Description

The chemotherapeutic Paclitaxel is listed by the World Health Organization as an essential medicine¹ and is one of the most intensively used anti-tumor drugs, yet is notoriously difficult to produce. Paclitaxel has historically been extracted with toxic solvents from the endangered Pacific Yew tree (Taxus brevifolia) with environmental consequences, low yields, and high cost. Extraction from trees still remains the most popular method³. More recently, research groups have begun to engineer microbes such as yeast (S. cerevisiae) and E. coli to contain at least some of the 19 enzymes required for Taxol production^4,5. These microbes, however, are insufficient hosts for this complicated pathway that includes many hydroxylations and organelle-specific enzymes. To date, no bioengineering attempts of microbes have successfully produced large enough quantities of the intermediate Taxadiene-5α-ol to continue with the rest of the pathway³.

The CU-Boulder team aims to genetically engineer the cost-effective model crop plant soybeans (Glycine. max) to produce precursors of Paclitaxel, which can then be completed by an easy chemical semi-synthesis into Paclitaxel. By producing Paclitaxel in efficiently farmed soybeans, the CU-Boulder team hopes to provide a sustainable and relatively inexpensive method of manufacturing this essential chemotherapeutic. To accomplish this, the CU-Boulder team is introducing enzymes of the Paclitaxel biosynthesis pathway from the Yew tree into soybeans. Soybeans are efficiently farmed and have high potential as a synthetic biology chassis for sustainable manufacturing of complex molecules.

Although Paclitaxel (brand name: taxol) is an essential medicine, many people around the world do not have access to it. Cancer doesn’t discriminate, but for people living below the poverty line or without adequate insurance, this medicine is almost inaccessible. Our work in soybeans aims to bring more access and equality to those who need the drug. Since soybeans are so simple and inexpensive to grow, this could be a great method for manufacturing this medicine in resource-poor countries, cutting out the need for purchasing it from predatory pharmaceutical companies. Since the production costs would be much less than the cost of producing it through current methods, the cost of the medicine would also be less.

As the first team to develop soybeans as a chassis for iGEM, we are also seeking to improve the safety of soybeans as a platform for bioengineering. As recipients of the iGEM 2022 Safety and Security grant, we are performing additional work to engineer soybeans whose flowers remain closed (cleistogamous) during the pollination period, eliminating the possibility of cross-pollination between engineered and non-engineered cultivars. Learn more about our safety and security work here.

To date, we have successfully tested a promoter (BBa_K4201000) and terminator (BBa_K4201009) for use in soybeans, and have repeated three cycles of the engineering process to determine a suitable reporter gene (BBa_K4201010,BBa_K4201011, BBa_K4201012). One of our composite parts, Crte-cytoTDS-MBP_RUBY, is currently transformed into soybeans and should allow the soybeans to begin producing the first two intermediates of the paclitaxel pathway. Prior to the Jamboree, we should have quantitative data on this composite part compared to non-transformed soybeans. We are in the process of constructing many other composite parts for further testing.

We are excited to use a new reporter gene that will allow us to see the results of our transformation with no additional equipment required. RUBY (BBa_K4201012) expresses a series of enzymes which are responsible for transforming tyrosine into betalain, which has a striking red color. By eliminating the need for fluorescent proteins or similar reporters, we can see when our plants are expressing our constructs without having to destroy the plant to analyze the parts under a microscope.

CU-Boulder iGem 2022 (The Bean Team) is very excited to be attending Paris 2022. We can’t wait to answer everyone’s questions and be inspired by all the great projects!

References

1. WHO Model Lists of Essential Medicines. https://www.who.int/groups/expert-committee-on-selection-and-use-of-essential-medicines/essential-medicines-lists.
2. He, Y., Zhang, T., Sun, H. et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res 7, 152 (2020).
3. Li, D. et al. Isolation, Purification, and Identification of Taxol and Related Taxanes from Taxol-Producing Fungus Aspergillus niger subsp. taxi. J. Microbiol. Biotechnol. 27, 1379–1385 (2017).
4. Mutanda, I., Li, J., Xu, F. & Wang, Y. Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol. Front. Bioeng. Biotechnol. 9, (2021).
5. Wang, T. et al. Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases. Molecules 26, 2855 (2021).