Tropane Alkaloid Biosynthesis in Atropa Belladonna

A chemically diverse set of specialized flavorings, fragrances, and medicinal metabolites are produced by plants to modulate their interactions with pollinators, herbivores, and other biotic and abiotic stresses. Tropane alkaloids are one group of these specialized metabolites which are produced in phylogenetically distinct plant families and include the narcotic cocaine and the pharmaceutical compounds hyoscyamine and scopolamine. Scopolamine is produced in the roots of plants in the Solanaceae family and serves as the precursor to semi-synthetic tropane pharmaceuticals. A set of eleven tissue-specific transcriptomes was generated from the tender perennial Solanaceous plant Atropa belladonna, deadly nightshade, to fill gaps in the biosynthetic pathway from putrescine to scopolamine. This dissertation describes the identification of enzymes which complete three missing portions of the scopolamine biosynthetic pathway. These are the biosynthesis of tropinone, the diversion of phenylalanine into production of phenyllactic acid, and the activation and conjugation of phenyllactic acid with tropine to form littorine, a late pathway precursor to scopolamine. Tropinone is the first metabolite in this pathway with the characteristic 8-azabicyclo[3.2.1]octane tropane core. This pharmacore was synthesized in a classic biomimetic chemical synthesis, but the mechanism for tropinone biosynthesis has remained an open question in all species which produce these compounds. The A. belladonna lateral root transcriptome revealed that tropinone biosynthesis proceeds through an atypical polyketide synthase which uses an imine as its starter, and that the polyketide is cyclized to tropinone by a cytochrome P450. Tropic acid, the specialized acyl group for both hyoscyamine and scopolamine, is produced from the primary metabolite phenylalanine through the intermediate phenyllactic acid. Aromatic aminotransferases are one route by which amino acids can be dedicated to specialized metabolism. Six aromatic aminotransferases are present in the A. belladonna transcriptome, and one of these, ArAT4, is a root-specific phenylalanine aminotransferase required for biosynthesis of phenyllactic acid and ultimately, scopolamine. In contrast to other aminotransferases which equilibrate multiple amino acids, this enzyme is highly directional for the consumption of phenylalanine and production of tyrosine. Littorine, a precursor of hyoscyamine and scopolamine, is the ester of tropine and phenyllactic acid. The mechanism for phenyllactic acid activation and esterification have remained as an open question of tropane alkaloid biosynthesis since the discovery of littorine. Two routes exist for activation and conjugation in plants, through either coenzyme A thioesters or glucose esters. In A. belladonna, littorine biosynthesis proceeds through a glucose ester of phenyllactic acid produced by a glucosyltransferase, UGT84A27, and an acyltransferase, LITTORINE SYNTHASE. This contrasts with cocaine acylation in the Erythroxylaceae, which uses a different route highlighting the repeated, independent origin of tropane alkaloid biosynthesis in plants. The enzymes identified in this dissertation have completed three missing sections of the tropane alkaloid biosynthetic pathway in A. belladonna, resulting in a nearly complete pathway, suitable for engineering.Thesis (Ph.D.)--Michigan State University. Plant Breeding, Genetics and Biotechnology - Horticulture - Doctor of Philosophy, 2021Includes bibliographical reference

Chemically induced dimerization modules as a platform for plant biosensor engineering

Protein biosensors for small molecules have important applications in agriculture, medicine, and security, but it remains difficult to rapidly produce a high-affinity sensor for a given ligand. This is partly due to two major challenges. First, most small molecule ligands have only a small number of residues with which a protein can make energetically favorable contacts, making it difficult to engineer high-affinity binding. Second, even if a high-affinity binding protein is engineered, it is difficult to transduce the binding event into an output. The majority of plant hormone perception occurs by chemically induced dimerization, where binding of the hormone to a soluble receptor causes a conformational change that allows the receptor to form a heterodimer with an interaction partner. These CID modules make an ideal platform for engineering small molecule biosensors because they naturally address the two primary challenges above: their unique architecture allows sensitive biosensors to be constructed from low-affinity receptors and protein dimerization provides a natural method of ligand binding transduction. The ability to engineer CID modules would lead directly to in planta biosensors and would also have broader applications to biosensor design in other biological systems. Here we describe the development of a general biosensor engineering platform using the abscisic acid receptor PYR1 of Arabidopsis thaliana, which was previously engineered to sense the agrochemical mandipropamid.1 We combine comprehensive mutagenesis2,3, high-throughput screening, deep sequencing, and machine learning to rapidly construct a model of the fitness landscape for binding of PYR1 to a specific ligand. We then use this model to design a targeted library to screen for higher affinity sensors. For high-throughput screening, we use both an established yeast two-hybrid (Y2H) screen and a novel yeast surface display (YSD) system. These techniques offer complementary advantages: Y2H is straightforward to implement and requires no purified protein, while YSD offers higher throughput and more stringent quantification of protein-protein interactions. Finally, we describe early development of two additional CID modules from the gibberellin and strigolactone sensing networks of A. thaliana. (1) Park, S.-Y.; Peterson, F. C.; Mosquna, A.; Yao, J.; Volkman, B. F.; Cutler, S. R. Agrochemical Control of Plant Water Use Using Engineered Abscisic Acid Receptors. Nature 2015, 520 (7548), 545–548. https://doi.org/10.1038/nature14123. (2) Wrenbeck, E. E.; Klesmith, J. R.; Stapleton, J. A.; Adeniran, A.; Tyo, K. E. J.; Whitehead, T. A. Plasmid-Based One-Pot Saturation Mutagenesis. Nat. Methods 2016, 13 (11), 928–930. https://doi.org/10.1038/nmeth.4029. (3) Medina-Cucurella, A. V.; Steiner, P. J.; Faber, M. S.; Beltrán, J.; Borelli, A. N.; Kirby, M. B.; Cutler, S. R.; Whitehead, T. A. User-Defined Single Pot Mutagenesis Using Unpurified Oligo Pools. Re

The scaffold-forming steps of plant alkaloid biosynthesis

Alkaloids from plants are characterised by structural diversity and bioactivity, and maintain a privileged position in both modern and traditional medicines. In recent years, there have been significant advances in elucidating the biosynthetic origins of plant alkaloids. In this review, I will describe the progress made in determining the metabolic origins of the so-called true alkaloids, specialised metabolites derived from amino acids containing a nitrogen heterocycle. By identifying key biosynthetic steps that feature in the majority of pathways, I highlight the key roles played by modifications to primary metabolism, iminium reactivity and spontaneous reactions in the molecular and evolutionary origins of these pathways

Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization

Tropinone is an intermediate in the biosynthesis of tropane alkaloids. Here, the authors discovered the enzymes AbPYKS and AbCYP82M3, a non-canonical polyketide synthase and a cytochrome P450, that work sequentially to form tropinone from N-methyl-Δ1-pyrrolinium cation

Metabolomics-guided discovery of cytochrome P450s involved in pseudotropine-dependent biosynthesis of modified tropane alkaloids

AbstractPlant alkaloids constitute an important class of bioactive chemicals with applications in medicine and agriculture. However, the knowledge gap of the diversity and biosynthesis of phytoalkaloids prevents systematic advances in biotechnology for engineered production of these high-value compounds. In particular, the identification of cytochrome P450s driving the structural diversity of phytoalkaloids has remained challenging. Here, we use a combination of reverse genetics with discovery metabolomics and multivariate statistical analysis followed by in planta transient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes from Atropa belladonna without a priori knowledge of their functions or information regarding the identities of key pathway intermediates. This approach uncovered a largely unexplored root localized alkaloid sub-network that relies on pseudotropine as precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverse N-demethylated modified tropane alkaloids.</jats:p

High-Performance Cannabinoid Sensor Empowered by Plant Hormone Receptors and Antifouling Magnetic Nanorods.

The misuse of cannabinoids and their synthetic variants poses significant threats to public health, necessitating the development of advanced techniques for detection of these compounds in biological or environmental samples. Existing methods face challenges like lengthy sample pretreatment and laborious antifouling steps. Herein, we present a novel sensing platform using magnetic nanorods coated with zwitterionic polymers for the simple, rapid, and sensitive detection of cannabinoids in biofluids. Our technique utilizes the engineered derivatives of the plant hormone receptor Pyrabactin Resistance 1 (PYR1) as drug recognition elements and employs the chemical-induced dimerization (CID) mechanism for signal development. Additionally, the magnetic nanorods facilitate efficient target capture and reduce the assay duration. Moreover, the zwitterionic polymer coating exhibits excellent antifouling capability, preserving excellent sensor performance in complex biofluids. Our sensors detect cannabinoids in undiluted biofluids like serum, saliva, and urine with a low limit of detection (0.002 pM in saliva and few pM in urine and serum) and dynamic ranges spanning up to 9 orders of magnitude. Moreover, the PYR1 derivatives demonstrate high specificity even in the presence of multiple interfering compounds. This work opens new opportunities for sensor development, showcasing the excellent performance of antifouling magnetic nanorods that can be compatible with different recognition units, including receptors and antibodies, for detecting a variety of targets