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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Computer-aided, resistance gene-guided genome mining for proteasome and HMG-CoA reductase inhibitors

Secondary metabolites (SMs) are biologically active small molecules, many of which are medically valuable. Fungal genomes contain vast numbers of SM biosynthetic gene clusters (BGCs) with unknown products, suggesting that huge numbers of valuable SMs remain to be discovered. It is challenging, however, to identify SM BGCs, among the millions present in fungi, that produce useful compounds. One solution is resistance gene-guided genome mining, which takes advantage of the fact that some BGCs contain a gene encoding a resistant version of the protein targeted by the compound produced by the BGC. The bioinformatic signature of such BGCs is that they contain an allele of an essential gene with no SM biosynthetic function, and there is a second allele elsewhere in the genome. We have developed a computer-assisted approach to resistance gene-guided genome mining that allows users to query large databases for BGCs that putatively make compounds that have targets of therapeutic interest. Working with the MycoCosm genome database, we have applied this approach to look for SM BGCs that target the proteasome β6 subunit, the target of the proteasome inhibitor fellutamide B, or HMG-CoA reductase, the target of cholesterol reducing therapeutics such as lovastatin. Our approach proved effective, finding known fellutamide and lovastatin BGCs as well as fellutamide- and lovastatin-related BGCs with variations in the SM genes that suggest they may produce structural variants of fellutamides and lovastatin. Gratifyingly, we also found BGCs that are not closely related to lovastatin BGCs but putatively produce novel HMG-CoA reductase inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N 2 . Nitrogenase is most commonly associated with the molybdenum–iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two “alternative nitrogenase” systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bacterial xylan utilization regulons: systems for coupling depolymerization of methylglucuronoxylans with assimilation and metabolism

Abstract Bioconversion of lignocellulosic resources offers an economically promising path to renewable energy. Technological challenges to achieving bioconversion include the development of cost-effective processes that render the cellulose and hemicellulose components of these resources to fermentable hexoses and pentoses. Natural bioprocessing of the hemicellulose fraction of lignocellulosic biomass requires depolymerization of methylglucuronoxylans. This requires secretion of endoxylanases that release xylooligosaccharides and aldouronates. Physiological, biochemical, and genetic studies with selected bacteria support a process in which a cell-anchored multimodular GH10 endoxylanase catalyzes release of the hydrolysis products, aldotetrauronate, xylotriose, and xylobiose, which are directly assimilated and metabolized. Gene clusters encoding intracellular enzymes, including α-glucuronidase, endoxylanase, β-xylosidase, ABC transporter proteins, and transcriptional regulators, are coordinately responsive to substrate induction or repression. The rapid rates of glucuronoxylan utilization and microbial growth, along with the absence of detectable products of depolymerization in the medium, indicate that assimilation and depolymerization are coupled processes. Genomic comparisons provide evidence that such systems occur in xylanolytic species in several genera, including Clostridium, Geobacillus, Paenibacillus, and Thermotoga. These systems offer promise, either in their native configurations or through gene transfer to other organisms, to develop biocatalysts for efficient production of fuels and chemicals from the hemicellulose fractions of lignocellulosic resources.

Biotechnology & Applied Microbiology↗
DOE OSTI2026

Structure and sequence evolution in the pennycress ( Thlaspi arvense ) pangenome

Eukaryotic genomes harbor many forms of variation, including nucleotide diversity and structural polymorphisms, which experience natural selection and contribute to genome evolution and biodiversity. Harnessing this variation for agriculture hinges on our ability to detect, quantify, catalog, and deploy genetic diversity. Here, we explore seven complete genomes of the emerging biofuel crop pennycress ( Thlaspi arvense ) drawn from across the species' current genetic diversity to catalog variation in genome structure and content. Across this new pangenome resource, we find contrasting evolutionary modes in different genomic zones. Gene-poor, repeat-rich pericentromeric regions experience frequent rearrangements, including repeated centromere repositioning. By contrast, conserved gene-dense chromosome arms maintain large-scale synteny across accessions even in fast-evolving NOD-like receptor immune genes, where microsynteny breaks down across species, but gene cluster positioning macrosynteny is maintained. Our findings highlight that multiple elements of the genome experience dynamic evolution that conserves functional content on the chromosome scale but allows repositioning and presence–absence variation on a local scale. This diversity is invisible to classical reference-based strategies and highlights the strength and utility of pangenomic resources. These results provide a valuable case study of rapid genomic structural evolution within a species and powerful resources for crop development in an emerging biofuel crop.

Thlaspi arvense↗
DOE OSTI2022

Identification of Novel, Replicable Genetic Risk Loci for Suicidal Thoughts and Behaviors Among US Military Veterans

Importance: Suicide is a leading cause of death; however, the molecular genetic basis of suicidal thoughts and behaviors (SITB) remains unknown. Objective: To identify novel, replicable genomic risk loci for SITB. Design, Setting, and Participants: This genome-wide association study included 633 778 US military veterans with and without SITB, as identified through electronic health records. GWAS was performed separately by ancestry, controlling for sex, age, and genetic substructure. Cross-ancestry risk loci were identified through meta-analysis. Study enrollment began in 2011 and is ongoing. Data were analyzed from November 2021 to August 2022. Main Outcome and Measures: SITB. Results: A total of 633 778 US military veterans were included in the analysis (57 152 [9%] female; 121 118 [19.1%] African ancestry, 8285 [1.3%] Asian ancestry, 452 767 [71.4%] European ancestry, and 51 608 [8.1%] Hispanic ancestry), including 121 211 individuals with SITB (19.1%). Meta-analysis identified more than 200 GWS (P < 5 × 10 -8 ) cross-ancestry risk single-nucleotide variants for SITB concentrated in 7 regions on chromosomes 2, 6, 9, 11, 14, 16, and 18. Top single-nucleotide variants were largely intronic in nature; 5 were independently replicated in ISGC, including rs6557168 in ESR1, rs12808482 in DRD2, rs77641763 in EXD3, rs10671545 in DCC, and rs36006172 in TRAF3. Associations for FBXL19 and AC018880.2 were not replicated. Gene-based analyses implicated 24 additional GWS cross-ancestry risk genes, including FURIN, TSNARE1, and the NCAM1-TTC12-ANKK1-DRD2 gene cluster. Cross-ancestry enrichment analyses revealed significant enrichment for expression in brain and pituitary tissue, synapse and ubiquitination processes, amphetamine addiction, parathyroid hormone synthesis, axon guidance, and dopaminergic pathways. Seven other unique European ancestry–specific GWS loci were identified, 2 of which (POM121L2 and METTL15/LINC02758) were replicated. Two additional GWS ancestry-specific loci were identified within the African ancestry (PET112/GATB) and Hispanic ancestry (intergenic locus on chromosome 4) subsets, both of which were replicated. Further, no GWS loci were identified within the Asian ancestry subset; however, significant enrichment was observed for axon guidance, cyclic adenosine monophosphate signaling, focal adhesion, glutamatergic synapse, and oxytocin signaling pathways across all ancestries. Within the European ancestry subset, genetic correlations (r > 0.75) were observed between the SITB phenotype and a suicide attempt-only phenotype, depression, and posttraumatic stress disorder. Additionally, polygenic risk score analyses revealed that the Million Veteran Program polygenic risk score had nominally significant main effects in 2 independent samples of veterans of European and African ancestry.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

New approaches to secondary metabolite discovery from anaerobic gut microbes

The animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilize in silico tools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Statistical 3D morphology characterization of vaterite microspheres produced by engineered Escherichia coli

Hollow vaterite microspheres are important materials for biomedical applications such as drug delivery and regenerative medicine owing to their biocompatibility, high specific surface area, and ability to encapsulate a large number of bioactive molecules and compounds. We demonstrated that hollow vaterite microspheres are produced by an Escherichia coli strain engineered with a urease gene cluster from the ureolytic bacteria Sporosarcina pasteurii in the presence of bovine serum albumin. We characterized the 3D nanoscale morphology of five biogenic hollow vaterite microspheres using 3D high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) tomography. Using automated high-throughput HAADF-STEM imaging across several sample tilt orientations, we show that the microspheres evolved from a smaller more ellipsoidal shape to a larger more spherical shape while the internal hollow core increased in size and remained relatively spherical, indicating that the microspheres produced by this engineered strain likely do not contain the bacteria. The statistical 3D morphology information demonstrates the potential for using biogenic calcium carbonate mineralization to produce hollow vaterite microspheres with controlled morphologies.

3D morphology↗
DOE OSTI2024

Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Genetics & Heredity↗
DOE OSTI2025

A cobalamin-dependent pathway of choline demethylation from the human gut acetogen Eubacterium limosum

Elevated serum levels of trimethylamine N-oxide (TMAO) are reported to promote the development of atherosclerosis. TMAO is produced by hepatic oxidation of trimethylamine (TMA) produced by the gut microbiome from dietary quaternary amines such as choline. Net TMA production in the gut depends on microbial enzymes that either produce or consume TMA and its precursors. Here we report the elucidation of a novel microbial pathway consuming choline without TMA production. The human gut acetogen Eubacterium limosum grows by demethylating choline to N-N-dimethylaminoethanol. Quantitative mass spectral analysis of the proteome revealed a multi-protein choline to tetrahydrofolate (THF) methyltransferase system present only in choline-grown cells. The components are encoded in a gene cluster on the genome and include MthB, an MttB superfamily member; MthC, homologous to methylotrophic cobalamin-binding proteins; MthA, homologous to cobalamin:THF methyltransferases; and MthK, a protein related to serine kinases. Together, MthB, MthC, and MthA methylate THF with phosphocholine, but not choline or other quaternary amines. MthB specifically methylates Co(I)-MthC with phosphocholine. MthK acts as a bifunctional choline kinase which can utilize ATP or the MthB demethylation product, N,N-dimethylaminoethanol phosphate, to phosphorylate choline. Together, MthK, MthB, MthC, and MthA are proposed to carry out the methylation of THF with choline. These results outline a THF methylation pathway in which choline is first activated with ATP to phosphocholine prior to demethylation to form N,N-dimethylaminoethanol phosphate. Furthermore, the latter can be recycled by MthK to form more phosphocholine without expending additional ATP, thus minimizing energy utilization during choline-dependent acetogenesis.

acetogenesis↗
DOE OSTI2021

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. Here, to characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Engineering Escherichia coli for Urease-Driven Synthesis of Metal Oxide Nanomaterials

The development of functional nanomaterials with controlled morphologies is essential for advancements in medicine, electronics and computing, energy, catalysis, and environmental applications. However, conventional synthesis methods often demand high energy input and pose significant environmental challenges. Urease-based biomineralization presents an efficient, eco-friendly alternative for nanomaterial production under mild conditions. In this study, we engineered Escherichia coli ( E. coli ) to express a urease gene cluster from Sporosarcina pasteurii using CRAGE-Duet technology. The engineered strain successfully synthesized calcium carbonate and calcium phosphate crystals. Expanding the approach, we synthesized metal oxide nanoparticles, including hematite (Fe 2 O 3 ), and nanocrystalline anatase titanium dioxide (TiO 2 ). These nanomaterials were characterized by electron microscopy, demonstrating the potential of E. coli as a sustainable and versatile platform for green nanomaterial synthesis.

bacteria↗
DOE OSTI2022

Intramolecular C–C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the alpha/beta-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. Here, this aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the alpha/ beta-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota

Abstract Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 μm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

59 BASIC BIOLOGICAL SCIENCES↗