Discovery and reconstitution of securinine alkaloid biosynthesis

Lead Research Organisation: University of York
Department Name: Biology


Chemicals derived from plant extracts, known as natural products, have been used by humankind for millennia, for applications including dyes, diets and drugs. These chemicals still have great relevance today. In fact, three modern crises can be addressed by the discovery of new plant natural products and discovering the genes the plant uses to make them. These crises are: (i) the demand for new drugs to treat the wide range of contemporary ailments, (ii) the loss of biodiversity caused by human action and (iii) the requirement to achieve net-zero to prevent a climate catastrophe. Natural products have historically be the source of inspiration for drugs, and this could be accelerated with modern genetic and robotic technologies. The loss of biodiversity risks us losing plants with potential cures - the modern genetic approach means we can transfer their talents into a new plant not at risk of extinction. Lastly, discovering the genes for plant natural products will let us make useful chemicals using biology, ending our reliance on petrochemical-derived technologies.
It is in this context that we are investigating how a plant called Flueggea suffructicosa, a deciduous shrub native to East Asia, makes a very interesting chemical called securinine. Securinine exhibits anticancer, antifungal, neuromodulatory, cognition enhancing, and neuroprotective activities. Whilst it has not currently in clinical use, there is ongoing interest in the potential of securinine and compounds closely related to securinine, both natural products and chemical modifications.
We aim to discover how the F. suffructicosa makes securinine: what genes does it use to make this complex and potent chemical? First, we will obtain genetic information from F. suffructicosa and then put together a list of the genes most likely involved in making securinine by search for those similar to well-understood genes, and then looking at when and where in the plant they are most active. These candidate genes are then to be tested. We do this using a species of tobacco Nicotiana benthamiana and getting that plant to produce the candidate genes. We then analyse the tobacco to see if new chemicals are being made. With this process we can build a pathway step-by-step. When a securinine-related gene is discovered we will analyse them and their enzyme products closely, to understand how securinine is made an atomic scale.
The overall aim is to show that we can make securinine in tobacco: this will be proof that we understand the pathway. This work will reveal new enzymes can that be used to make bioactive chemicals. Success will also allow us to make securinine and related chemicals outside the chemistry lab or the native plant. This may help develop new classes of molecules that might be the drugs of the future, benefitting human health.

Technical Summary

Securinine is a highly bioactive GABA-receptor antagonist derived from Flueggea suffructicosa, a deciduous shrub native to East Asia and one of the fifty fundamental herbs in traditional Chinese medicine. Securinine and related securinega alkaloids are of interest due to their bioactivities, tetracyclic structure and stereochemical variety. The elucidation and heterologous reconstitution of securinine biosynthesis would provide access to bioactive compounds and enable rapid biological or chemical semi-synthesis to generate natural-product like libraries for bioactivity screening. The metabolic pathway is likely to contain unusual and unique enzymes catalysing stereoselective reactions of mechanistic and biocatalytic significance.
In this project, we aim to discover the biosynthetic route to securinega alkaloids and reconstitute it in a heterologous system. This will be achieved with four measurable objectives, focused logically on the four rings of securinine: the piperideine A ring, the structure defining B ring, and the C/D rings derived ultimately from tyrosine. We have generated a transcriptome of F. suffructicosa and will use functional annotation and co-expression to identify gene candidates. The candidates will be expressed in Nicotiana benthamiana and activities detected by mass-spectrometry. Candidates demonstrating relevant conversions will be examined as purified enzymes, and, where possible, X-ray crystal structures will be solved. The whole project will be underpinned by stereoselective syntheses of key substrates, intermediates and products. Through this logical approach we will be able to discover and reconstitute securinine biosynthesis in N. benthamiana to set the stage for future heterologous production of securinine, its isomers and bioactive derivatives.


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