Engineering Success

Design

Our research aims to heterologously express steviol glycosides in Escherichia coli. We divided the project into three modules: the DMAPP/IPP module, the steviol module, and the glycosyltransferase module. We synthesized the genes for each module and assembled them using a method like NEB BioBrick assembly. These modules were then inserted into vectors pACYCduet-1, PET21a, and PET28a. Under induction with IPTG, we expect to detect the product of rebaudiosides.

BUILD

  1. The backbone of rebaudiosides is a terpenoid. Naturally, all terpenoids are built from C5 blocks: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In eukaryotes, IPP and DMAPP are produced through the mevalonate (MVA) pathway while in prokaryotes they are produced through the methylerythritol-phosphate (MEP) pathway in. To increase the supply of isoprenoid we enhanced the expression of endogenous MEP pathway, three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphatesynthase (IspA) and isopentenyl diphosphate isomerase (IDI) were over overexpressed. (Kong et al., 2015)

  2. Since the terpenoids share the common C5 isoprenoid precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the commonly used methods include enhancing the endogenous MEP pathway or introducing exogenous MVA pathway were applied to enhance precursor supply. These natural pathways have many limitations, such as feedback inhibition of pathway intermediates or products and competing cofactor ATP, CTP, NADH, and NADPH, as well as precursors for IPP synthesis with central metabolism. To overcome these limitations, the isopentenol utilization pathway (IUP) were introduced. As shown the figure below, Isopentenol isomers were firstly phosphorylated to form isopentenyl monophosphate (IP) or dimethylallyl monophosphate (DMAP), and then they were convert to IPP or DMAPP by isopentenyl phosphate kinase (IPK). We incorporate the three genes, including SccK, AtiPK, idi into pACYCduet-1 for the biosynthesis of IPP and DMAPP from isopentenol. (Chatzivasileiou et al., 2018)

  3. The steviol module consisting of six genes which are geranylgeranyl diphosphatase (GGPPS), ent-copayl diphosphate synthase (CDPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), NADPH-dependent cytochrome P450 reductase (CPR) and kaurenoic acid 13α-hydroxylase (KAH). We assemble them into Pet21a through NEB BioBrick assembly as shown below.(Wang et al., 2015)

  4. The steviol is further converted to different kinds of rebaudiosides through five glycosyl transferase (UGT82C2, UGT74G1, UGT76G1, UGT91D2w and EUGT11M(F379)). These genes were assembled into Pet28a and incorporated with the isoprenoid modules and steviol modules to achieve the biosynthesis of Rebaudiosides.(Wang et al., 2015, Lin et al., 2020)

TEST

  1. To quantitatively analyze the production of steviol glycosides, we prepared a standard curve for Rebaudioside A, D and M. The analysis conditions are as follows: ThermoScientific Q Exactive Plus Mass Spectrometer; Column:  Agilent Zorbax C18, 5μm, 150 x 4.6 mm; Solvent:A, H2O+folic acid (0.1%); B, CH3CN + folic acid (0.1%), Gradient: 0 to 9 min, 20% to 50% solvent B; 9 to 10 min, 50% to 90% solvent B; 10 to 11 min, 90% to 20% solvent B; 11 to 12 min, 20% B.

  2. As a proof of concept, we construct three strains, all the strains derived from E.coli BL21(DE3) harboring different combination of plasmids. The plasmids are pACYCDuet-ispA-dxs-idi（MEP module） and pACYCDuet-Atipk-scck-idi（IUP module）， pET28a-UGT82C2-UGT74G1-UGT76G1-UGT91D2w-EUGT11M(F379)（glycosyl transferase module）, pET21a-GGPPS-KO-KS-CDPS-CPR-17αtr29CYP714A2（steviol module）. Strain A is a blank control, Strain B contains MEP module and steviol module, strain C contains IUP module and steviol module, strain D harbors the complete biosynthetic pathway for rebaudiosides which are IUP module, steviol module and glycosyl transferase module. After induction by 0.05 mM IPTG, these strain were cultivated in shake flask at 28℃ for 3 days. The culture were extracted by equivalent volume of n-butanol. After extraction, the organic phase was collected and dried under vacuum. Then they were dissolved by dimethyl sulfoxide and filtered. The samples were analyzed using ThermoScientific Q Exactive Plus Mass Spectrometer.

Learn

  1. Through experimentation, we discovered that increasing the precursor process can effectively improve the yield of steviol glycosides. Introduction of IUP pathway is better than overexpression of the MEP pathway due to the influence of E. coli's inherent metabolic regulation. However, however both method can enhance the supply of precursor.

  2. The activity of the glycosyltransferase needs improvement. Through experimentation, we observed the production of not only our intended product, Rebaudioside M, but also Rebaudioside A and Rebaudioside D as the final products.

Reference

  1. Kong, M., Kang, H. J., Kim, J. H., Oh, S. H., & Lee, P. C. (2015). Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli. Journal of Biotechnology, 214, 95–102. https://doi.org/10.1016/j.jbiotec.2015.09.016

  2. Chatzivasileiou, A. O., Ward, V. C., Edgar, S., & Stephanopoulos, G. (2018). Two-step pathway for isoprenoid synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(2), 506–511. https://doi.org/10.1073/pnas.1812935116

  3. Wang, J., Li, S., Xiong, Z., & Wang, Y. (2015). Pathway mining-based integration of critical enzyme parts for de novo biosynthesis of steviolglycosides sweetener in Escherichia coli. Cell Research, 26(2), 258–261. https://doi.org/10.1038/cr.2015.111

  4. Lin, M., Wang, F., & Zhu, Y. (2020). Modeled structure-based computational redesign of a glycosyltransferase for the synthesis of rebaudioside D from rebaudioside A. Biochemical Engineering Journal, 159, 107626. https://doi.org/10.1016/j.bej.2020.107626