Re-engineering the tropane alkaloid biosynthesis pathway in potato
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Tropane alkaloids are a group of nitrogenous compounds that have a role in the treatment of multiple medical conditions. Many members of the Solanaceae produce these high valued compounds, such as the commercial source Duboisia. However, potato (Solanum tuberosum) does not produce tropane alkaloids. High levels of compounds which share tropane alkaloid precursors do accumulate in potato tissues (the nortropane alkaloids). The aim of this project is to investigate whether the range of alkaloids synthesised in potato can be enhanced by utilising biotechnological approaches to re-engineer the biochemical pathways, overcoming lesion points within potato. Transgenic lines have been developed to investigate whether metabolic flux can be diverted away from the nortropane alkaloids and towards the precursors of the high value tropane alkaloids. Transgenic lines which over-express tropinone reductase I, have been shown to accumulate the precursor tropine, after the application of exogenous tropinone, and within leaf disc assays tropine accumulation reached 161.00 ± 3.61 μg/g FW in transgenic lines compared to 0.63 ± 0.05 μg/g FW in Désirée controls. Similarly, tropinone reductase II down-regulating lines have shown a decrease in the accumulation of pseudotropine, a nortropane alkaloid precursor; however, there was no statistically significant increase in tropine accumulation. Pseudotropine accumulation in down-regulating transgenic lines reached 1.64 ± 0.27 μg/g FW, compared to 5.24 ± 0.44 μg/g FW in Désirée controls. Other regions of the biosynthetic pathway were investigated through recombinant protein expression assays to generate a more in-depth understanding and identify further lesions in this pathway within potato. These assays showed that the annotated hyoscyamine-6β-hydroxylase within the potato genome browsers were not able to convert hyosycamine to the downstream tropane alkaloids, when compared to a bona-fide h6h enzyme from Datura.