Team:Victoria Wellington/Description

Description

The overall scope of the project is to heterologously produce tropane alkaloids in the bacterial hosts Eschericha coli and Synechococcus elongatus. Specifically, we hypothesize that the tropane alkaloid intermediate (tropine) can be efficiently produced from the precursor putrescine, which is naturally produced in S. elongatus. This idea is derived from the pathway previously used for tropine biosynthesis in Saccharomyces cerevisiae (Srinivasan & Smolke 2019). To achieve this, our plan was to conjugate the gene pathway into the S. elongatus UTEX2973 (Yu et al. 2015). However, this strain doesn’t have very good natural competence, which we were hoping to overcome using triparental conjugation. Initial attempts were planned with the helper plasmid pRL623 (Elhai et al. 1997) conjugated into previously used E. coli helper strains (HB101 or MC1061). To focus the metabolic flux of S. elongatus UTEX2973 towards this pathway, we also wanted to knock out the spermidine biosynthesis pathway using CRISPR, specifically the pCRISPomyces-2 plasmid (Cobb et al. 2015).

Due to the reasons outlined in the Project/Challenges section, we would like state our intention of making this a Two-Phase project and continuing this project next year

Figure 1 - The tropine biosynthesis pathway

Figure 2 - pAM4951 construct with promoters, RBS and terminators taken from et al. 20. Picture is a screenshot from Vasudevan et al. 2019. Picture is a screenshot from Geneious Prime

Inspiration

Tropane alkaloids are an important medicinal compound with applications in neurochemistry, as neurotransmitter inhibitors. They are also precursors in the synthesis of hyoscyamine and scopolamine, two plant proteins classified as essential medicines by WHO. Tropane alkaloids can be found in plants such as Brugmansia also known as Angel’s Trumpet. Our project goal is to remedy the impact that current world crises have had on the cultivation and exportation of the drug. Current production methods of these tropane alkaloids involve large scale cultivation of plants that produce these compounds. This leaves supply of the medicine dependent on factors such as climate, pests, and changes in land use. The Australian wildfires and COVID-19 have caused adverse effects on the global supply of drugs and so there is an increasing need for large-scale, climate-independent, and local production of these tropane alkaloids. We hope our project will make an impact in the medical field by introducing an effective and cheap alternative to tropine production, which can then be used to generate tropane alkaloids for various medicinal uses.

References

Charlier, Daniel & Bervoets, Indra. (2019). Regulation of arginine biosynthesis, catabolism and transport in Escherichia coli. Amino Acids. 51. 10.1007/s00726-019-02757-8.

Cobb, R. E., Wang, Y., & Zhao, H. (2015). High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS synthetic biology, 4(6), 723-728.

Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP. (1997) Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120 J Bacteriol., 179(6):1998-2005. PubMed 9068647

Kohnen-Johannsen, K. L., & Kayser, O. (2019). Tropane alkaloids: chemistry, pharmacology, biosynthesis and production. Molecules, 24(4), 796.

Srinivasan, P., & Smolke, C. D. (2019). Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids. Nature communications, 10(1), 1-15

Vasudevan, R., Gale, G. A., Schiavon, A. A., Puzorjov, A., Malin, J., Gillespie, M. D., ... & McCormick, A. J. (2019). CyanoGate: A modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), 39-55.

Wendt, K.E., Ungerer, J., Cobb, R.E. et al. (2016). CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb Cell Fact 15, 115. https://doi.org/10.1186/s12934-016-0514-7

Yu, J., Liberton, M., Cliften, P. F., Head, R. D., Jacobs, J. M., Smith, R. D., ... & Pakrasi, H. B. (2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO 2. Scientific reports, 5(1), 1-10.