• OVERVIEW

  • Peroxisomal Mevalonate Pathway Reconstitution

  • Oleanane-Type Triterpenoid Synthesis

  • Ginsenoside Ro Multi-Enzyme Glycosylation System

  • UDP-Glucose Supply Module

  • Future Perspectives

  • Reference

OVERVIEW

We employed a metabolic engineering strategy to deconstruct the biosynthesis of ginsenoside Ro into four independent modules. First, in the Peroxisomal Mevalonate Pathway Reconstruction module, we targeted ERG10, ERG13, and HMG1 to the peroxisome, thereby enabling efficient utilization of fatty acid-derived acetyl-CoA. Second, within the Oleanane-Type Triterpenoid Synthesis module, we introduced β-AS , CYP716A12 and MtCPR ,among other elements, to generate the oleanolic acid precursor. Third, in the Ginsenoside Ro Multi-Enzyme Glycosylation System, we integrated a suite of glycosyltransferases to achieve the selective glycosylation of Ro. Finally, in the UDP-Glucose Supply Module, we expanded the donor sugar pool by overexpressing FBPase1, PGM2, and UGP1.Through the synergistic operation of these four modules, we successfully reconstituted the biosynthesis of ginsenoside Ro.

Peroxisomal Mevalonate Pathway Reconstitution

In the peroxisomal mevalonate (MVA) pathway reconstruction module, we engineered plasmids to express Acetoacetyl-CoA thiolase (Erg10), Hydroxymethylglutaryl-CoA synthase (Erg13), and Hydroxymethylglutaryl-CoA reductase (HMG1) within the peroxisome by incorporating a peroxisomal targeting signal 1 (PTS1). This design established the metabolic route from acetyl-CoA to mevalonate (MVA), thereby enabling the utilization of acetyl-CoA derived from β-oxidation for MVA biosynthesis.

Figure 1. Schematic representation of the plasmid pRS314-Erg10-Erg13-HMG1

Figure 2. PCR verification of the three genes encoded on the plasmid
(A) Erg10, showing the expected band size of 390 bp.
(B) Erg13, showing the expected band size of 1201 bp.
(C) HMG1, showing the expected band size of 366 bp.

Figure 3. Western blot verification of the three plasmid-encoded proteins, detected with tag-specific antibodies

Figure 4. Growth curve of S.cerevisiae on different single-fatty-acid media before and after plasmid transformation, monitored over time

Figure 5. Growth curve of plasmid-transformed S.cerevisiae on different mixed-fatty-acid media,monitored over time

Oleanane-Type Triterpenoid Synthesis

In the oleanane-type triterpenoid synthetic module, we successfully reconstituted the pathway in Saccharomyces cerevisiae by heterologously expressing β-amyrin synthase (β-AS) from Glycyrrhiza glabra, cytochrome P450 monooxygenase (CYP716A12) from Medicago truncatula, and cytochrome P450 reductase 1 (MtCPR). Functional expression of these enzymes enabled the complete conversion from 2,3-oxidosqualene(2,3-OSQ) to β-amyrin and subsequently to oleanolic acid (OA), providing the key triterpenoid precursor OA for the biosynthesis of ginsenoside Ro.

Figure 6. Schematic representation of the plasmid pRS314-βAS-CYP716A12-MtCPR

Figure 7. PCR verification of the three genes encoded on the plasmid
(A) MtCPR, showing the expected band size of 403 bp.
(B) CYP716A12, showing the expected band size of 328 bp.
(C) β-AS, showing the expected band size of 378 bp.

Figure 8. SDS-PAGE analysis of the three plasmid-encoded proteins visualized by Coomassie Brilliant Blue staining

Figure 9. Growth curve of plasmid-transformed S.cerevisiae on SC-Trp selective medium, monitored over time

Figure 10. High-performance liquid chromatography (HPLC) analysis of the oleanolic acid (OA) product peak

Ginsenoside Ro Multi-Enzyme Glycosylation System

In the multi-enzyme ginsenoside Ro glycosylation system, we constructed plasmids for heterologous expression in Saccharomyces cerevisiae of UGT73P40, UGT73F3, and Pn022859 from Panax notoginseng. This reconstituted the ginsenoside Ro glycosylation module, enabling a multi-step glycosylation network converting oleanolic acid to ginsenoside Ro.

Figure 11. Schematic representation of the plasmid pRS315-UGT73P40-UGT73F3-Pn022859

Figure 12. PCR verification of the three genes encoded on the plasmid
(A) UGT73P40, showing the expected band size of 400 bp.
(B) UGT73F3, showing the expected band size of 612 bp.
(C) Pn022859, showing the expected band size of 387 bp.

Figure 13. SDS-PAGE analysis of the three plasmid-encoded proteins visualized by Coomassie Brilliant Blue staining

Figure 14. Growth curve of plasmid-transformed S.cerevisiae in SC-Trp-Leu selective medium, monitored over time

Figure 15. High-performance liquid chromatography (HPLC) analysis of ginsenoside Ro produced by S.cerevisiae

UDP-Glucose Supply Module

In the UDP-glucose supply module, we constructed plasmids for overexpression in S.cerevisiae of fructose-1,6-bisphosphatase (FBPase1), phosphoglucomutase (PGM2), and UTP-glucose-1-phosphate uridylyltransferase (UGP1). This enhances sugar phosphate flux and UDP-Glc biosynthesis, providing sufficient sugar donors for the glycosylation reactions of ginsenoside Ro.

Figure 16. Schematic representation of the plasmid pRS314-FBPase1-PGM2-UGP1

Figure 17. PCR verification of the three genes encoded on the plasmid
(A) FBPase1, showing the expected band size of 355 bp.
(B) PGM2, showing the expected band size of 381 bp.
(C) UGP1, showing the expected band size of 360 bp.

Figure 18. Verification of the three plasmid-encoded proteins by Western blot using tag-specific antibodies

Future Perspectives

Future work will focus on scaling up and optimization. We plan to implement glycerol/fatty acid feeding strategies and fine-tune pH, oxygen levels, and feed rates to transfer the pathway into bioreactor fermentation. Adaptive laboratory evolution or dynamic regulation strategies, such as inducible systems, will be explored to enhance robustness on non-glucose carbon sources. Additionally, we aim to screen more UGT variants to achieve higher titers. Finally, the pathway could be extended to other oleanane-type saponins, such as rhamnosylated derivatives, to broaden its applications. Overall, this project opens multiple avenues for synthetic biology, green chemistry, and collaborations with biotechnology companies.

Reference

1. Guo, X. X., Li, Y., Sun, C., Jiang, D., Lin, Y. J., Jin, F. X., Lee, S. K., & Jin, Y. H. (2014). p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells. Protein & cell, 5(3), 224–234. https://doi.org/10.1007/s13238-014-0027-2
2. Yang, X. D., Yang, Y. Y., Ouyang, D. S., & Yang, G. P. (2015). A review of biotransformation and pharmacology of ginsenoside compound K. Fitoterapia, 100, 208–220. https://doi.org/10.1016/j.fitote.2014.11.019
3. Zhao, F., Bai, P., Liu, T., Li, D., Zhang, X., Lu, W., & Yuan, Y. (2016). Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnology and bioengineering, 113(8), 1787–1795. https://doi.org/10.1002/bit.25934
4. Han, J. Y., Kim, H. J., Kwon, Y. S., & Choi, Y. E. (2011). The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant & cell physiology, 52(12), 2062–2073. https://doi.org/10.1093/pcp/pcr150
5. Chu, L. L., Montecillo, J. A. V., & Bae, H. (2020). Recent Advances in the Metabolic Engineering of Yeasts for Ginsenoside Biosynthesis. Frontiers in bioengineering and biotechnology, 8, 139. https://doi.org/10.3389/fbioe.2020.00139
6. Zheng, S. W., Xiao, S. Y., Wang, J., Hou, W., & Wang, Y. P. (2019). Inhibitory Effects of Ginsenoside Ro on the Growth of B16F10 Melanoma via Its Metabolites. Molecules (Basel, Switzerland), 24(16), 2985. https://doi.org/10.3390/molecules24162985
7. Li, X., Wang, Y., Fan, Z., Wang, Y., Wang, P., Yan, X., & Zhou, Z. (2021). High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae. Metabolic engineering, 66, 87–97. https://doi.org/10.1016/j.ymben.2021.04.006
8. Paddon, C. J., Westfall, P. J., Pitera, D. J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M. D., Tai, A., Main, A., Eng, D., Polichuk, D. R., Teoh, K. H., Reed, D. W., Treynor, T., Lenihan, J., Fleck, M., Bajad, S., Dang, G., Dengrove, D., Diola, D., … Newman, J. D. (2013). High-level semi-synthetic production of the potent antimalarial artemisinin. Nature, 496(7446), 528–532. https://doi.org/10.1038/nature12051
9. Krivoruchko, A., & Nielsen, J. (2015). Production of natural products through metabolic engineering of Saccharomyces cerevisiae. Current opinion in biotechnology, 35, 7–15. https://doi.org/10.1016/j.copbio.2014.12.004
10. Dai, Z., Wang, B., Liu, Y., Shi, M., Wang, D., Zhang, X., Liu, T., Huang, L., & Zhang, X. (2014). Producing aglycons of ginsenosides in bakers' yeast. Scientific reports, 4, 3698. https://doi.org/10.1038/srep03698
11. Wang, P., Wei, Y., Fan, Y., Liu, Q., Wei, W., Yang, C., Zhang, L., Zhao, G., Yue, J., Yan, X., & Zhou, Z. (2015). Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metabolic engineering, 29, 97–105. https://doi.org/10.1016/j.ymben.2015.03.003
12. Wei, W., Wang, P., Wei, Y., Liu, Q., Yang, C., Zhao, G., Yue, J., Yan, X., & Zhou, Z. (2015). Characterization of Panax ginseng UDP-Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rh1 in Metabolically Engineered Yeasts. Molecular plant, 8(9), 1412–1424. https://doi.org/10.1016/j.molp.2015.05.010
13. Tang, J. R., Chen, G., Lu, Y. C., Tang, Q. Y., Song, W. L., Lin, Y., Li, Y., Peng, S. F., Yang, S. C., Zhang, G. H., & Hao, B. (2021). Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major. Planta, 253(5), 91. https://doi.org/10.1007/s00425-021-03617-0
14. Fan, G., Liu, X., Sun, S., Shi, C., Du, X., Han, K., Yang, B., Fu, Y., Liu, M., Seim, I., Zhang, H., Xu, Q., Wang, J., Su, X., Shao, L., Zhu, Y., Shao, Y., Zhao, Y., Wong, A. K., Zhuang, D., … Lee, S. M. (2020). The Chromosome Level Genome and Genome-wide Association Study for the Agronomic Traits of Panax Notoginseng. iScience, 23(9), 101538. https://doi.org/10.1016/j.isci.2020.101538
15. Zhao, Y., Fan, J., Wang, C., Feng, X., & Li, C. (2018). Enhancing oleanolic acid production in engineered Saccharomyces cerevisiae. Bioresource technology, 257, 339–343. https://doi.org/10.1016/j.biortech.2018.02.096 16. Kawasaki, A., Chikugo, A., Tamura, K., Seki, H., & Muranaka, T. (2021). Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis. Plant biotechnology (Tokyo, Japan), 38(2), 205–218. https://doi.org/10.5511/plantbiotechnology.21.0222a
17. Morgan, J. L., Strumillo, J., & Zimmer, J. (2013). Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature, 493(7431), 181–186. https://doi.org/10.1038/nature11744
18. McFarlane, H. E., Döring, A., & Persson, S. (2014). The cell biology of cellulose synthesis. Annual review of plant biology, 65, 69–94. https://doi.org/10.1146/annurev-arplant-050213-040240
19. Zhang, L., Ren, S., Liu, X., Liu, X., Guo, F., Sun, W., Feng, X., & Li, C. (2020). Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids. Biotechnology and bioengineering, 117(12), 3651–3663. https://doi.org/10.1002/bit.27518
20. Wang, X., Pereira, J. H., Tsutakawa, S., Fang, X., Adams, P. D., Mukhopadhyay, A., & Lee, T. S. (2021). Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450. Metabolic engineering, 64, 41–51. https://doi.org/10.1016/j.ymben.2021.01.004
21. Bian, G., Deng, Z., & Liu, T. (2017). Strategies for terpenoid overproduction and new terpenoid discovery. Current opinion in biotechnology, 48, 234–241. https://doi.org/10.1016/j.copbio.2017.07.002
22. Dai, Z., Liu, Y., Zhang, X., Shi, M., Wang, B., Wang, D., Huang, L., & Zhang, X. (2013). Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metabolic engineering, 20, 146–156. https://doi.org/10.1016/j.ymben.2013.10.004
23. Jozwiak, A., Sonawane, P. D., Panda, S., Garagounis, C., Papadopoulou, K. K., Abebie, B., Massalha, H., Almekias-Siegl, E., Scherf, T., & Aharoni, A. (2020). Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery. Nature chemical biology, 16(7), 740–748. https://doi.org/10.1038/s41589-020-0541-x