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Summary Plant‐specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant‐specialized metabolic networks are poorly characterized.TheN‐methyl Δ¹‐pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots ofAtropa belladonna(Deadly Nightshade) reduces tropane alkaloid abundance and causes highN‐methyl Δ¹‐pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled withAbPyKSsilencing to reveal major changes in the root alkaloid metabolome ofA. belladonna.We discovered and annotated almost 40 pyrrolidine alkaloids that increase whenAbPyKSactivity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineeringin planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich‐like decarboxylative condensation of theN‐methyl Δ¹‐pyrrolinium cation with 2‐O‐malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono‐ and dipyrrolidine alkaloid diversity.This study reveals the previously unknown complexity of theA. belladonnaroot metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids. 

Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally by plants involving a hormone receptor which binds hormone, triggering a conformational change in the receptor which enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are well characterized by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio between the hormone receptor and binding protein. Thus, the same molecular ratchet can have a picomolar or micromolar EC50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including ELISA-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation. 

Abstract Plant 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 byin plantatransient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes fromAtropa belladonnawithout 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 catalyzeN-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverseN-demethylated modified tropane alkaloids. 

Abstract A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.