Composite

Part:BBa_K5059001

Designed by: Isaac Chang   Group: iGEM24_Cornell   (2024-09-17)

TDH3-AAS-TPS1

This is a composite part that integrates a strong constitutive promoter (BBa_K124002), Kozak sequence (BBa_K165002), Team Cornell's BBA_K5059000 BioBrick that encodes for alpha-amyrin synthase (AAS), and a strong terminator (BBa_K2926005). Implementing these BioBricks together allows for increased expression of AAS, outputting more alpha-amyrin for ursolic acid production.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Ursolic acid has gained traction recently as a potential therapeutic agent. Preliminary studies have been done on ursolic acid, determining its therapeutic potential for cancer, liver disease, and obesity, among other health benefits [2]. There is particular interest in ursolic acid's properties in fighting cancer, as it is an antioxidant and anti-inflammatory agent. Clinical trials are currently underway to test its use in cancer-treating drugs. However, the current method for ursolic acid extraction from fruits, such as apples and loquats, is inefficient, environmentally taxing, and expensive. By engineering S. cerevisiae to produce ursolic acid, the traditional method for its extraction can be bypassed by utilizing the pathway shown below.

Figure 1: Metabolic Pathway for Producing Ursolic Acid in Yeast

Within this pathway, TDH3-AAS-TPS1 is the first enzyme required to catalyze ursolic acid production. Specifically, it catalyzes the conversion of 2,3-oxidosqualene to alpha-amyrin, which is the direct precursor for ursolic acid through the action of Cytochrome P450, which is activated via Cytochrome P450 reductase donating an electron from NADPH to it.

After designing BBA_K5059000 and BBa_K5059001, a gel confirmation was done to confirm AAS Thing 1 and AAS Thing 2 separately, which was successful, as shown below.

Figure 2: Gel Confirmation of AAS Thing 1 and AAS Thing 2. Left to right: Lane 1 — Ladder, Lane 2-3 — AAS Thing 1 (2.3kb), Lane 4-7 — AAS Thing 2 (1.1kb)


Next, AAS Thing 1 and AAS Thing 2 would need to be digested and ligated to form the complete AAS sequence, which would be validated with another gel confirmation.

Functionality

Catalytic Efficiency

Substrate Product Km (µM) kcat (min^-1) kcat/Km (min^-1/µM)
1. 2,3-oxidosqualene alpha-amyrin 50.07 43.4 0.87
2. alpha-amyrin Ursolic Acid 24.5 35 1.43
This data comes from Dr. Yu et al.[3].

References

[1] Jia, N., Li, J., Zang, G., Yu, Y., Jin, X., He, Y., Feng, M., Na, X., Wang, Y., & Li, C. (2024). Engineering Saccharomyces cerevisiae for high-efficient production of ursolic acid via cofactor engineering and acetyl-CoA optimization. Biochemical Engineering Journal, 203, 109189. https://doi.org/10.1016/j.bej.2023.109189


[2] Alam, M., Ali, S., Ahmed, S., Elasbali, A. M., Adnan, M., Islam, A., Hassan, Md. I., & Yadav, D. K. (2021). Therapeutic Potential of Ursolic Acid in Cancer and Diabetic Neuropathy Diseases. International Journal of Molecular Sciences, 22(22), 12162. https://doi.org/10.3390/ijms222212162


[3] Yu, Y., Chang, P., Yu, H., Ren, H., Hong, D., Li, Z., Wang, Y., Song, H., Huo, Y., & Li, C. (2018). Productive Amyrin Synthases for Efficient α-Amyrin Synthesis in Engineered Saccharomyces cerevisiae. ACS Synthetic Biology, 7(10), 2391–2402. https://doi.org/10.1021/acssynbio.8b00176

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