Elizabeth Sattely

All Publications

• Plants Utilize a Protection/Deprotection Strategy in Limonoid Biosynthesis: A "Missing Link" Carboxylesterase Boosts Yields and Provides Insights into Furan Formation. Journal of the American Chemical Society Hodgson, H., Stephenson, M. J., Kikuchi, S., Martin, L. B., Liu, J. C., Casson, R., Rejzek, M., Sattely, E. S., Osbourn, A. 2024

  Abstract

  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. This has allowed, for the first time, the isolation and structural elucidation of unknown intermediates, refining our understanding of furan formation in limonoid biosynthesis.

  View details for DOI 10.1021/jacs.4c11213

  View details for PubMedID 39418479

• Transcriptome analysis reveals role of transcription factor WRKY70 in early N-hydroxy-pipecolic acid signaling. Plant physiology Foret, J., Kim, J. G., Sattely, E. S., Mudgett, M. B. 2024

  Abstract

  N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and amplifying systemic acquired resistance (SAR) following pathogen attack. Early phases of NHP signaling leading to immunity have remained elusive. Here, we report the early transcriptional changes mediated by NHP and the role salicylic acid (SA) plays during this response in Arabidopsis (Arabidopsis thaliana). We show that distinct waves of expression within minutes to hours of NHP treatment include increased expression of WRKY transcription factor genes as the primary transcriptional response, followed by the induction of WRKY-regulated defense genes as the secondary response. Most genes induced by NHP within minutes were SA-dependent, whereas those induced within hours were SA-independent. These data suggest that NHP induces the primary transcriptional response under basal levels of SA and that new SA biosynthesis via ISOCHORISMATE SYNTHASE 1/SA-INDUCTION DEFICIT 2 (ICS1/SID2) is dispensable for inducing the secondary transcriptional response. We demonstrate that WRKY70 is required for the induced expression of a set of genes defining some of the secondary transcriptional response, SAR protection, and NHP-dependent enhancement of ROS production in response to flagellin treatment. Our study highlights the key genes and pathways defining early NHP responses and the role of WRKY70 in regulating NHP-dependent transcription.

  View details for DOI 10.1093/plphys/kiae544

  View details for PubMedID 39404105

• A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes. Cell Mehta, N., Meng, Y., Zare, R., Kamenetsky-Goldstein, R., Sattely, E. 2024

  Abstract

  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. A similar trend is found in the production of steroidal alkaloids (e.g., cyclopamine) in corn lily. This model of active biosynthesis enabled the elucidation of a complete set of biosynthetic genes that can be used to produce AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodils. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes, where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as above-ground tissue develops.

  View details for DOI 10.1016/j.cell.2024.08.027

  View details for PubMedID 39276773

• Discovery and characterization of dietary antigens in oral tolerance. bioRxiv : the preprint server for biology Blum, J. E., Kong, R., Schulman, E. A., Chen, F. M., Upadhyay, R., Romero-Meza, G., Littman, D. R., Fischbach, M. A., Nagashima, K., Sattely, E. S. 2024

  Abstract

  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. All of the epitopes derive from seed storage proteins that are resistant to degradation and abundant in the edible portion of the plant. Multiple unrelated T cell clones were specific for an epitope at the C-terminus of 19 kDa alpha-zein, a protein from maize kernel. An MHC tetramer loaded with this antigen revealed that zein-specific T cells are predominantly Tregs localized to the intestine. These cells, which develop concurrently with weaning, constitute up to 2% of the peripheral Treg pool. Bulk and single-cell RNA sequencing revealed that these cells express higher levels of immunosuppressive markers and chemokines compared to other Tregs. These data suggest that immune tolerance to plant-derived foods is focused on a specific class of antigens with common features, and they reveal the functional properties of naturally occurring food-specific Tregs.

  View details for DOI 10.1101/2024.05.26.593976

  View details for PubMedID 38853977

  View details for PubMedCentralID PMC11160622

• Reconstitution of early paclitaxel biosynthetic network. Nature communications Liu, J. C., De La Peña, R., Tocol, C., Sattely, E. S. 2024; 15 (1): 1419

  Abstract

  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. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

  View details for DOI 10.1038/s41467-024-45574-8

  View details for PubMedID 38360800

  View details for PubMedCentralID PMC10869802

• Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis. Nature Nett, R. S., Dho, Y., Tsai, C., Passow, D., Martinez Grundman, J., Low, Y. Y., Sattely, E. S. 2023

  Abstract

  Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

  View details for DOI 10.1038/s41586-023-06716-y

  View details for PubMedID 37938780

  View details for PubMedCentralID 6863165

• Reconstitution of Early Paclitaxel Biosynthetic Network. bioRxiv : the preprint server for biology Chun-Ting Liu, J., De La Pena, R., Tocol, C., Sattely, E. S. 2023

  Abstract

  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. Here, we structurally characterized four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression, levels of these proposed byproducts decrease with a concomitant increase in the accumulation of taxadien-5α-ol by four-fold. This engineered system enabled the reconstitution of a six step biosynthetic pathway to produce isolatable 5α,10β-diacetoxy-taxadien-13α-ol. Furthermore, we showed that this pathway may function as a metabolic network rather than a linear pathway. The engineering of the paclitaxel biosynthetic network demonstrates that Taxus genes can coordinatively function for the biosynthetic production of key early stage paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

  View details for DOI 10.1101/2023.09.27.559859

  View details for PubMedID 37808792

  View details for PubMedCentralID PMC10557666

• Mapping the T cell repertoire to a complex gut bacterial community. Nature Nagashima, K., Zhao, A., Atabakhsh, K., Bae, M., Blum, J. E., Weakley, A., Jain, S., Meng, X., Cheng, A. G., Wang, M., Higginbottom, S., Dimas, A., Murugkar, P., Sattely, E. S., Moon, J. J., Balskus, E. P., Fischbach, M. A. 2023

  Abstract

  Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. 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. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

  View details for DOI 10.1038/s41586-023-06431-8

  View details for PubMedID 37587342

  View details for PubMedCentralID 4128479

• Multiplicity of the Agrobacterium Infection of Nicotiana benthamiana for Transient DNA Delivery ACS SYNTHETIC BIOLOGY Carlson, E. D., Rajniak, J., Sattely, E. S. 2023: 2329-2338

  Abstract

  Biological DNA transfer into plant cells mediated by Agrobacterium 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. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Here, we analyze the Poisson probability distribution of the T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/Nicotiana benthamiana. These data delineate the relationship between an individual Agrobacterium strain infiltration OD600, plant cell perimeter, and leaf age, as well as plant cell coinfection rates. Our analysis establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf at infiltration OD600 above ∼0.2 for individual strains. In contrast, single-strain T-DNA delivery can be achieved at low strain infiltration OD600: at OD600 0.02, we observe that ∼40% of plant cells are infected, with 80% of those infected cells containing T-DNA product from just a single strain. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.

  View details for DOI 10.1021/acssynbio.3c00148

  View details for Web of Science ID 001045173400001

  View details for PubMedID 37558215

• A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes. bioRxiv : the preprint server for biology Mehta, N., Meng, Y., Zare, R., Kamenetsky-Goldstein, R., Sattely, E. 2023

  Abstract

  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. A similar trend is found for the production of steroidal alkaloids (e.g. cyclopamine) in corn lily. This model of active biosynthesis enabled elucidation of a complete set of biosynthetic genes for the production of AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodil. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as aboveground tissue develops.

  View details for DOI 10.1101/2023.05.12.540595

  View details for PubMedID 37214939

  View details for PubMedCentralID PMC10197729

• Complex scaffold remodeling in plant triterpene biosynthesis. Science (New York, N.Y.) De La Pena, R., Hodgson, H., Liu, J. C., Stephenson, M. J., Martin, A. C., Owen, C., Harkess, A., Leebens-Mack, J., Jimenez, L. E., Osbourn, A., Sattely, E. S. 2023; 379 (6630): 361-368

  Abstract

  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., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

  View details for DOI 10.1126/science.adf1017

  View details for PubMedID 36701471

• Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets. Analytical chemistry Khariton, M., McClune, C. J., Brower, K. K., Klemm, S., Sattely, E. S., Fordyce, P. M., Wang, B. 2023

  Abstract

  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. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.

  View details for DOI 10.1021/acs.analchem.2c03475

  View details for PubMedID 36598332

• Author Correction: Modular, programmable RNA sensing using ADAR editing in living cells. Nature biotechnology Kaseniit, K. E., Katz, N., Kolber, N. S., Call, C. C., Wengier, D. L., Cody, W. B., Sattely, E. S., Gao, X. J. 2022

  View details for DOI 10.1038/s41587-022-01617-3

  View details for PubMedID 36418591

• Modular, programmable RNA sensing using ADAR editing in living cells. Nature biotechnology Kaseniit, K. E., Katz, N., Kolber, N. S., Call, C. C., Wengier, D. L., Cody, W. B., Sattely, E. S., Gao, X. J. 2022

  Abstract

  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). We validate, and then expand, our basic design, characterize its performance, and analyze its compatibility with human and mouse transcriptomes. We identify strategies to boost output levels and improve the dynamic range. Additionally, we show that RADAR enables compact AND logic. In addition to responding to transcript levels, RADAR can distinguish disease-relevant sequence alterations of transcript identities, such as point mutations and fusions. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

  View details for DOI 10.1038/s41587-022-01493-x

  View details for PubMedID 36198772

• Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana. ACS synthetic biology Kim, S. S., Wengier, D. L., Ragland, C. J., Sattely, E. S. 2022

  Abstract

  Nicotiana benthamiana 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. Here, we use transient transcriptional reprogramming of endogenous N. benthamiana metabolism to drastically improve flux through the etoposide pathway derived from the medicinal plant Podophyllum spp. Specifically, coexpression of a single lignin-associated transcription factor, MYB85, with pathway genes results in unprecedented levels of heterologous product accumulation in N. benthamiana leaves: 1 mg/g dry weight (DW) of the etoposide aglycone, 35 mg/g DW (-)-deoxypodophyllotoxin, and 3.5 mg/g DW (-)-epipodophyllotoxin─up to two orders of magnitude above previously reported biosynthetic yields for the etoposide aglycone and eight times higher than what is observed for (-)-deoxypodophyllotoxin in the native medicinal plant. Unexpectedly, transient activation of lignin metabolism by transcription factor overexpression also reduces the production of undesired side products that likely result from competing N. benthamiana metabolism. Our work demonstrates that synthetic activation of lignin biosynthesis in leaf tissue is an effective strategy for optimizing the production of medicinal compounds derived from phenylpropanoid precursors in the plant chassis N. benthamiana. Furthermore, our results highlight the engineering value of MYB85, an early switch in lignin biosynthesis, for on-demand modulation of monolignol flux and support the role of MYB46 as a master regulator of lignin polymer deposition.

  View details for DOI 10.1021/acssynbio.2c00289

  View details for PubMedID 36122905

• A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes. Communications biology Khlystov, N. A., Yoshikuni, Y., Deutsch, S., Sattely, E. S. 2021; 4 (1): 1027

  Abstract

  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. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

  View details for DOI 10.1038/s42003-021-02464-9

  View details for PubMedID 34471192

• A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis. Proceedings of the National Academy of Sciences of the United States of America Nett, R. S., Dho, Y., Low, Y., Sattely, E. S. 2021; 118 (24)

  Abstract

  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. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer's disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

  View details for DOI 10.1073/pnas.2102949118

  View details for PubMedID 34112718

• Engineering post-translational regulation of glutamine synthetase for controllable ammonia production in the plant-symbiont A. brasilense. Applied and environmental microbiology Schnabel, T., Sattely, E. 2021

  Abstract

  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. Here we design and test a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that post-translationally modifies, and deactivates glutamine synthase, a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly and we leverage the inherent self-contained nature of our post-translational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 muM h-1 OD600 -1 When grown in co-culture with the model monocot Setaria viridis, we demonstrate that these strains increases the biomass and chlorophyll content of plants up to 54% and 71% respectively relative to WT. Furthermore, we rigorously demonstrate direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of co-cultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings is derived from diazotrophically fixed dinitrogen, whereas no nitrogen is incorporated in plants co-cultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision could be deployable in a consortium of nitrogen fixing symbiotic diazotrophs for plant fertilization.Importance StatementNitrogen is the most limiting nutrient in modern agriculture. Free living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here we use a rational engineering approach to generate ammonia excreting strains of Azospirillum Our design features post-translational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We show that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrate in comparison to WT controls that our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

  View details for DOI 10.1128/AEM.00582-21

  View details for PubMedID 33962983

• Integrated project-based learning (IPBL) implementation for first year chemical engineering student: DIY hydraulic jack project EDUCATION FOR CHEMICAL ENGINEERS Burkholder, E., Hwang, L., Sattely, E., Holmes, N. 2021; 35: 69-80

  View details for DOI 10.1016/j.ece.2021.01.002

  View details for Web of Science ID 000640776000008

• Dirigent Proteins Guide Asymmetric Heterocoupling for the Synthesis of Complex Natural Product Analogues. Journal of the American Chemical Society Kim, S. S., Sattely, E. S. 2021

  Abstract

  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 C2 symmetric pathway intermediate. Despite the power of this coupling reaction for the elegant and rapid assembly of the etoposide scaffold, dirigent proteins have not been utilized to generate other complex lignan natural products. Here, we demonstrate that dirigent proteins from Podophyllum hexandrum in combination with a laccase guide the heterocoupling of natural and synthetic coniferyl alcohol analogues for the enantioselective synthesis of pinoresinol analogues. This route for complexity generation is remarkably direct and efficient: three new bonds and four stereocenters are produced from two different achiral monomers in a single step. We anticipate our results will enable biocatalytic routes to difficult-to-access non-natural lignan analogues and etoposide derivatives. Furthermore, these dirigent protein and laccase-promoted reactions of coniferyl alcohol analogues represent new regio- and enantioselective oxidative heterocouplings for which no other chemical methods have been reported.

  View details for DOI 10.1021/jacs.0c13164

  View details for PubMedID 33780244

• Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato. The Plant cell Holmes, E. C., Chen, Y. C., Mudgett, M. B., Sattely, E. S. 2021; 33 (3): 750–65

  Abstract

  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. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

  View details for DOI 10.1093/plcell/koaa052

  View details for PubMedID 33955491

• Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine. Journal of the American Chemical Society Nett, R. S., Sattely, E. S. 2021

  Abstract

  Colchicine (1) is a bioactive plant alkaloid from Colchicum and Gloriosa 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. Here, we report the discovery of multiple pathway enzymes from Gloriosa superba that allows for the reconstitution of a complete metabolic route to 1. This includes three enzymes that process a previously established tropolone-containing intermediate into 1 via tailoring of the nitrogen atom. We further demonstrate the total biosynthesis of enantiopure (-)-1 from primary metabolites via heterologous production in a model plant, thus enabling future efforts for the metabolic engineering of this medicinal alkaloid. Additionally, our results provide insight into the timing and tissue specificity for the late stage modifications required in colchicine biosynthesis, which are likely connected to the biological functions for this class of medicinal alkaloids in native producing plants.

  View details for DOI 10.1021/jacs.1c08659

  View details for PubMedID 34780686

• Improved Stability of Engineered Ammonia Production in the Plant-Symbiont Azospirillum brasilense. ACS synthetic biology Schnabel, T., Sattely, E. 2021

  Abstract

  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. Efforts to engineer diazotrophs for plant nitrogen provision as an alternative to chemical fertilization have yielded several strains that transiently release ammonia. However, these strains suffer from selection pressure for nonproducers, which rapidly deplete ammonia accumulating in culture, likely limiting their potential for plant growth promotion (PGP). Here we report engineered Azospirillum brasilense strains with significantly extend ammonia production lifetimes of up to 32 days in culture. Our approach relies on multicopy genetic redundancy of a unidirectional adenylyltransferase (uAT) as a posttranslational mechanism to induce ammonia release via glutamine synthetase deactivation. Testing our multicopy stable strains with the model monocot Setaria viridis in hydroponic monoassociation reveals improvement in plant growth promotion compared to single copy strains. In contrast, inoculation of Zea mays in nitrogen-poor, nonsterile soil does not lead to increased PGP relative to WT, suggesting strain health, resource competition, or colonization capacity in soil may also be limiting factors. In this context, we show that while engineered strains fix more nitrogen per cell compared to WT strains, the expression strength of multiple uAT copies needs to be carefully balanced to maximize ammonia production rates and avoid excessive fitness defects caused by excessive glutamine synthetase shutdown.

  View details for DOI 10.1021/acssynbio.1c00287

  View details for PubMedID 34591447

• Rerouting plant terpene biosynthesis enables momilactone pathway elucidation. Nature chemical biology De La Pena, R., Sattely, E. S. 2020

  Abstract

  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. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).

  View details for DOI 10.1038/s41589-020-00669-3

  View details for PubMedID 33106662

• Engineering Plant Synthetic Pathways for the Biosynthesis of Novel Antifungals. ACS central science Calgaro-Kozina, A., Vuu, K. M., Keasling, J. D., Loque, D., Sattely, E. S., Shih, P. M. 2020; 6 (8): 1394–1400

  Abstract

  Plants produce a wealth of biologically active compounds, many of which are used to defend themselves from various pests and pathogens. We explore the possibility of expanding upon the natural chemical diversity of plants and create molecules that have enhanced properties, by engineering metabolic pathways new to nature. We rationally broaden the set of primary metabolites that can be utilized by the core biosynthetic pathway of the natural biopesticide, brassinin, producing in planta a novel class of compounds that we call crucifalexins. Two of our new-to-nature crucifalexins are more potent antifungals than brassinin and, in some instances, comparable to commercially used fungicides. Our findings highlight the potential to push the boundaries of plant metabolism for the biosynthesis of new biopesticides.

  View details for DOI 10.1021/acscentsci.0c00241

  View details for PubMedID 32875080

• Discovery and engineering of colchicine alkaloid biosynthesis. Nature Nett, R. S., Lau, W., Sattely, E. S. 2020

  Abstract

  Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-completebiosynthetic pathway tocolchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.

  View details for DOI 10.1038/s41586-020-2546-8

  View details for PubMedID 32699417

• A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont. Cell Liou, C. S., Sirk, S. J., Diaz, C. A., Klein, A. P., Fischer, C. R., Higginbottom, S. K., Erez, A., Donia, M. S., Sonnenburg, J. L., Sattely, E. S. 2020; 180 (4): 717

  Abstract

  Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B.thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 invitro. Monocolonization of mice with mutant BtDelta2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

  View details for DOI 10.1016/j.cell.2020.01.023

  View details for PubMedID 32084341

• Root-Secreted Coumarins and the Microbiota Interact to Improve Iron Nutrition in Arabidopsis. Cell host & microbe Harbort, C. J., Hashimoto, M. n., Inoue, H. n., Niu, Y. n., Guan, R. n., Rombolà, A. D., Kopriva, S. n., Voges, M. J., Sattely, E. S., Garrido-Oter, R. n., Schulze-Lefert, P. n. 2020

  Abstract

  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. Disrupting this pathway alters the microbiota and impairs plant growth in iron-limiting soil. Furthermore, the microbiota improves iron-limiting plant performance via a mechanism dependent on plant iron import and secretion of the coumarin fraxetin. This beneficial trait is strain specific yet functionally redundant across phylogenetic lineages of the microbiota. Transcriptomic and elemental analyses revealed that this interaction between commensals and coumarins promotes growth by relieving iron starvation. These results show that coumarins improve plant performance by eliciting microbe-assisted iron nutrition. We propose that the bacterial root microbiota, stimulated by secreted coumarins, is an integral mediator of plant adaptation to iron-limiting soils.

  View details for DOI 10.1016/j.chom.2020.09.006

  View details for PubMedID 33027611

• A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato. Cell Jeon, J. E., Kim, J. G., Fischer, C. R., Mehta, N. n., Dufour-Schroif, C. n., Wemmer, K. n., Mudgett, M. B., Sattely, E. n. 2020; 180 (1): 176–87.e19

  Abstract

  In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

  View details for DOI 10.1016/j.cell.2019.11.037

  View details for PubMedID 31923394

• Total Biosynthesis for Milligram-Scale Production of Etoposide Intermediates in a Plant Chassis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Schultz, B. J., Kim, S., Lau, W., Sattely, E. S. 2019; 141 (49): 19231–35

  Abstract

  Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more dependable production method to replace the semisynthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report milligram-scale production of (-)-deoxypodophyllotoxin, a late-stage biosynthetic precursor to the etoposide aglycone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobacco metabolite and monolignol precursor to the etoposide aglycone. We show that transient expression of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the accumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chromatography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from difficult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.

  View details for DOI 10.1021/jacs.9b10717

  View details for Web of Science ID 000502687800008

  View details for PubMedID 31755709

• An engineered pathway for N-hydroxy-pipecolic acid synthesis enhances systemic acquired resistance in tomato. Science signaling Holmes, E. C., Chen, Y., Sattely, E. S., Mudgett, M. B. 2019; 12 (604)

  Abstract

  Systemic acquired resistance (SAR) is a powerful immune response that triggers broad-spectrum disease resistance throughout a plant. In the model plant Arabidopsis thaliana, long-distance signaling and SAR activation in uninfected tissues occur without circulating immune cells and instead rely on the metabolite N-hydroxy-pipecolic acid (NHP). Engineering SAR in crop plants would enable external control of a plant's ability to mount a global defense response upon sudden changes in the environment. Such a metabolite-engineering approach would require the molecular machinery for producing and responding to NHP in the crop plant. Here, we used heterologous expression in Nicotiana benthamiana leaves to identify a minimal set of Arabidopsis genes necessary for the biosynthesis of NHP. Local expression of these genes in tomato leaves triggered SAR in distal tissues in the absence of a pathogen, suggesting that the SAR trait can be engineered to enhance a plant's endogenous ability to respond to pathogens. We also showed tomato produces endogenous NHP in response to a bacterial pathogen and that NHP is present across the plant kingdom, raising the possibility that an engineering strategy to enhance NHP-induced defenses could be possible in many crop plants.

  View details for DOI 10.1126/scisignal.aay3066

  View details for PubMedID 31641079

• The alkynes we eat: Where do they come from and how do we identify them? Fischer, C., Jeon, J., Smith, K., Sattely, E. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525055500789

• Developing Plant Synthetic Biology Tools for Complex Metabolic Engineering. Shih, P. M., Calgaro-Kozina, A., Khanh Vuu, Keasling, J. D., Loque, D., Sattely, E. S. SPRINGER. 2019: S2

  View details for Web of Science ID 000467573200004

• Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production. Proceedings of the National Academy of Sciences of the United States of America Hodgson, H. n., De La Peña, R. n., Stephenson, M. J., Thimmappa, R. n., Vincent, J. L., Sattely, E. S., Osbourn, A. n. 2019

  Abstract

  Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3β-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3β-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

  View details for DOI 10.1073/pnas.1906083116

  View details for PubMedID 31371503

• Plant-derived coumarins shape the composition of an Arabidopsis synthetic root microbiome. Proceedings of the National Academy of Sciences of the United States of America Voges, M. J., Bai, Y. n., Schulze-Lefert, P. n., Sattely, E. S. 2019

  Abstract

  The factors that contribute to the composition of the root microbiome and, in turn, affect plant fitness are not well understood. Recent work has highlighted a major contribution of the soil inoculum in determining the composition of the root microbiome. However, plants are known to conditionally exude a diverse array of unique secondary metabolites, that vary among species and environmental conditions and can interact with the surrounding biota. Here, we explore the role of specialized metabolites in dictating which bacteria reside in the rhizosphere. We employed a reduced synthetic community (SynCom) of Arabidopsis thaliana root-isolated bacteria to detect community shifts that occur in the absence of the secreted small-molecule phytoalexins, flavonoids, and coumarins. We find that lack of coumarin biosynthesis in f6'h1 mutant plant lines causes a shift in the root microbial community specifically under iron deficiency. We demonstrate a potential role for iron-mobilizing coumarins in sculpting the A. thaliana root bacterial community by inhibiting the proliferation of a relatively abundant Pseudomonas species via a redox-mediated mechanism. This work establishes a systematic approach enabling elucidation of specific mechanisms by which plant-derived molecules mediate microbial community composition. Our findings expand on the function of conditionally exuded specialized metabolites and suggest avenues to effectively engineer the rhizosphere with the aim of improving crop growth in iron-limited alkaline soils, which make up a third of the world's arable soils.

  View details for DOI 10.1073/pnas.1820691116

  View details for PubMedID 31152139

• D2O Labeling to Measure Active Biosynthesis of Natural Products in Medicinal Plants. AIChE journal. American Institute of Chemical Engineers Nett, R. S., Guan, X., Smith, K., Faust, A. M., Sattely, E. S., Fischer, C. R. 2018; 64 (12): 4319-4330

  Abstract

  Plant natural products have served as a prominent source of medicines throughout human history, and are still used today as clinically-approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow-growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically-relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids, and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short time scales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes.

  View details for DOI 10.1002/aic.16413

  View details for PubMedID 31235979

  View details for PubMedCentralID PMC6590064

• N-hydroxy-pipecolic acid is a mobile metabolite that induces systemic disease resistance in Arabidopsis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, Y., Holmes, E. C., Rajniak, J., Kim, J., Tang, S., Fischer, C. R., Mudgett, M., Sattely, E. S. 2018; 115 (21): E4920–E4929

  Abstract

  Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.

  View details for PubMedID 29735713

• Biosynthesis of redox-active metabolites in response to iron deficiency in plants NATURE CHEMICAL BIOLOGY Rajniak, J., Giehl, R. H., Chang, E., Murgia, I., von Wiren, N., Sattely, E. S. 2018; 14 (5): 442-+

  Abstract

  Iron is an essential but poorly bioavailable nutrient because of its low solubility, especially in alkaline soils. Here, we describe the discovery of a previously undescribed redox-active catecholic metabolite, termed sideretin, which derives from the coumarin fraxetin and is the primary molecule exuded by Arabidopsis thaliana roots in response to iron deficiency. We identified two enzymes that complete the biosynthetic pathway of fraxetin and sideretin. Chemical characterization of fraxetin and sideretin, and biological assays with pathway mutants, suggest that these coumarins are critical for iron nutrition in A. thaliana. Further, we show that sideretin production also occurs in eudicot species only distantly related to A. thaliana. Untargeted metabolomics of the root exudates of various eudicots revealed production of structurally diverse redox-active molecules in response to iron deficiency. Our results indicate that secretion of small-molecule reductants by roots may be a widespread and previously underappreciated component of reduction-based iron uptake.

  View details for PubMedID 29581584

• A lignin-epoxy resin derived from biomass as an alternative to formaldehyde-based wood adhesives GREEN CHEMISTRY Li, R., Gutierrez, J., Chung, Y., Frank, C. W., Billington, S. L., Sattely, E. S. 2018; 20 (7): 1459–66

  View details for DOI 10.1039/c7gc03026f

  View details for Web of Science ID 000432567300005

• HEx: A heterologous expression platform for the discovery of fungal natural products SCIENCE ADVANCES Harvey, C. B., Tang, M., Schlecht, U., Horecka, J., Fischer, C. R., Lin, H., Li, J., Naughton, B., Cherry, J., Miranda, M., Li, Y., Chu, A. M., Hennessy, J. R., Vandova, G. A., Inglis, D., Aiyar, R. S., Steinmetz, L. M., Davis, R. W., Medema, M. H., Sattely, E., Khosla, C., St Onge, R. P., Tang, Y., Hillenmeyer, M. E. 2018; 4 (4): eaar5459

  Abstract

  For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

  View details for PubMedID 29651464

• Biosynthesis of cabbage phytoalexins from indole glucosinolate. Proceedings of the National Academy of Sciences of the United States of America Klein, A. P., Sattely, E. S. 2017; 114 (8): 1910-1915

  Abstract

  Brassica crop species are prolific producers of indole-sulfur phytoalexins that are thought to have an important role in plant disease resistance. These molecules are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymatic steps that assemble the key precursor brassinin. Here, we report the minimum set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochemical characterization revealed an additional role for the previously described carbon-sulfur lyase SUR1 in processing cysteine-isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Additionally, the β-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole-sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole-sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.

  View details for DOI 10.1073/pnas.1615625114

  View details for PubMedID 28154137

  View details for PubMedCentralID PMC5338394

• Metabolism of the xenobiotic compound benzotriazole in Arabidopsis plants LeFevre, G., Lipsky, A., Mueller, C., Sattely, E., Luthy, R. AMER CHEMICAL SOC. 2016

  View details for Web of Science ID 000431460200721

• Plant Assimilation Kinetics and Metabolism of 2-Mercaptobenzothiazole Tire Rubber Vulcanizers by Arabidopsis ENVIRONMENTAL SCIENCE & TECHNOLOGY Lefevre, G. H., Portmann, A. C., Muller, C. E., Sattely, E. S., Luthy, R. G. 2016; 50 (13): 6762-6771

  Abstract

  2-Mercaptobenzothiazole (MBT) is a tire rubber vulcanizer found in potential sources of reclaimed water where it may come in contact with vegetation. In this work, we quantified the plant assimilation kinetics of MBT using Arabidopsis under hydroponic conditions. MBT depletion kinetics in the hydroponic medium with plants were second order (t1/2 = 0.52 to 2.4 h) and significantly greater than any abiotic losses (>18 times faster; p = 0.0056). MBT depletion rate was related to the initial exposure concentration with higher rates at greater concentrations from 1.6 μg/L to 147 μg/L until a potentially inhibitory level (1973 μg/L) lowered the assimilation rate. 9.8% of the initial MBT mass spike was present in the plants after 3 h and decreased through time. In-source LC-MS/MS fragmentation revealed that MBT was converted by Arabidopsis seedlings to multiple conjugated-MBT metabolites of differential polarity that accumulate in both the plant tissue and hydroponic medium; metabolite representation evolved temporally. Multiple novel MBT-derived plant metabolites were detected via LC-QTOF-MS analysis; proposed transformation products include glucose and amino acid conjugated MBT metabolites. Elucidating plant transformation products of trace organic contaminants has broad implications for water reuse because plant assimilation could be employed advantageously in engineered natural treatment systems, and plant metabolites in food crops could present an unintended exposure route to consumers.

  View details for DOI 10.1021/acs.est.5b04716

  View details for Web of Science ID 000379366300022

  View details for PubMedID 26698834

• Competing mechanisms for perfluoroalkyl acid accumulation in plants revealed using an Arabidopsis model system. Environmental toxicology and chemistry Müller, C. E., Lefevre, G. H., Timofte, A. E., Hussain, F. A., Sattely, E. S., Luthy, R. G. 2016; 35 (5): 1138-1147

  Abstract

  Perfluoroalkyl acids (PFAAs) bioaccumulate in plants, presenting a human exposure route if present in irrigation water. Curiously, accumulation of PFAAs in plant tissues is greatest for both the short-chain and long-chain PFAAs, generating a U-shaped relationship with chain length. In the present study, the authors decouple competing mechanisms of PFAA accumulation using a hydroponic model plant system (Arabidopsis thaliana) exposed to a suite of 10 PFAAs to determine uptake, depuration, and translocation kinetics. Rapid saturation of root concentrations occurred for all PFAAs except perfluorobutanoate, the least-sorptive (shortest-chain) PFAA. Shoot concentrations increased continuously, indicating that PFAAs are efficiently transported and accumulate in shoots. Tissue concentrations of PFAAs during depuration rapidly declined in roots but remained constant in shoots, demonstrating irreversibility of the translocation process. Root and shoot concentration factors followed the U-shaped trend with perfluoroalkyl chain length; however, when normalized to dead-tissue sorption, this relationship linearized. The authors therefore introduce a novel term, the "sorption normalized concentration factor," to describe PFAA accumulation in plants; because of their hydrophobicity, sorption is the determining factor for long-chain PFAAs, whereas the shortest-chain PFAAs are most effectively transported in the plant. The present study provides a mechanistic explanation for previously unexplained PFAA accumulation trends in plants and suggests that shorter-chained PFAAs may bioaccumulate more readily in edible portions.

  View details for DOI 10.1002/etc.3251

  View details for PubMedID 26383989

• Discovery of benzotriazole and novel plant metabolites in Arabidopsis and food crops LeFevre, G., Lipsky, A., Sattely, E., Higgins, C., Hyland, K., Luthy, R. AMER CHEMICAL SOC. 2016

  View details for Web of Science ID 000431905700575

• Discovery and engineering of plant chemistry for plant and human health Sattely, E. AMER CHEMICAL SOC. 2016

  View details for Web of Science ID 000431903800710

• Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis. Nature chemical biology Klein, A. P., Sattely, E. S. 2015; 11 (11): 837-839

  Abstract

  Phytoalexins are abundant in edible crucifers and have important biological activities, yet no dedicated gene for their biosynthesis is known. Here, we report two new cytochromes P450 from Brassica rapa (Chinese cabbage) that catalyze unprecedented S-heterocyclizations in cyclobrassinin and spirobrassinin biosynthesis. Our results provide genetic and biochemical insights into the biosynthesis of a prominent pair of dietary metabolites and have implications for pathway discovery across >20 recently sequenced crucifers.

  View details for DOI 10.1038/nchembio.1914

  View details for PubMedID 26389737

• A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defence. Nature Rajniak, J., Barco, B., Clay, N. K., Sattely, E. S. 2015; 525 (7569): 376-379

  Abstract

  Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, which suggests that there are numerous molecules contributing to plant fitness that have not yet been discovered. Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens. Here we start with a single pathogen-induced P450 (ref. 5), CYP82C2, and use a combination of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a previously unknown Arabidopsis metabolite. This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature; furthermore, the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivo and validate the functions of its enzymes. Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defence. Arabidopsis has been the pre-eminent model system for studying the role of small molecules in plant innate immunity; our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defence response. These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.

  View details for DOI 10.1038/nature14907

  View details for PubMedID 26352477

• Rapid Phytotransformation of Benzotriazole Generates Synthetic Tryptophan and Auxin Analogs in Arabidopsis. Environmental science & technology Lefevre, G. H., Müller, C. E., Li, R. J., Luthy, R. G., Sattely, E. S. 2015; 49 (18): 10959-10968

  Abstract

  Benzotriazoles (BTs) are xenobiotic contaminants widely distributed in aquatic environments and of emerging concern due to their polarity, recalcitrance, and common use. During some water reclamation activities, such as stormwater bioretention or crop irrigation with recycled water, BTs come in contact with vegetation, presenting a potential exposure route to consumers. We discovered that BT in hydroponic systems was rapidly (approximately 1-log per day) assimilated by Arabidopsis plants and metabolized to novel BT metabolites structurally resembling tryptophan and auxin plant hormones; <1% remained as parent compound. Using LC-QTOF-MS untargeted metabolomics, we identified two major types of BT transformation products: glycosylation and incorporation into the tryptophan biosynthetic pathway. BT amino acid metabolites are structurally analogous to tryptophan and the storage forms of auxin plant hormones. Critical intermediates were synthesized (authenticated by (1)H/(13)C NMR) for product verification. In a multiple-exposure temporal mass balance, three major metabolites accounted for >60% of BT. Glycosylated BT was excreted by the plants into the hydroponic medium, a phenomenon not observed previously. The observed amino acid metabolites are likely formed when tryptophan biosynthetic enzymes substitute synthetic BT for native indolic molecules, generating potential phytohormone mimics. These results suggest that BT metabolism by plants could mask the presence of BT contamination in the environment. Furthermore, BT-derived metabolites are structurally related to plant auxin hormones and should be evaluated for undesirable biological effects.

  View details for DOI 10.1021/acs.est.5b02749

  View details for PubMedID 26301449

• Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science Lau, W., Sattely, E. S. 2015; 349 (6253): 1224-1228

  Abstract

  Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.

  View details for DOI 10.1126/science.aac7202

  View details for PubMedID 26359402

• Key applications of plant metabolic engineering. PLoS biology Lau, W., Fischbach, M. A., Osbourn, A., Sattely, E. S. 2014; 12 (6)

  Abstract

  Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field's progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field.

  View details for DOI 10.1371/journal.pbio.1001879

  View details for PubMedID 24915445

  View details for PubMedCentralID PMC4051588

• The chemical logic of plant natural product biosynthesis. Current opinion in plant biology Anarat-Cappillino, G., Sattely, E. S. 2014; 19: 51-58

  Abstract

  Understanding the logic of plant natural product biosynthesis is important for three reasons: it guides the search for new natural products and pathways, illuminates the function of existing pathways in the context of host biology, and builds an enabling 'parts list' for plant and microbial metabolic engineering. In this review, we highlight the chemical themes that underlie a broad range of plant pathways, dividing pathways into two parts: scaffold-generating steps that draw on a limited set of chemistries, and tailoring reactions that produce a wide range of end products from a small number of common scaffolds.

  View details for DOI 10.1016/j.pbi.2014.03.007

  View details for PubMedID 24727074

• Minimum set of cytochromes p450 for reconstituting the biosynthesis of camalexin, a major Arabidopsis antibiotic. Angewandte Chemie (International ed. in English) Klein, A. P., Anarat-Cappillino, G., Sattely, E. S. 2013; 52 (51): 13625-13628

  Abstract

  Bringing it all together: The missing key step in the biosynthesis of camalexin was uncovered by in vitro biochemical characterization. The coupling of Trp- and Cys-derived fragments through CS bond formation is promoted by an unusual cytochrome P450 CYP71A13. The in vitro reconstitution of the camalexin biosynthesis (left) from Trp and Cys was achieved using just three cytochromes P450. IAN=indole-3-acetonitrile.

  View details for DOI 10.1002/anie.201307454

  View details for PubMedID 24151049

  View details for PubMedCentralID PMC3867539

• A Renewable Lignin-Lactide Copolymer and Application in Biobased Composites ACS SUSTAINABLE CHEMISTRY & ENGINEERING Chung, Y., Olsson, J. V., Li, R. J., Frank, C. W., Waymouth, R. M., Billington, S. L., Sattely, E. S. 2013; 1 (10): 1231-1238

  View details for DOI 10.1021/sc4000835

  View details for Web of Science ID 000325512000004

• Graft-polymerization of poly(lactic acid) onto lignin with application to biobased composites Chung, Y., Olsson, J. V., Li, R., Billington, S., Frank, C. W., Waymouth, R. M., Sattely, E. AMER CHEMICAL SOC. 2013

  View details for Web of Science ID 000323851301718

• Identification and characterization of key enzymes and multi-enzyme complexes in plant secondary metabolism Anarat-Cappillino, G., Sattely, E. S. AMER CHEMICAL SOC. 2013

  View details for Web of Science ID 000323851300824

• Three Cytochromes P450 are Sufficient to Reconstitute the Biosynthesis of Camalexin, a Major Arabidopsis Antibiotic Angew. Chem. Int. Ed. Klein, A., P., Anarat-Cappillino, G., Sattely, E., S. 2013; 52: 13625-13628
• Design and Stereoselective Preparation of a New Class of Chiral Olefin Metathesis Catalysts and Application to Enantioselective Synthesis of Quebrachamine: Catalyst Development Inspired by Natural Product Synthesis J. Am. Chem. Soc. Sattely, E., S., Meek, S., J., Malcolmson, S., J., Schrock, R., R., Hoveyda, A., H. 2009; 131: 943- 953
• Three Siderophores from One Assembly Line Enzyme J. Am. Chem. Soc. Wuest, W., M., Sattely, E., S., Walsh, C., T. 2009; 131: 5056-5057
• Enzymatic Tailoring of Ornithine in the Biosynthesis of the Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin J. Am. Chem. Soc. Heemstra Jr., J., R., Walsh, C., T., Sattely, E., S. 2009; 131: 15317-15329
• A Latent Oxazoline Electrophile for N-O-C Bond Formation in Pseudomonine Biosynthesis J. Am. Chem. Soc. Sattely, E., S., Walsh, C., T. 2008; 130: 12282-12284
• Total Biosynthesis: in vitro Reconstitution of Polyketide and Nonribosomal Peptide Pathways Nat. Prod. Rep. Sattely, E., S., Fischbach, M., A., Walsh, C., T. 2008; 25: 757-793
• Highly Efficient Molybdenum-Based Catalysts for Enantioselective Alkene Metathesis Nature Malcolmson, S., J., Meek, S., J., Sattely, E., S., Schrock, R., R., Hoveyda, A., H. 2008; 456: 933-937
• Enantioselective Synthesis of Cyclic Amines and Amides through Mo-Catalyzed Asymmetric Ring-Closing Metathesis J. Am. Chem. Soc. Sattely, E., S., Cortez, G., A., Moebius, D., C., Schrock, R., R., Hoveyda, A., H. 2005; 127: 8526-8533
• Efficient Catalytic Enantioselective Synthesis of Unsaturated Amines: Preparation of Small- and Medium- Ring Cyclic Amines through Mo-Catalyzed Asymmetric Ring-Closing Metathesis in the Absence of Solvent J. Am. Chem. Soc. Dolman, S., J., Sattely, E., S., Hoveyda, A., H., Schrock, R., R. 2002; 124: 6991-6997
• Catalytic Asymmetric Ring-Opening Metathesis/Cross Metathesis (AROM/CM) Reactions. Mechanism and Application to Enantioselective Synthesis of Functionalized Cyclopentanes J. Am. Chem. Soc. La, D., S., Sattely, E., S., Ford, J., G., Schrock, R., R., Hoveyda, A., H. 2001; 123: 7767-7778
• Tandem Catalytic Asymmetric Ring-Opening Metathesis/Cross Metathesis J. Am. Chem. Soc. La, D., S., Ford, J., G., Sattely, E., S., Bonitatebus, P., J., Schrock, R., R., Hoveyda, A., H. 1999; 121: 11603-11604