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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Dataset Repository for Investigating Suicide Risk Using Social and Environmental Determinants of Health

Suicide is frequently modeled as a function of genetics and environment, where the latter refers to factors other than direct biological consequences, such as air quality, financial level, social connectivity, transportation and food access, and homelessness status. According to the World Health Organization, clean air, a stable climate, adequate water, sanitation and hygiene, safe chemical use, radiation protection, healthy and safe workplaces, sound agricultural practices, health-supportive cities and built environments, and a preserved natural environment are all prerequisites for good health. Understanding the relationships between these determinants and mental health outcomes requires standardized data that can be included in healthcare programs and health outcome models. There is a wealth of publicly available data on social and environmental factors provided by various US organizations that can benefit the design of health care systems and public health interventions, as well as improve our comprehension of factors that impact health. Such information would not only help improve the understanding of individual and community risk but also identify new risk factors that have not previously been therapeutically targeted, especially in terms of their impact on mental health. However, curating and standardizing such datasets is challenging because they are often recorded at numerous geographical and temporal resolutions and with varying spatial and temporal granularities. To address this challenge, we launched an endeavor in conjunction with the Veterans Health Administration to collect publicly available socioeconomic and environmental determinants of health statistics in the US. In this manuscript, we describe a social and environmental determinants of health (SEDH) datasets repository, data curation documentation, and a pipeline framework for data generation; This effort started in 2020, when we began constructing a scalable pipeline to automate the download, extraction, preparation, analysis, and production of datasets. These datasets have been made available to the VHA and may be shared upon agreement with collaborating organizations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Registration of the sorghum carbon–partitioning nested association mapping (CP–NAM) population

The sorghum [ Sorghum bicolor (L.) Moench] carbon-partitioning nested association mapping (CP_NAM) (Reg. no. MP-4, NSL 542189 MAP) population was developed at Clemson University, SC, using 11 diverse, male founder accessions, each crossed with a recurrent female parent ‘Grassl’. The male parents represent all five major botanical races and the four major agronomic types: cellulosic (5), sweet (3), grain (2) and forage (1). A set of 11 recombinant inbred line (RIL) families CP_NAM01 to CP_NAM011 were maintained, which consisted of 2,484 (F 6 ) individuals. Each RIL family contained a minimum of 193 individuals (CP_NAM01) and a maximum of 287 individuals (CP_NAM06). For the development of this population, the founder lines were judiciously selected from the sorghum Bioenergy Association Panel based on carbon-partitioning phenotypes that make this population an ideal genetic resource for dissecting a wide range of agronomic and compositional traits for basic and applied research. The founder accessions of the CP_NAM were phenotypically characterized for various traits, including agronomic, biomass and related components, and additional compositional components. Each of the 11 F 6 RIL families of the CP_NAM were genotyped using genotyping-by-sequencing analysis, and 144,087 single nucleotide polymorphisms were generated for each individual. Genotypic information along with phenotypic data were used for the characterization of this population and to explore the range of phenotypes that permits the understanding of carbon-partitioning dynamics. This population is a unique resource for researchers to study a wide range of contrasting carbon-partitioning characteristics in sorghum to understand the genetic architecture underlying whole-plant carbon partitioning and allocation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Full ribosomal operon sequencing of anaerobic gut fungi (phylum Neocallimastigomycota ): insights on its markers and phylogenetic resolution

The phylogenetic affiliations of anaerobic gut fungi (Neocallimastigomycota) are typically evaluated using single-gene markers. However, this approach often fails to resolve relationships between closely related lineages. To address this issue and identify alternative markers, we created a curated database comprising the complete ribosomal operon sequences of 156 isolates, representing 20 of the 22 recognized genera and two new genus-level clades. Using long-read sequencing, we obtained ~9 kbp operon sequences and developed a robust analysis pipeline. Incorporating both coding genes and non-coding regions (excluding IGS1) improved phylogenetic resolution. This phylogenetic approach successfully resolved the Cyllamyces and Caecomyces clades (hard-to-distinguish genetically), as well as seven analysed Piromyces species. We also scanned the operon for markers that are suitable for short-read sequencing platforms, with the aim of enhancing biodiversity and phylogenetic studies. Notably, the ETS1 genetic region also enabled the distinction between these lineages, indicating its phylogenetic value within the ribosomal operon. The resulting database is a valuable resource for expanding and strengthening phylogenetic frameworks.

High-throughput sequencing↗
DOE OSTI2024

Biosynthesis of Strained Amino Acids by a PLP‐Dependent Enzyme through Cryptic Halogenation

Abstract Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non‐ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non‐canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C 4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal‐5′‐phosphate‐dependent enzyme, PazB, via an S N 2‐like attack at C 4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans , show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane‐containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Sosa, Max B.↗
DOE OSTI2025

MPK6-mediated phosphorylation destabilizes MYC2 and attenuates its transcriptional activity in jasmonate signaling

Given the role of MYC2 as a pivotal regulator in the jasmonate (JA) signaling pathway, influencing the expression of a multitude of downstream genes (Zander et al., 2020), understanding the regulatory dynamics of MYC2 is essential for unraveling plant responses to stress conditions and hormonal signals. Previous studies on the regulation of MYC2 by MPK6 have reported conflicting findings. Takahashi et al. (2007) reported that MPK6 acts as a negative regulator of MYC2, whereas Sethi et al. (2014) proposed a positive regulatory role. Despite these findings, the precise genetic and biochemical mechanisms underlying the MPK6–MYC2 interaction remain poorly understood, highlighting the need for further investigation.

Im, Jong Hee [Michigan State University, East Lans↗
DOE OSTI2023

TCF High Efficiency Anaerobic Electroporation (CRADA Final Report)

The Joint BioEnergy Institute (JBEI) researchers were co-inventors of the technology that will be used on this project and have developed a more current version of the chip and controller. JBEI will also assist with the design of the pathways and implementation of pathways on the chip. LanzaTech has developed novel gas fermentation technology that captures and utilizes greenhouse gases for production of fuels and chemicals. In contrast to traditional fermentation that uses sugars as substrate (and releases CO2 as a byproduct), gas fermentation utilizes C1 substrates carbon monoxide (CO) or CO2. This enables a diverse range of feedstock options including waste gases from industrial sources (e.g., steel mills and processing plants) or syngas generated from any biomass resource (e.g., agricultural waste, municipal solid waste, or organic industrial waste). Biomass is then gasified, allowing for maximum yields and complete carbon utilization including the recalcitrant lignocellulosic fraction that cannot be utilized in traditional sugar fermentation. To maximize the value that can be added to the array of gas resources that the LanzaTech process can use as an input, LanzaTech has pioneered genetic modification of acetogens and developed a comprehensive set of genetic tools to perform routine strain modification, including genome editing tools as CRISPR/Cas9 and libraries of validated genetic parts as promoters and terminators. Using this platform, production of over 50 new molecules have been demonstrated directly from gas. For a few selected molecules production rates and yields have been optimized and surpass production of native producers and engineered E. coli or yeast strains, but a higher throughput approach for combinatorial optimization of pathways is required to further advance synthesis of additional products in parallel.

09 BIOMASS FUELS↗
DOE OSTI2024

Building a FAIR data ecosystem for incorporating single-cell transcriptomics data into agricultural genome to phenome research

Introduction The agriculture genomics community has numerous data submission standards available, but the standards for describing and storing single-cell (SC, e.g., scRNA- seq) data are comparatively underdeveloped. Methods To bridge this gap, we leveraged recent advancements in human genomics infrastructure, such as the integration of the Human Cell Atlas Data Portal with Terra, a secure, scalable, open-source platform for biomedical researchers to access data, run analysis tools, and collaborate. In parallel, the Single Cell Expression Atlas at EMBL-EBI offers a comprehensive data ingestion portal for high-throughput sequencing datasets, including plants, protists, and animals (including humans). Developing data tools connecting these resources would offer significant advantages to the agricultural genomics community. The FAANG data portal at EMBL-EBI emphasizes delivering rich metadata and highly accurate and reliable annotation of farmed animals but is not computationally linked to either of these resources. Results Herein, we describe a pilot-scale project that determines whether the current FAANG metadata standards for livestock can be used to ingest scRNA-seq datasets into Terra in a manner consistent with HCA Data Portal standards. Importantly, rich scRNA-seq metadata can now be brokered through the FAANG data portal using a semi-automated process, thereby avoiding the need for substantial expert curation. We have further extended the functionality of this tool so that validated and ingested SC files within the HCA Data Portal are transferred to Terra for further analysis. In addition, we verified data ingestion into Terra, hosted on Azure, and demonstrated the use of a workflow to analyze the first ingested porcine scRNA-seq dataset. Additionally, we have also developed prototype tools to visualize the output of scRNA-seq analyses on genome browsers to compare gene expression patterns across tissues and cell populations. This JBrowse tool now features distinct tracks, showcasing PBMC scRNA-seq alongside two bulk RNA-seq experiments. Discussion We intend to further build upon these existing tools to construct a scientist-friendly data resource and analytical ecosystem based on Findable, Accessible, Interoperable, and Reusable (FAIR) SC principles to facilitate SC-level genomic analysis through data ingestion, storage, retrieval, re-use, visualization, and comparative annotation across agricultural species.

Genetics & Heredity↗
DOE OSTI2025

Modulation of lignin and anthocyanin homeostasis by GTP cyclohydrolase1 in maize

Summary Maize is a key biomass resource with wide agricultural applications. Anthocyanins, potent antioxidants, offer health benefits like reducing oxidative stress. The biosynthesis of anthocyanins competes with that of lignin for shared metabolic precursors, which can lead to trade‐offs in plant growth and feed quality. Higher lignin content can decrease silage digestibility, posing challenges for livestock feed. The maizebrown midrib 6(bm6) mutant, known for reduced lignin, has an unclear genetic basis. Here, we identifyZmGCH1as the candidate gene forbm6through fine mapping. Mutations inZmGCH1shift precursors from lignin to anthocyanin biosynthesis. Furthermore, we show that ZmGCH1 interacts with ZmPEBP15 to modulate chalcone synthase activity, thereby stabilizing the allocation of precursors between lignin and anthocyanin pathways. To evaluate the practical implications of our findings, we introduced thebm6mutation into Zhengdan958 and Xianyu335. In vitro rumen digestion assays confirmed that the introduction of thebm6mutation significantly improved silage digestibility. This discovery not only holds great potential for enhancing silage digestibility but also provides a broader strategy for optimizing maize production to better meet the increasing demands of both the food and livestock feed.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Host-specific adaptation in Fusarium oxysporum correlates with distinct accessory chromosome content in human and plant pathogenic strains

ABSTRACT Fusarium oxysporumis a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations ofF. oxysporumto animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). Infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal disease in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato plants.In vitroassays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant to osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) that encode genes with different functions and have distinct transposon profiles between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotypes with phenotype and for developing targeted antifungals for agricultural and clinical uses. IMPORTANCE Fusarium oxysporumis a cross-kingdom fungal pathogen that infects both plants and animals. In addition to causing many devastating wilt diseases, this group of organisms was recently recognized by the World Health Organization as a high-priority threat to human health. Climate change has increased the risk ofFusariuminfections, asFusariumstrains are highly adaptable to changing environments. Deciphering fungal adaptation mechanisms is crucial to developing appropriate control strategies. We performed a comparative analysis ofFusariumstrains using an animal (mouse) and plant (tomato) host andin vitroconditions that mimic abiotic stress. We also performed comparative genomics analyses to highlight the genetic differences between human and plant pathogens and correlate their phenotypic and genotypic variations. We uncovered important functional hubs shared by plant and human pathogens, such as chromatin modification, transcriptional regulation, and signal transduction, which could be used to identify novel antifungal targets.

Microbiology↗
DOE OSTI2023

Model Organisms To Study Methanogenesis, a Uniquely Archaeal Metabolism

Methanogenic archaea are the only organisms that produce CH 4 as part of their energy-generating metabolism. They are ubiquitous in oxidant-depleted, anoxic environments such as aquatic sediments, anaerobic digesters, inundated agricultural fields, the rumen of cattle, and the hindgut of termites, where they catalyze the terminal reactions in the degradation of organic matter. Methanogenesis is the only metabolism that is restricted to members of the domain Archaea. Here, we discuss the importance of model organisms in the history of methanogen research, including their role in the discovery of the archaea and in the biochemical and genetic characterization of methanogenesis. Finally, we also discuss outstanding questions in the field and newly emerging model systems that will expand our understanding of this uniquely archaeal metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Arabidopsis thaliana zinc accumulation in leaf trichomes is correlated with zinc concentration in leaves

Zinc (Zn) is a key micronutrient for plants and animals, and understanding Zn homeostasis in plants can improve both agriculture and human health. While root Zn transporters in plant model species have been characterized in detail, comparatively little is known about shoot processes controlling Zn concentrations and spatial distribution. Previous work showed that Zn hyperaccumulator species such as Arabidopsis halleri accumulate Zn and other metals in leaf trichomes. To date there is no systematic study regarding Zn accumulation in the trichomes of the non-accumulating, genetic model species A. thaliana. Here, we used Synchrotron X-Ray Fluorescence mapping to show that Zn accumulates at the base of trichomes of A. thaliana. Using transgenic and natural accessions of A thaliana that vary in bulk leaf Zn concentration, we demonstrate that higher leaf Zn increases total Zn found at the base of trichome cells. Our data indicates that Zn accumulation in trichomes is a function of the Zn status of the plant, and provides the basis for future studies on a genetically tractable plant species to understand the molecular steps involved in Zn spatial distribution in leaves.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Root angle in maize influences nitrogen capture and is regulated by calcineurin B-like protein (CBL)-interacting serine/threonine-protein kinase 15 ( ZmCIPK15 )

Crops with reduced nutrient and water requirements are urgently needed in global agriculture. Root growth angle plays an important role in nutrient and water acquisition. A maize diversity panel of 481 genotypes was screened for variation in root angle employing a high-throughput field phenotyping platform. Genome-wide association mapping identified several single nucleotide polymorphisms (SNPs) associated with root angle, including one located in the root expressed CBL-interacting serine/threonine-protein kinase 15 (ZmCIPK15) gene (LOC100285495). Reverse genetic studies validated the functional importance of ZmCIPK15, causing a approximately 10° change in root angle in specific nodal positions. A steeper root growth angle improved nitrogen capture in silico and in the field. OpenSimRoot simulations predicted at 40 days of growth that this change in angle would improve nitrogen uptake by 11% and plant biomass by 4% in low nitrogen conditions. In field studies under suboptimal N availability, the cipk15 mutant with steeper growth angles had 18% greater shoot biomass and 29% greater shoot nitrogen accumulation compared to the wild type after 70 days of growth. We propose that a steeper root growth angle modulated by ZmCIPK15 will facilitate efforts to develop new crop varieties with optimal root architecture for improved performance under edaphic stress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Newly developed sugarbeet lines with altered postharvest respiration rates differ in transcription factor and glycolytic enzyme expression

Respiration is the principal cause for postharvest sucrose loss in sugarbeet (Beta vulgaris L.) roots. Although reductions in respiration rate could mitigate these losses, developing sugarbeet cultivars and storage procedures that reduce respiration are hindered by a lack of knowledge of the genetic and metabolic factors that control storage respiration rate. Research was conducted to identify genes and gene products that affect storage respiration rate by creating two sugarbeet lines that differ in respiration rate and characterizing gene expression differences between these lines. Sugarbeet lines F1056 and F1057, which differ by up to 42% in respiration rate, were created by divergent selection of a sugarbeet population using root respiration rate after 30 d in storage as the principal selection criterion. RNA sequencing identified 287 differentially expressed genes (DEGs) between these lines on the day of harvest and after 28-d storage. Of these DEGs, nine encoded transcription factors and five encoded enzymes involved in the respiratory pathway. Other DEGs contributed to a variety of biological and molecular functions based on gene ontology classifications. Of respiratory pathway DEGS, genes for NAD+- and NADP+-dependent forms of glyceraldehyde-3-phosphate dehydrogenase were of note due to their high upregulation in the high respiring line and for their established role in glycolysis, a pathway identified as a likely bottleneck in respiratory substrate production. Overall, lines F1056 and F1057 provide new tools for investigating genetic and physiological differences in storage respiration rate, and their DEGs identify candidates for genes affecting sugarbeet root respiration rate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Phenylpropanoid methyl esterase unlocks catabolism of aromatic biological nitrification inhibitors

Microbial nitrification of fertilizers represents is a significant global source of greenhouse gas emissions. This process increases emissions, fosters toxic algal blooms, and raises crop production costs. Some plants naturally release biological nitrification inhibitors to suppress ammonium-oxidizing microbes and reduce nitrification. Engineering nitrification inhibitor production into food and bioenergy crops via synthetic biology offers a promising mitigation strategy, but its success depends on addressing gaps in our understanding of inhibitor degradation in soil. This study begins to fill this gap by identifying a previously unknown microbial pathway for degrading phenylpropanoid methyl esters, a key class of aromatic nitrification inhibitors. Using transcriptomics and high-throughput functional genomics, we discovered genes essential for phenylpropanoid methyl ester degradation. Genetic and biochemical analyses revealed two novel enzymes, including a newly identified phenylpropanoid methyl esterase, that direct phenylpropanoid methyl esters into known metabolic pathways. Importantly, transferring these genes into bacteria capable of metabolizing other phenylpropanoids enabled them to use the methyl esters as a carbon source. This work provides critical insights into microbial nitrification inhibitor degradation, a poorly understood element of the nitrification cycle.

Genetic Engineering↗
DOE OSTI2023

Unlocking the magic in mycelium: Using synthetic biology to optimize filamentous fungi for biomanufacturing and sustainability

Filamentous fungi drive carbon and nutrient cycling across our global ecosystems, through its interactions with growing and decaying flora and their constituent microbiomes. The remarkable metabolic diversity, secretion ability, and fiber-like mycelial structure that have evolved in filamentous fungi have been increasingly exploited in commercial operations. The industrial potential of mycelial fermentation ranges from the discovery and bioproduction of enzymes and bioactive compounds, the decarbonization of food and material production, to environmental remediation and enhanced agricultural production. Despite its fundamental impact in ecology and biotechnology, molds and mushrooms have not, to-date, significantly intersected with synthetic biology in ways comparable to other industrial cell factories (e.g. Escherichia coli,Saccharomyces cerevisiae, and Komagataella phaffii). In this review, we summarize a suite of synthetic biology and computational tools for the mining, engineering and optimization of filamentous fungi as a bioproduction chassis. A combination of methods across genetic engineering, mutagenesis, experimental evolution, and computational modeling can be used to address strain development bottlenecks in established and emerging industries. These include slow mycelium growth rate, low production yields, non-optimal growth in alternative feedstocks, and difficulties in downstream purification. In the scope of biomanufacturing, we then detail previous efforts in improving key bottlenecks by targeting protein processing and secretion pathways, hyphae morphogenesis, and transcriptional control. Bringing synthetic biology practices into the hidden world of molds and mushrooms will serve to expand the limited panel of host organisms that allow for commercially-feasible and environmentally-sustainable bioproduction of enzymes, chemicals, therapeutics, foods, and materials of the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Variation in morpho-physiological and metabolic responses to low nitrogen stress across the sorghum association panel

Abstract Background Access to biologically available nitrogen is a key constraint on plant growth in both natural and agricultural settings. Variation in tolerance to nitrogen deficit stress and productivity in nitrogen limited conditions exists both within and between plant species. However, our understanding of changes in different phenotypes under long term low nitrogen stress and their impact on important agronomic traits, such as yield, is still limited. Results Here we quantified variation in the metabolic, physiological, and morphological responses of a sorghum association panel assembled to represent global genetic diversity to long term, nitrogen deficit stress and the relationship of these responses to grain yield under both conditions. Grain yield exhibits substantial genotype by environment interaction while many other morphological and physiological traits exhibited consistent responses to nitrogen stress across the population. Large scale nontargeted metabolic profiling for a subset of lines in both conditions identified a range of metabolic responses to long term nitrogen deficit stress. Several metabolites were associated with yield under high and low nitrogen conditions. Conclusion Our results highlight that grain yield in sorghum, unlike many morpho-physiological traits, exhibits substantial variability of genotype specific responses to long term low severity nitrogen deficit stress. Metabolic response to long term nitrogen stress shown higher proportion of variability explained by genotype specific responses than did morpho-pysiological traits and several metabolites were correlated with yield. This suggest, that it might be possible to build predictive models using metabolite abundance to estimate which sorghum genotypes will exhibit greater or lesser decreases in yield in response to nitrogen deficit, however further research needs to be done to evaluate such model.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Factors underlying a latitudinal gradient in the S/G lignin monomer ratio in natural poplar variants

The chemical composition of wood plays a pivotal role in the adaptability and structural integrity of trees. However, few studies have investigated the environmental factors that determine lignin composition and its biological significance in plants. Here, we examined the lignin syringyl-to-guaiacyl (S/G) ratio in members of a Populus trichocarpa population sourced from their native habitat and conducted a genome wide association study to identify genes linked to lignin formation. Our results revealed many significant associations, suggesting that lignin biosynthesis is a complex polygenic trait. Additionally, we found an increase in the S/G ratio from northern to southern geographic origin of the trees sampled, along with a corresponding metabolic and transcriptional reprogramming of xylem cell wall biosynthesis. Further molecular analysis identified a mutation in a cell wall laccase genetically associated with higher S/G ratios that predominate in trees from warmer lower latitudes. Collectively, our findings suggest that lignin heterogeneity arises from an evolutionary process enabling poplar adaptation to different climatic challenges.

adaptation↗
DOE OSTI2024

Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an in vivo genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in Escherichia coli. We characterized the enzyme variants in vitro and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. Furthermore, this work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗