Lupeol has a variety of biological activities and has important application value in the field of food and medicine.Escherichia coli is a model microorganism widely used in the synthesis of active compounds due to its simple culture conditions and short fermentation cycle.In this study, the lupeol synthesis pathway was constructed in E.coli by co-expressing human squalene synthase (hSQS), squalene epoxidase (YlSQE) in Yarrowia lipolytica and lupeol synthase (BgLUS) in Bruguiera gymnorrhiza.By comparing the three NADPH-cytochrome P450 reductase/squalene epoxidase (CPR/SQE) oxidation systems, it was found that AtCPR from Arabidopsis thaliana / McSQE from Methylococcus capsularis was most conducive to the synthesis of lupeol.Overexpressing the rate-limiting enzymes 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (IDI) to increase the upstream methylerythritol phosphate (MEP) pathway flow, the engineered strain LUEAMU could produce 155.25 mg/L lupeol, which was 174.44 times that of the original strain LUE.By optimizing the medium and glycerol addition, it was found that TB medium containing 64 g/L glycerol could increase the production of lupeol by 1.38 times to 214.87 mg/L.The highest production of lupeol by E.coli LUEAMU was 313.95 mg/L by fed-batch culture in this medium.This is the highest yield of lupeol synthesized by prokaryotes as chassis.This study laid the foundation for the efficient synthesis of lupeol.
[1] SIDDIQUE H R, SALEEM M.Beneficial health effects of lupeol triterpene:A review of preclinical studies[J].Life Sciences, 2011, 88(7-8):285-293.
[2] FUKUSHIMA E O, SEKI H, OHYAMA K, et al.CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis[J].Plant & Cell Physiology, 2011, 52(12):2050-2061.
[3] PAPI REDDY K, SINGH A B, PURI A, et al.Synthesis of novel triterpenoid (lupeol) derivatives and their in vivo antihyperglycemic and antidyslipidemic activity[J].Bioorganic & Medicinal Chemistry Letters, 2009, 19(15):4463-4466.
[4] RAMOS-HERNÁNDEZ J A, CALDERÓN-SANTOYO M, NAVARRO-OCAÑA A, et al.Use of emerging technologies in the extraction of lupeol, α-amyrin and β-amyrin from sea grape (Coccoloba uvifera L.)[J].Journal of Food Science and Technology, 2018, 55(7):2377-2383.
[5] LIU S Y, XU F, LIU L J, et al.A convenient synthesis of lupeol from betulin[J].Industrial Crops and Products, 2015, 74:494-496.
[6] PADDON C J, WESTFALL P J, PITERA D J, et al.High-level semi-synthetic production of the potent antimalarial artemisinin[J].Nature, 2013, 496(7446):528-532.
[7] WESTFALL P J, PITERA D J, LENIHAN J R, et al.Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin[J].Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(3):E111-E118.
[8] ZHANG J L, BAI Q Y, PENG Y Z, et al.High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components[J].Biotechnology for Biofuels, 2020, 13:133.
[9] BRÖKER J N, MÜLLER B, VAN DEENEN N, et al.Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes[J].Applied Microbiology and Biotechnology, 2018, 102(16):6923-6934.
[10] LIN X N, AN T Y, FU D N, et al.Optimization of central carbon metabolism by Warburg effect of human cancer cell improves triterpenes biosynthesis in yeast[J].Advanced Biotechnology, 2023, 1(4):4.
[11] JIANG M, STEPHANOPOULOS G, PFEIFER B A.Toward biosynthetic design and implementation of Escherichia coli -derived-derived paclitaxel and other heterologous polyisoprene compounds[J].Applied and Environmental Microbiology, 2012, 78(8):2497-2504.
[12] LEONARD E, KOFFAS M A G.Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli[J].Applied and Environmental Microbiology, 2007, 73(22):7246-7251.
[13] QIAO W B, ZHOU Z L, LIANG Q, et al.Improving lupeol production in yeast by recruiting pathway genes from different organisms[J].Scientific Reports, 2019, 9:2992.
[14] BASYUNI M, OKU H, TSUJIMOTO E, et al.Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae[J].The FEBS Journal, 2007, 274(19):5028-5042.
[15] LIU Z, ZHANG Y N, SUN J N, et al.A novel soluble squalene-hopene cyclase and its application in efficient synthesis of hopene[J].Frontiers in Bioengineering and Biotechnology, 2020, 8:426.
[16] RENAULT H, BASSARD J E, HAMBERGER B, et al.Cytochrome P450-mediated metabolic engineering:Current progress and future challenges[J].Current Opinion in Plant Biology, 2014, 19:27-34.
[17] HAUSJELL J, HALBWIRTH H, SPADIUT O.Recombinant production of eukaryotic cytochrome P450 s in microbial cell factories[J].Bioscience Reports, 2018, 38(2):BSR20171290.
[18] DAS A, YOON S H, LEE S H, et al.An update on microbial carotenoid production:Application of recent metabolic engineering tools[J].Applied Microbiology and Biotechnology, 2007, 77(3):505-512.
[19] LIU H W, WANG Y, TANG Q, et al.MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli[J].Microbial Cell Factories, 2014, 13(1):135.
[20] LI D S, ZHANG Q, ZHOU Z J, et al.Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered Escherichia coli[J].Biotechnology Letters, 2016, 38(4):603-609.
[21] JADAUN J S, SANGWAN N S, NARNOLIYA L K, et al.Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented Geranium and Withania somnifera:Active involvement of plastid isoprenogenic pathway in their biosynthesis[J].Physiologia Plantarum, 2017, 159(4):381-400.
[22] HARADA H, MISAWA N.Novel approaches and achievements in biosynthesis of functional isoprenoids in Escherichia coli[J].Applied Microbiology and Biotechnology, 2009, 84(6):1021-1031.
[23] TAKEMURA M, TANAKA R, MISAWA N.Pathway engineering for the production of β-amyrin and cycloartenol in Escherichia coli:A method to biosynthesize plant-derived triterpene skeletons in E.coli[J].Applied Microbiology and Biotechnology, 2017, 101(17):6615-6625.
[24] STUDIER F W.Protein production by auto-induction in high-density shaking cultures[J].Protein Expression and Purification, 2005, 41(1):207-234.
