Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 μg/L) and 7-fold (172 μg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

Nematicidal activity of the tropane alkaloids hyoscyamine and scopolamine against the root-knot nematode Meloidogyne incognita

Summary Alkaloids and alkaloid-producing plants have the potential to reduce crop damage by plant-parasitic nematodes. In a series of in vitro experiments, the nematicidal activity of the tropane alkaloids, hyoscyamine and scopolamine, and a mixture of both on the root-knot nematode, Meloidogyne incognita, was tested. Solutions of 16 mg of compound per ml of solvent were used in concentrations of 80-1280 μg ml−1 of water. Inactivity of second-stage juveniles of M. incognita increased with increasing concentration and exposure time. Lethal concentrations (LC50) after 120 min of exposure ranged from 182.4 μg ml−1 for scopolamine to 318.4 μg ml−1 for hyoscyamine and 332.8 μg ml−1 for the combination of both alkaloids. Similarly, the same concentrations of scopolamine inhibited hatching of M. incognita to a greater extent than hyoscyamine. In a glasshouse experiment, M. incognita was not able to penetrate the roots, induce galls and reproduce on Datura stramonium, D. innoxia and D. tatula. Results indicate that the tropane alkaloids hyoscyamine and scopolamine contained in Datura plants express a strong nematicidal activity against M. incognita and could possibly be used for an alternative and sustainable nematode management.

Altitudinal Variation in Alkaloid Composition of Hyoscyamus niger: A study with reference to Kashmir region of Himalayas

The present study has been undertaken to understand the impact of altitude on the synthesis of alkaloids in Hyoscyamus niger found in Kashmir valley of J&K state in India. Given its wide distributional range, the study aimed to quantify the alkaloids (hyoscyamine and scopolamine) in Hyoscyamus niger grown at various altitudes in Kashmir valley. The sampling of populations at varying altitudes allowed assessing the intraspecieac variation and ecological trends of accumulation of alkaloids in the plant. Furthermore the resource allocation in the parts of the plant has also been shown. The four different sites were Gulmarg, Pahalgam, Gurez and Qazigund in Kashmir valley. The study was able to identify the elite genotype and the best suited habitat (altitude) for commercial cultivation of the species with higher quantity of alkaloids.