• Introduction

  • Chassis Selection: S.cerevisiae

  • Module I - Peroxisomal Conversion of Fatty Acids to MVA

  • Module II - MVA Pathway toward Oleanolic Acid

  • Module III - Oleanolic Acid to Ginsenoside Ro

  • Module IV - Optimization of UDP-Glucose Supply

  • References

Introduction

Our project BiOilRo aims to achieve the complete biosynthesis of ginsenoside Ro in Saccharomyces cerevisiae using glycerol and fatty acids derived from waste oils as carbon sources. We have selected the Saccharomyces cerevisiae strain w303 as the chassis for this project. Within yeast, fatty acids undergo β-oxidation within peroxisomes; however, the resulting intermediate acetyl-CoA cannot be directly utilised in the cytoplasm. To address this, we have relocated key enzymes of the mevalonate (MVA) pathway to the peroxisome. This enables fatty acid-derived intermediates to be locally converted into MVA, which subsequently diffuses into the cytoplasm. Concurrently, glycerol enters the cytoplasmic MVA pathway via natural metabolic routes, establishing MVA as a convergence point for both substrates. At this central node, downstream modules convert MVA into oleanolic acid through oxidation and glycosylation steps, ultimately yielding ginsenoside Ro.

The experimental design comprises four plasmids:

1. One constructing a peroxisome-targeted pathway from fatty acids to mevalonic acid (MVA).

2. One converting MVA into the triterpene skeleton oleanolic acid.

3. A plasmid converting oleanolic acid into ginsenoside Ro via glycosylation.

4. A plasmid engineered to optimise uridine diphosphate glucose supply and enhance glycosylation efficiency.

Chassis Selection: S.cerevisiae

We chose Saccharomyces cerevisiae as the chassis for ginsenoside Ro biosynthesis. Several key factors support this decision:

1. Endogenous mevalonate pathway 1 Yeast naturally harbors the mevalonate (MVA) pathway for sterol biosynthesis, making it an ideal host for engineering extended triterpenoid pathways[1].

2. Previous studies have demonstrated that S. cerevisiae can successfully express plant cytochrome P450 monooxygenases, P450 reductases, and UDP-glycosyltransferases with functional activity. These enzymes are indispensable for the oxidation and glycosylation steps in ginsenoside biosynthesis[2].

3. Engineered yeast strains have already been used to synthesize ginsenosides such as Rh2 and Rg3, achieving significant titers in both shake-flask and fed-batch fermentation. These results confirm the feasibility of yeast-based ginsenoside production and highlight its potential for scale-up[3,4].

Module I - Peroxisomal Conversion of Fatty Acids to MVA

To channel fatty acid-derived acetyl-CoA into the mevalonate (MVA) pathway, we reconstructed the upstream MVA enzymes inside the peroxisome. A peroxisomal targeting signal (PTS1:GGC GGC GGC GGC TCT) was fused to each enzyme, ensuring localization to the peroxisome[5]. The plasmid carries Erg10 (acetyl-CoA acetyltransferase), Erg13 (HMG-CoA synthase), and HMG1 (HMG-CoA reductase). Together, these enzymes enable in situ conversion of fatty acid-derived acetyl-CoA into MVA. Additionally, the plasmid includes a Trp biosynthesis gene as an auxotrophic marker for plasmid maintenance.

Module II - MVA Pathway toward Oleanolic Acid

Once MVA is generated, it converges with glycerol-derived flux in the cytosol. To extend this pathway toward triterpenoid backbone synthesis, we constructed a second plasmid encoding β-amyrin synthase (β-AS), CYP716A2 (a cytochrome P450 monooxygenase catalyzing the oxidation of β-amyrin), and its redox partner MtCPR (cytochrome P450 reductase). These enzymes convert 2,3-oxidosqualene into oleanolic acid, the key precursor for ginsenoside biosynthesis[6]. A Trp biosynthesis gene was included for selection.

Module III - Oleanolic Acid to Ginsenoside Ro

The conversion of oleanolic acid to ginsenoside Ro requires precise glycosylation and further modifications. For this purpose, we designed a third plasmid encoding UGT73P40, UGT73F3, and Pn022859, three UDP-glycosyltransferases capable of catalyzing the stepwise addition of glucose moieties to oleanolic acid. Since this plasmid is used in combination with Module II, it contains Leu biosynthesis genes as dual markers, ensuring stable co-selection with plasmid 2.This module establishes the downstream glycosylation steps necessary to yield ginsenoside Ro.

Module IV - Optimization of UDP-Glucose Supply

Efficient glycosylation is dependent on sufficient pools of UDP-glucose. To address this metabolic bottleneck, we constructed a fourth plasmid enhancing UDP-glucose biosynthesis. It carries FBPase-1 (fructose-1,6-bisphosphatase), UGP1 (UDP-glucose pyrophosphorylase), and PGM2 (phosphoglucomutase), which collectively increase the flux toward UDP-glucose formation[7-9]. A Trp biosynthesis gene was included for plasmid selection. This module ensures that adequate UDP-glucose is available for UGT-mediated glycosylation in Module III.

References

[1] Guo, H., Wang, H. & Huo, Y.-X. Engineering Critical Enzymes and Pathways for Improved Triterpenoid Biosynthesis in Yeast. ACS Synthetic Biology 9, 2214-2227 (2020). https://doi.org:10.1021/acssynbio.0c00124

[2] Ro, D.-K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943 (2006). https://doi.org:10.1038/nature04640

[3] Wang, P. et al. Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency. Cell Discovery 5, 5 (2019). https://doi.org:10.1038/s41421-018-0075-5

[4] Nan, W., Zhao, F., Zhang, C., Ju, H. & Lu, W. Promotion of compound K production in Saccharomyces cerevisiae by glycerol. Microbial Cell Factories 19, 41 (2020). https://doi.org:10.1186/s12934-020-01306-3

[5] Ma, Y., Shang, Y. & Stephanopoulos, G. Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica. Proc Natl Acad Sci U S A 121, e2314798121 (2024). https://doi.org:10.1073/pnas.2314798121

[6] Dale, M. P., Moses, T., Johnston, E. J. & Rosser, S. J. A systematic comparison of triterpenoid biosynthetic enzymes for the production of oleanolic acid in Saccharomyces cerevisiae. PLOS ONE 15, e0231980 (2020). https://doi.org:10.1371/journal.pone.0231980

[7] Becker, J., Klopprogge, C., Zelder, O., Heinzle, E. & Wittmann, C. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71, 8587-8596 (2005). https://doi.org:10.1128/aem.71.12.8587-8596.2005

[8] Decker, D. & Kleczkowski, L. A. UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions. Front Plant Sci 9, 1822 (2018). https://doi.org:10.3389/fpls.2018.01822

[9] Xu, Y. et al. De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts. Nature Communications 13, 3040 (2022). https://doi.org:10.1038/s41467-022-30826-2