Multiplexed perturbation of yew reveals cryptic proteins that enable a total biosynthesis of baccatin III and Taxol precursors.

Plants make complex and potent therapeutic molecules, but difficulties in sourcing from natural producers or chemical synthesis can challenge their use in the clinic. A prominent example is the anti-cancer therapeutic paclitaxel (Taxol). Identification of the full paclitaxel biosynthetic pathway would enable heterologous drug production, but it has eluded discovery despite a half century of intensive research.

Plants Utilize a Protection/Deprotection Strategy in Limonoid Biosynthesis: A "Missing Link" Carboxylesterase Boosts Yields and Provides Insights into Furan Formation.

The furan ring is a defining feature of limonoids, a class of highly rearranged and bioactive plant tetranortriterpenoids. We recently reported an apparent complete biosynthetic pathway to these important natural furanoids. Herein, we disclose the subsequent discovery of a yield-boosting "missing link" carboxylesterase that selectively deprotects a late-stage intermediate, so triggering more efficient furan biosynthesis.

A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes.

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A notable example is the >150 Amaryllidaceae alkaloids (AmAs), including galantamine, an FDA-approved treatment for Alzheimer's disease. We show that while AmAs accumulate to high levels in many daffodil tissues, biosynthesis is localized to nascent, growing tissue at the leaf base.

Discovery and characterization of dietary antigens in oral tolerance.

Food antigens elicit immune tolerance through the action of regulatory T cells (Tregs) in the intestine. Although antigens that trigger common food allergies are known, the epitopes that mediate tolerance to most foods have not been described. Here, we identified murine T cell receptors specific for maize, wheat, and soy, and used expression cloning to de-orphan their cognate epitopes.

Reconstitution of early paclitaxel biosynthetic network.

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products.

Reconstitution of Early Paclitaxel Biosynthetic Network.

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. This diverts metabolic flux away from the paclitaxel precursor, taxadien-5α-ol, thus previous attempts of reconstitution have not yielded sufficient material for characterization, regardless of the heterologous host.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain.

Multiplicity of the Infection of for Transient DNA Delivery.

Biological DNA transfer into plant cells mediated by represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single versus multiple transfection events per cell.

A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes.

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A lead example is the >150 Amaryllidaceae alkaloids (AmAs) including galantamine, an FDA-approved treatment for Alzheimer's disease. We show that while AmAs accumulate to high levels in many tissues in daffodils, biosynthesis is localized to nascent, growing tissue at the base of leaves.

Complex scaffold remodeling in plant triterpene biosynthesis.

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g.

Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets.

Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles.

Modular, programmable RNA sensing using ADAR editing in living cells.

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular, programmable system for live RNA sensing using adenosine deaminases acting on RNA (RADAR).

Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in .

is a valuable plant chassis for heterologous production of medicinal plant natural products. This host is well suited for the processing of organelle-localized plant enzymes, and the conservation of the primary metabolism across the plant kingdom often provides required plant-specific precursor molecules that feed a given pathway. Despite this commonality in metabolism, limited precursor supply and/or competing host pathways can interfere with yields of heterologous products.

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine.

Colchicine () is a bioactive plant alkaloid from and species that is used as a pharmaceutical treatment for inflammatory diseases, including gouty arthritis and familial Mediterranean fever. The activity of this alkaloid is attributed to its ability to bind tubulin dimers and inhibit microtubule assembly, which not only promotes anti-inflammatory effects, but also makes colchicine a potent mitotic poison. The biochemical origins of colchicine biosynthesis have been investigated for over 50 years, but only recently has the underlying enzymatic machinery become clear.

Improved Stability of Engineered Ammonia Production in the Plant-Symbiont .

Bioavailable nitrogen is the limiting nutrient for most agricultural food production. Associative diazotrophs can colonize crop roots and fix their own bioavailable nitrogen from the atmosphere. Wild-type (WT) associative diazotrophs, however, do not release fixed nitrogen in culture and are not known to directly transfer fixed nitrogen resources to plants.

A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes.

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology.

Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato.

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants.

Dirigent Proteins Guide Asymmetric Heterocoupling for the Synthesis of Complex Natural Product Analogues.

Phenylpropanoids are a class of abundant building blocks found in plants and derived from phenylalanine and tyrosine. Phenylpropanoid polymerization leads to the second most abundant biopolymer lignin while stereo- and site-selective coupling generates an array of lignan natural products with potent biological activity, including the topoisomerase inhibitor and chemotherapeutic etoposide. A key step in etoposide biosynthesis involves a plant dirigent protein that promotes selective dimerization of coniferyl alcohol, a common phenylpropanoid, to form (+)-pinoresinol, a critical C symmetric pathway intermediate.

Rerouting plant terpene biosynthesis enables momilactone pathway elucidation.

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host.

Root-Secreted Coumarins and the Microbiota Interact to Improve Iron Nutrition in Arabidopsis.

Plants benefit from associations with a diverse community of root-colonizing microbes. Deciphering the mechanisms underpinning these beneficial services are of interest for improving plant productivity. We report a plant-beneficial interaction between Arabidopsis thaliana and the root microbiota under iron deprivation that is dependent on the secretion of plant-derived coumarins.