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Revision as of 20:25, 1 October 2024

Alpha-Amyrin Synthase (AAS)

This sequence resembles the MdOSC1 variant of the alpha-amyrin synthase from Malus domestica. Alpha-amyrin synthase (AAS) converts 2,3-oxidosqualene to alpha-amyrin, the precursor for ursolic acid in the mevalonate pathway [1]. Since S. cerevisiae doesn't contain AAS, we integrated this sequence into its genome to successfully produce ursolic acid. We codon-optimized the sequence using Benchling to be compatible with iGEM Assembly Standards. Since the sequence is over 2000 base pairs, we split the sequence into two parts, Thing 1 and Thing 2, for easier construction. We designed an overlapping EcoRI region so the sequences would be digested and then ligated together using T4 ligase. We also attached a 6xHis tag at the end of the sequence to enable Ni-NTA affinity chromatography for convenient enzyme isolation.

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, AAS 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, a gel confirmation was done to confirm Thing 1 and 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 and 3 - AAS Thing 1 (2.3kb), Lane 4-7 - AAS Thing 2(1.1kb).


Next, we performed a gel confirmation to validate the digestion and ligation of AAS Thing 1 and AAS Thing 2, which was unsuccessful.


Figure 3: Gel Confirmation of Digestion and Ligation of AAS Thing 1 and AAS Thing 2.

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