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DOE OSTI2022

Room-Temperature Optical Detection of 14 CO 2 below the Natural Abundance with Two-Color Cavity Ring-Down Spectroscopy

Radiocarbon’s natural production, radiative decay, and isotopic rarity make it a unique tool to probe carbonaceous systems in the life and earth sciences. However, the difficulty of current radiocarbon ( 14 C) detection methods limits scientific adoption. Here, two-color cavity ring-down spectroscopy detects 14 CO 2 in room-temperature samples with an accuracy of one-tenth the natural abundance in 3 min. The intracavity pump–probe measurement uses two cavity-enhanced lasers to cancel out cavity ring-down rate fluctuations and strong one-photon absorption interference (>10 000 1/s) from hot-band transitions of CO 2 isotopologues. Selective, room-temperature detection of small 14 CO 2 absorption signals (<1 1/s) reduces the technical and operational burdens for cavity-enhanced measurements of radiocarbon, which can benefit a wide range of applications like biomedical research and field-detection of combusted fossil fuels.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A 2 and B 2 bound to duplex 5'-TAGTT sites

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA 2 “green” (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5'-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA 2 into preformed host–guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA 2 was found to be similar to those reported earlier at the same DNA site for BLMB 2 , the intercalated bithiazole of BLMB 2 is “flipped” 180° relative to DNA-bound BLMA 2 . Further, this finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Leveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.

molecular biology↗

DOE OSTI2020

Combinatorial Biosynthesis of Sulfated Benzenediol Lactones with a Phenolic Sulfotransferase from Fusarium graminearum PH-1

Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus Fusarium graminearum PH-1. We expressed FgSULT1 in a Saccharomyces cerevisiae chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of Fusarium spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Advances in supramolecular host-mediated reactivity

Since the trailblazing discoveries of Lehn, Cram and Pedersen, supramolecular chemistry has established itself as a cornerstone of organic chemistry. Supramolecular hosts offer defined microenvironments that mimic the active sites of enzymes, utilizing specific host–guest interactions to enable remarkable rate enhancements and product selectivity. The development of a diverse array of self-assembled hosts, coupled with the increased demand for shorter and greener synthetic routes, have spurred significant progress in the field of supramolecular catalysis. This Review Article covers recent advances in the field, ranging from novel organic reactivity aided by supramolecular hosts to catalytic cooperation between hosts and organometallic compounds or metal nanoparticles. Furthermore, strides have also been made in the synthetic application of these hosts in site-selective substrate modifications and challenging photochemical reactions. These efforts have enabled the incorporation of non-covalent macromolecular catalysis in natural product syntheses, evidencing their unique advantages as a synthetic tool, and their powerful potential for practical applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-$α$-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Characterization of Oil and Gas Drill Cuttings for Critical Mineral Recovery and Reuse Potential as Soil Supplements

Expansion of unconventional oil and natural gas production over the last decade in the United States has resulted in record production of natural gas and oil in 2024. Millions of tons of drill cuttings generated from shale gas development are currently disposed of in landfills. Converting these cuttings into value product(s) such as critical minerals (CMs) and soil supplements, has the potential of reducing environmental impact of fossil fuel exploration. In this study we characterize the CM distribution and extractability from collected drill cuttings and core samples from major U.S. shale formations. A four-step sequential extraction, consisting of a sonicated soap step, sonicated EDTA step , mildly reducing agent, and a oxidizing agent, was developed to simultaneously extract CMs and convert the drill cuttings into soil supplements. Viability of converted drill cuttings as soil supplement was determined with a seedling growth experiment. Results show high concentrations of vanadium (V) (up to 1575 ppm, barite (up to 5 wt. %), and rare earth elements (REE) concentrations (up to 253 ppm). High extractability of selected critical minerals such as REE (19-50%) and Ba (10-58%) show promising results. Preliminary results show seedling growth in a mixture of converted drill cuttings with soil. These results show the potential for recovery of CMs from drill cuttings as an alternative domestic and the reduction of the environmental impact of fossil fuel production.

Barczok, Maximilian↗

DOE OSTI2020

Role of interaction between hydraulic and natural fractures on production

One of the main purposes of hydraulic fracturing in unconventional resources such as shale is to improve the connectivity between existing natural fractures and the production well. Since the inherent permeability of shale is extremely low, this newly formed fracture connectivity provides faster paths for the hydrocarbon stored in preexisting natural fractures. In this study, we performed numerical simulations of the free gas production at large scale fractured reservoir with different pre-existing natural fracture network intensities and with variable hydraulic fracture sizes and staging. We explored production characteristics as a function of the properties of both, natural fractures (intensity) and hydraulic fractures (size and staging). Our hypothesis is that once maximum fracture connectivity is achieved, additional stimulation does not increase production significantly. In this numerical study we use an advanced modeling tool to study fractures connectivity, Discrete Fracture Network (DFN) approach, which can represent fracture networks similar to those observed on individual reservoir sites. In our model the horizontal production well and vertical hydraulic fractures were deterministically defined and surrounded by stochastically generated natural fracture networks. The performed numerical experiments show how fracture connectivity depends on hydraulic fracture settings in fractured reservoir with different preexisting fracture densities. We numerically showed that cumulative free gas production is not affected significantly by hydraulic fractures size (25 m–150 m) or spacing (15 m–45 m) for dense natural fracture networks. However, the hydraulic fracture size is important in reservoirs with sparse natural fractures, although spacing (number of stages) does not play a significant role.

58 GEOSCIENCES↗

DOE OSTI2022

Hanford Reach Fall Chinook Salmon Productivity Analysis, Brood Years 1975–2016

Updating the Hanford Reach fall Chinook salmon productivity analysis to include seven additional years of data (BY 2010–2016), including three years of record escapements (>150,000 adults), reaffirmed the highly productive nature of this population and the positive effect of flow fluctuation constraints as implemented at Priest Rapids Dam by the VBSA and HRFCPP. The addition of several years of high escapements also revealed that competition among juveniles (and not spawning habitat limitations) is likely the mechanism driving density dependence in the Hanford Reach. The addition of the high escapement brood years also provided evidence for the high recruitment capacity of the population. As such, the WDFW may consider increasing the maximum escapement goal from 42,000 adults to 49,000 adults, which would be more in-line with SMSY as estimated from this update. Given the in-river protections that are in place as part of the HRFCPP, ocean conditions appear to be the primary factor controlling adult production. Therefore, the success of any additional management actions taken in freshwater are likely to be dependent on survival in the ocean. As such, the most effective management actions may be those that limit juvenile competition and density dependent growth effects in the Hanford Reach. Limiting competition would be expected to increase growth, resulting in larger smolts that would be less likely to suffer size-dependent mortality in the marine environment, even during years of poor ocean conditions. Increasing terminal harvest rates when high escapements are anticipated (from downstream dam counts) is one management option that could help to curtail juvenile competition.

Harnish, Ryan A.↗

DOE OSTI2025

Oxidative Dehydrogenation of N ‐Heteroaromatic Alkyl Alcohols and Amines Facilitated by Dearomative Tautomerization

The oxidation of alcohols, amines, and halides is a fundamental transformation in organic chemistry with significant applications in the synthesis of fine chemicals, pharmaceuticals, and natural products. Here we show that a broad variety of N-heteroarenes bearing hydroxymethyl, aminomethyl, or halomethyl groups are oxidatively dehydrogenated to their respective aldehydes by simply heating them in acidic or basic aqueous solution under ambient atmosphere. The quantitative oxidation of 9-acridinemethanol to 9-acridinecarboxaldehyde serves as an illustrative example, proceeding to completion within 3 hours in refluxing 5% aqueous acetic acid or even household vinegar. Quinoline derivatives may be similarly oxidized but require higher temperatures and longer reaction times, while indole derivatives are oxidized under basic conditions. Based on comprehensive regioselectivity screens, internal kinetic isotope competition, and density functional theory (DFT) calculations, we propose a mechanism in which migration of a methylene hydrogen to the pyridinic nitrogen by acid-catalyzed dearomative tautomerization yields an unstable enol or enamine intermediate that then irreversibly loses two hydrogen atoms to atmospheric oxygen. In addition to the simplicity and environmentally benign nature of our method, we observe no indication of any over-oxidation to carboxylic acids. Finally, we demonstrate the synthetic utility of this reaction through two different one-pot formylations of acridine.

Chemistry↗

DOE OSTI2020

Genomics-enabled analysis of specialized metabolism in bioenergy crops: Current progress and challenges

Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production have intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of ‘user-friendly’ biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin

The siderophore rhizoferrin (N 1 ,N 4 -dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Microfluidic droplets with amended culture media cultivate a greater diversity of soil microorganisms

ABSTRACT Uncultivated but abundant soil microorganisms have untapped potential for producing broad ranges of natural products, as well as for bioremediation. However, cultivating soil microorganisms while maintaining a broad microorganism diversity to enable phenotyping and functional analysis of as diverse individual isolates as possible remains challenging. In this study, we developed and tested the ability of several culture media formulations that contain defined soil metabolites or soil extracts to maintain microorganism diversity during culture. We also assessed their performance in microfluidic droplet cultivation where single-soil microorganism isolates were encapsulated and cultivated in picoliter-volume water-in-oil emulsion droplets to enable clonal growth needed for downstream functional analyses. Our results show that droplet cultivation with media supplemented by soil extract or soil metabolites enables the recovery of soil microorganisms with higher diversity (up to 1.5-fold higher richness) compared to bulk cultivation methods. Importantly, 1.7-fold more of less abundant (<1%) phyla and 11-fold more of unique genera were recovered, demonstrating the utility of this method for interrogating highly diverse soil microorganisms for broad ranges of applications. IMPORTANCE Although soil microorganisms hold a significant value in bioproduction and bioremediation, only a small fraction—less than 1%—can be cultured under specific media and cultivation conditions. This indicates that there are ample opportunities in harvesting the diverse environmental microorganisms if isolating and recovering these uncultured microorganisms are possible. This paper presents a new cultivation technique composed of isolating single-soil microorganism cell from anin situsoil microorganism community in microfluidic droplets and conducting in-droplet cultivation in media supplemented by soil extract or soil metabolites. This method enables the recovery of a broader diversity of the original microorganism community, laying the groundwork for a high-throughput phenotyping of these diverse microorganisms from their natural habitats.

Biotechnology & Applied Microbiology↗

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 OSTI2024

Copper‐Catalyzed C(sp 3 )−H α‐Acetylation: Generation of Quaternary Centers

Abstract α‐substituted ketones are important chemical targets as synthetic intermediates as well as functionalities in natural products and pharmaceuticals. We report the α‐acetylation of C(sp 3 )−H substrates R−H with arylmethyl ketones ArC(O)Me to provide α‐alkylated ketones ArC(O)CH 2 R at RT with t BuOO t Bu as oxidant via copper(I) ‐diketiminato catalysts. Proceeding via alkyl radicals R•, this method enables α‐substitution with bulky substituents without competing elimination that occurs in more traditional alkylation reactions between enolates and alkyl electrophiles. DFT studies suggest the intermediacy of copper(II) enolates [Cu II ](CH 2 C(O)Ar) that capture alkyl radicals R• to give R−CH 2 C(O)Ar outcompeting dimerization of the copper(II) enolate to give the 1,4‐diketone ArC(O)CH 2 CH 2 C(O)Ar.

Okoromoba, Otome E.↗

DOE OSTI2024

Copper‐Catalyzed C(sp 3 )−H α‐Acetylation: Generation of Quaternary Centers

Okoromoba, Otome E.↗

DOE OSTI2023

Cannabis monoterpene synthases: evaluating structure–function relationships

Terpene synthases catalyze the first committed step in the biosynthesis of terpenes, a structurally diverse class of natural products that also encompasses volatiles derived from precursors in the C10 to C15 range (termed monoterpenes and sesquiterpenes, respectively). In the review section of this article, we are providing information about all functionally characterized monoterpene synthases (MTSs) and sesquiterpene synthases (STSs) of Cannabis sativa L. We are also exploring the locations of MTSs and STSs in the chromosome-level assembly of the reference chemovar CBDRx. A follow-up computational structure–function analysis focuses on MTSs, as there is already a rich literature available on the topic. More specifically, by employing sequence comparisons and homology structural modeling, we infer which amino acid residues are likely to constrain the available space in the active site of cannabis MTSs. The emphasis of these studies was to investigate why some MTSs accept only a C10 diphosphate as substrate, while mixed MTS/STS enzymes also accommodate a C15 diphosphate. Here, by combining a literature review and computational analyses in a hybrid format, we are laying the foundation for future studies to better understand the determinants of substrate and product specificity in these fascinating enzymes.

59 BASIC BIOLOGICAL SCIENCES↗