYP716A12 and opAtCPR1 integrated into the genome of Yl201249,was constructed and 0.32 mg/L betulinic acid was produced.Fusion of opMtCYP716A12 and t46optAtCPR1 increased the efficiency of P450 electron transfer,making Yl-BA2 with 1.53 mg/L betulinic acid.Key genes tHMG1,erg9,and opAtLUP1 were integrated into the genome of Yl-BA2,making strains Y1 to Y13.The engineered strain Y13 produced 9.41 mg/L of betulinic acid,6.15-fold to that of Yl-BA2.Fermentation with 200 g/L glycerol as carbon source in 5 L fermentor conducted 30.96 mg/L betulinic acid,which was 3.29-fold to 9.41 mg/L.Y13 produced 26.63 mg/L betulinic acid with 40 g/L glycerol as carbon source in shake flaskes,and the yield is higher than that of engineered Saccharomyces cerevisiae with 50 g/L glucose as carbon source in shake flaskes,but the production in 5 L fermentor is needed to be improved.