Construction Of Cell Factories For Production Of Medical Triterpenoid Acids

Triterpenoid acids, such as betulinic acid and oleanolic acid, are widely used in medical industries for their diverse pharmacological properties with anti-tumor, anti-HIV and liver protective activities, et al. Direct extraction from natural plants and chemical synthesis of triterpenoid acids have many problems, such as environmental pollution, low productivity and high energy consumption, Recently, microbial fermentation method has attracted much attention due to its low production cost and environmentally friendly production process. Since the physiological and genetic characteristics of Saccharomyces cerevisiae, which is a safe model eukaryote, have been widely investigated and mature genetic engineering tools and large scale fermentation technology have been developed, fermentative production of plant-derived natural products by recombinant Saccharomyces cerevisiae has attracted much attention.To obtain a high betulinic acid producing cell factory, the LUP (encoding lupeol synthase from Glycyrrhiza uralensis) and tHMG1 (encoding a truncted 3-hydroxyl-3-methylglutaryl-CoA reductase which is the rate-limiting enzyme of the MVA pathway) genes were firstly co-expressed at rDNA multicopy sites of a chassis S. cerevisiae strain BY-T3 for triterpenoid prodution. The resulting strain BY-LUP02 produced 117.4 mg/L lupeol when cultivated in YPD medium for 6 days, which can be used for downstream betulinic acid production. Cell factories for production of betulinic acid were then constructed by introducing Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) and 3 cytochrome P450s from different sources, among which the cytochrome P450 from Vitis vinifera (CYP15) had the highest betulinic acid production. For further improving betulinic acid production,7 cytochrome P450 reductases (CPRs) from different sources were screened and finally a high betulinic acid producing Saccharomyces cerevisiae cell factory BY-BA42 was obtained, which produced 12.7 mg/L betulinic acid.In previous studies, our laboratory had successfully constructed a cell factory BY-OA for production of oleanolic acid. However, the precursor squalene still had a high accumulation (141.2 mg/L). For further increasing the conversion rate of squalene to oleanolic acid, one copy of Glycyrrhiza glabra β-amyrin synthase gene (GgbAS), Medicago truncatula oleanolic acid synthase gene (MtOAS) and AtCPR1 were introduced into strain BY-OA, resulting in the second generation cell factory BY-20A. When cultivated in YPD medium with 20 g/L glucose for 7 days, β-amyrin increased 54% to 136.5 mg/L and oleanolic acid increased 30% to 92.5 mg/L. By changing the glucose concentration, the titer of oleanolic acid increased to 165.7 mg/L when cultivated in YPD medium with 40 g/L glucose for 7 days.In this study, cell factories for production of betulinic acid and oleanolic acid were constructed and optimized, which provide a basis for creating an alternative way for production of triterpenoid acids in replace of extraction from natural plant or chemical synthesis.