CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A non-canonical type III polyketide synthase (PKS), pyrrolidine ketide synthase (PYKS), catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

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.

Datura genome reveals duplications of psychoactive alkaloid biosynthetic genes and high mutation rate following tissue culture

Background Datura stramonium (Jimsonweed) is a medicinally and pharmaceutically important plant in the nightshade family (Solanaceae) known for its production of various toxic, hallucinogenic, and therapeutic tropane alkaloids. Recently, we published a tissue-culture based transformation protocol for D. stramonium that enables more thorough functional genomics studies of this plant. However, the tissue culture process can lead to undesirable phenotypic and genomic consequences independent of the transgene used. Here, we have assembled and annotated a draft genome of D. stramonium with a focus on tropane alkaloid biosynthetic genes. We then use mRNA sequencing and genome resequencing of transformants to characterize changes following tissue culture. Results Our draft assembly conforms to the expected 2 gigabasepair haploid genome size of this plant and achieved a BUSCO score of 94.7% complete, single-copy genes. The repetitive content of the genome is 61%, with Gypsy-type retrotransposons accounting for half of this. Our gene annotation estimates the number of protein-coding genes at 52,149 and shows evidence of duplications in two key alkaloid biosynthetic genes, tropinone reductase I and hyoscyamine 6 β-hydroxylase. Following tissue culture, we detected only 186 differentially expressed genes, but were unable to correlate these changes in expression with either polymorphisms from resequencing or positional effects of transposons. Conclusions We have assembled, annotated, and characterized the first draft genome for this important model plant species. Using this resource, we show duplications of genes leading to the synthesis of the medicinally important alkaloid, scopolamine. Our results also demonstrate that following tissue culture, mutation rates of transformed plants are quite high (1.16 × 10− 3 mutations per site), but do not have a drastic impact on gene expression.