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SalL, an enzyme that catalyzes the synthesis of SAM from l-methionine and 5'-chloro-5'-deoxyoadenosine, is shown to accept 5'-chloro-5'-deoxythienoadenosine as a substrate and facilitate the synthesis of a synthetic SAM analog with an unnatural nucleobase. This synthetic cofactor is demonstrated to replace SAM in the DNA methylation reaction with M.TaqI.

OBJECTIVES:. The utility and risks to providers of performing cardiopulmonary resuscitation after in-hospital cardiac arrest in COVID-19 patients have been questioned. Additionally, there are discrepancies in reported COVID-19 in-hospital cardiac arrest survival rates. We describe outcomes after cardiopulmonary resuscitation for in-hospital cardiac arrest in two COVID-19 patient cohorts. DESIGN:. Retrospective cohort study. SETTING:. New York-Presbyterian Hospital/Columbia University Irving Medical Center in New York, NY. PATIENTS:. Those admitted with COVID-19 between March 1, 2020, and May 31, 2020, as well as between March 1, 2021, and May 31, 2021, who received resuscitation after in-hospital cardiac arrest. INTERVENTIONS:. None. MEASUREMENT AND MAIN RESULTS:. Among 103 patients with coronavirus disease 2019 who were resuscitated after in-hospital cardiac arrest in spring 2020, most self-identified as Hispanic/Latino or African American, 35 (34.0%) had return of spontaneous circulation for at least 20 minutes, and 15 (14.6%) survived to 30 days post-arrest. Compared with nonsurvivors, 30-day survivors experienced in-hospital cardiac arrest later (day 22 vs day 7; p = 0.008) and were more likely to have had an acute respiratory event preceding in-hospital cardiac arrest (93.3% vs 27.3%; p < 0.001). Among 30-day survivors, 11 (73.3%) survived to hospital discharge, at which point 8 (72.7%) had Cerebral Performance Category scores of 1 or 2. Among 26 COVID-19 patients resuscitated after in-hospital cardiac arrest in spring 2021, 15 (57.7%) had return of spontaneous circulation for at least 20 minutes, 3 (11.5%) survived to 30 days post in-hospital cardiac arrest, and 2 (7.7%) survived to hospital discharge, both with Cerebral Performance Category scores of 2 or less. Those who survived to 30 days post in-hospital cardiac arrest were younger (46.3 vs 67.8; p = 0.03), but otherwise there were no significant differences between groups. CONCLUSIONS:. Patients with COVID-19 who received cardiopulmonary resuscitation after in-hospital cardiac arrest had low survival rates. Our findings additionally show return of spontaneous circulation rates in these patients may be impacted by hospital strain and that patients with in-hospital cardiac arrest preceded by acute respiratory events might be more likely to survive to 30 days, suggesting Advanced Cardiac Life Support efforts may be more successful in this subpopulation.

Aromatic diisocyanates, invaluable commodity chemicals for polymer manufacturing, are produced annually on megaton scales from petroleum-derived diamines via phosgenation. Existing routes toward renewable alternatives are sparse and limited by access to functionalized aromatic starting materials, such as terephthalates. Herein, we report the development of a robust route to renewable terephthalates and aromatic diisocyanates from D-galactose via Eastwood olefination and Diels-Alder cycloaddition, followed by a mild electrochemical decarboxylative aromatization. This process was developed and applied on gram-scale to synthesize terephthalates, which were transformed into aromatic diisocyanates via Curtius rearrangement in flow. We demonstrate gram-scale preparation of 1,4-phenylene diisocyanate and 2,5-toluene diisocyanate and formulation of these monomers to prepare fully renewable thermoplastic polyurethanes. Preparation of these renewable aromatic diisocyanates proceeds without the use of high-pressure gases or costly transition-metals and represents a novel route to fully renewable aromatic diisocyanates., (© 2025 Wiley-VCH GmbH.)

Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called megasynthetases 1 . They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation 2 steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures 3 . Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains 4,5 . We apply tetrazine click chemistry to trap the condensation of two carrier protein substrates within the active site of the condensation domain that unites the first two modules of tyrocidine biosynthesis and report the high-resolution cryo-EM structure of this complex. Together with the X-ray crystal structure of the first carrier protein crosslinked to its epimerization domain, these structures highlight captured intermodular recognition events and define the processive movement of a carrier protein from one catalytic step to the next. Characterization of these structural relationships remains central to understanding the molecular details of these unique synthetases and critically informs future synthetic biology design of these pathways., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Mycobacterium tuberculosis (Mtb) is a leading cause of death, with an escalating global occurrence of drug-resistant infections that are partially attributed to cell wall mycolic acids derived from type II fatty acid biosynthesis (FAS-II). Here, the central acyl carrier protein, AcpM, contributes to the regulation of complex and specific protein-protein interactions (PPIs), though the orchestration of these events remain largely unresolved due to unique features of AcpM. Limitations include complexities in generating modified AcpM in a single state. Herein, we report a streamlined method to generate homogeneous samples of modified AcpM for applications in structure and functional studies. We apply these to generate solvatochromic labeled crypto -AcpM, where fluorescence response reports cargo sequestration and chain flipping upon interaction with four FAS-II enzymes. We find an increased fluorescence in a truncated form, AcpM80, indicating that the 35-residue C-terminus is involved in modulating the chemical environment surrounding the substrate and contributing to the regulation of PPIs. This study establishes an efficient chemo-enzymatic strategy to generate AcpM analogs for biophysical studies to aid in understanding the processes driving Mtb pathogenicity and drug resistance.

Microalgae offer a compelling platform for the production of commodity products, due to their superior photosynthetic efficiency, adaptability to nonarable lands and nonpotable water, and their capacity to produce a versatile array of bioproducts, including biofuels and biomaterials. However, the scalability of microalgae as a bioresource has been hindered by challenges such as costly biomass production related to vulnerability to pond crashes during large-scale cultivation. This study presents a pipeline for the genetic engineering and pilot-scale production of biodiesel and thermoplastic polyurethane precursors in the extremophile species Chlamydomonas pacifica . This extremophile microalga exhibits exceptional resilience to high pH (>11.5), high salinity (up to 2% NaCl), and elevated temperatures (up to 42 °C). Initially, we evolved this strain to also have a high tolerance to high light intensity (>2000 μE/m 2 /s) through mutagenesis, breeding, and selection. We subsequently genetically engineered C. pacifica to significantly enhance lipid production by 28% and starch accumulation by 27%, all without affecting its growth rate. We demonstrated the scalability of these engineered strains by cultivating them in pilot-scale raceway ponds and converting the resulting biomass into biodiesel and thermoplastic polyurethanes. This study showcases the complete cycle of transforming a newly discovered species into a commercially relevant commodity production strain. This research underscores the potential of extremophile algae, including C. pacifica , as a key species for the burgeoning sustainable bioeconomy, offering a viable path forward in mitigating environmental challenges and supporting global bioproduct demands., Competing Interests: The authors declare the following competing financial interest(s): Stephen Mayfield and Michael D. Burkart are co-founders of and holds equity in Algenesis Inc., a company that could potentially benefit from this research. Marissa Tessman is an employee and shareholder in Algenesis Inc. The other authors declare that their research was conducted without any commercial or financial relationships that could be perceived as potential conflicts of interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Glyoxal has been implicated as a significant contributor to the formation of secondary organic aerosols, which play a key role in our ability to estimate the impact of aerosols on climate. Elevated concentrations of glyoxal over open ocean waters suggest that there exists an additional source, different from urban and forest environments, which has yet to be identified., Methods: Based on mass spectrometric analyses of nascent sea spray aerosols (SSAs) and gas-phase molecules generated during the course of a controlled algal bloom, the work herein suggests that marine microorganisms are capable of excreting toluene in response to environmental stimuli. Additional culture flask experiments demonstrated that pathogenic attack could also serve as a trigger for toluene formation. Using solid-phase microextraction methods, the comparison of samples collected up-channel and over the breaking wave suggests it was transferred across the air-water interface primarily through SSA formation., Results: The presence and then absence of phenylacetic acid in the SSA days prior to the appearance of toluene support previous reports that proposed toluene is produced as a metabolite of phenylalanine through the Shikimate pathway. As a result, once in the atmosphere, toluene is susceptible to oxidation and subsequent degradation into glyoxal., Conclusions: This work adds to a minimal collection of literature that addresses the primary production of aromatic hydrocarbons from marine microorganisms and provides a potential missing source of glyoxal that should be considered when accounting for its origins in remote ocean regions.

Carrier protein-dependent synthases are ubiquitous enzymes involved both in primary and secondary metabolism. Biocatalysis within these synthases is governed by key interactions between the carrier protein, substrate, and partner enzymes. The weak and transient nature of these interactions has rendered them difficult to study. Here we develop a useful fluorescent solvatochromic probe, dapoxyl-pantetheinamide, to monitor and quantify carrier protein interactions in vitro . Upon loading with target carrier proteins, we observe dramatic shifts in fluorescence emission wavelength and intensity and further demonstrate that this tool has the potential to be applied across numerous biosynthetic pathways. The environmental sensitivity of this probe allows rapid characterization of carrier protein interactions, with the ability to quantitatively determine inhibition of protein-protein interactions. We anticipate future application of these probes for inhibitor screening and in vivo characterization., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems., (© 2024. The Author(s).)

Petroleum-based plastic waste plagues the natural environment, but plastics solve many high-performance solutions across industries. For example, porous polymer membranes are used for air filtration, advanced textiles, energy, and biomedical applications. Sustainable and biodegradable Bioplastic membranes can compete with nonrenewable materials in strength, durability, and functionality but biodegrade under select conditions after disposal. Membranes electrospun using a blend of bioderived thermoplastic polyurethane (TPU) and polylactic acid (PLA) perform effectively under tensile and cyclic loading, act adequately as an air filter media, and biodegrade in a home-compost environment, with the aliphatic formulation of TPU showing greater biodegradability compared to the formulation containing aromatic moieties. Blending TPU with PLA dramatically increases the strain at break of the PLA membrane, while the addition of PLA in TPU stiffens the material considerably. Measurements of the pressure drop and filtration efficiency deem this electrospun membrane an effective air filter. This membrane provides a solution to the need for quality air filtration while decreasing the dependence on petroleum feedstocks and addressing the issue of plastic disposal through biodegradation., Competing Interests: The authors declare the following competing financial interest(s): G.B.S., J.R., R.S., and K.O. are employees and shareholders in Algenesis Corporation, a company that could benefit from this research. M.D.B. and S.P.M. are founders and hold an equity position in Algenesis Corporation., (© 2024 The Authors. Published by American Chemical Society.)

The antibiotic cerulenin is a fungal natural product identified as a covalent inhibitor of ketosynthases within fatty acid and polyketide biosynthesis. Due to its selective and potent inhibitory activity, cerulenin has found significant utility in multidisciplinary biochemical, biomedical, and clinical studies. Although its covalent inhibition profile has been confirmed, cerulenin's mechanism has not been fully determined at a molecular level, frustrating the drug development of related analogues. Herein, we describe the use of stable isotopic tracking with NMR and MS methods to unravel the covalent mechanism of cerulenin against type II fatty acid ketosynthases. We detail the discovery of a unique C2-C3 retro-aldol bond cleavage and a structural rearrangement upon covalent inhibition of cerulenin at the active cysteine residue in E. coli type II fatty acid ketosynthases FabB and FabF.

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

The acyl carrier protein of Escherichia coli , termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15 N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.

The accumulation of microplastics in various ecosystems has now been well documented and recent evidence suggests detrimental effects on various biological processes due to this pollution. Accumulation of microplastics in the natural environment is ultimately due to the chemical nature of widely used petroleum-based plastic polymers, which typically are inaccessible to biological processing. One way to mitigate this crisis is adoption of plastics that biodegrade if released into natural environments. In this work, we generated microplastic particles from a bio-based, biodegradable thermoplastic polyurethane (TPU-FC1) and demonstrated their rapid biodegradation via direct visualization and respirometry. Furthermore, we isolated multiple bacterial strains capable of using TPU-FC1 as a sole carbon source and characterized their depolymerization products. To visualize biodegradation of TPU materials as real-world products, we generated TPU-coated cotton fabric and an injection molded phone case and documented biodegradation by direct visualization and scanning electron microscopy (SEM), both of which indicated clear structural degradation of these materials and significant biofilm formation., (© 2024. The Author(s).)

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Outside of their involvement in energy production, mitochondria play a critical role for the cell through their access to a discrete pathway for fatty acid biosynthesis. Despite decades of study in bacterial fatty acid synthases (the putative evolutionary mitochondrial precursor), our understanding of human mitochondrial fatty acid biosynthesis remains incomplete. In particular, the role of the key carrier protein, human mitochondrial acyl carrier protein (mACP), which shuttles the substrate intermediates through the pathway, has not been well-studied in part due to challenges in protein expression and purification. Herein, we report a reliable method for recombinant Escherichia coli expression and purification of mACP. Fundamental characteristics, including substrate sequestration and chain-flipping activity, are demonstrated in mACP using solvatochromic response. This study provides an efficient approach toward understanding the fundamental protein-protein interactions of mACP and its partner proteins, ultimately leading to a molecular understanding of human mitochondrial diseases such as mitochondrial fatty acid oxidation deficiencies.

Carrier-protein-dependent metabolic pathways biosynthesize fatty acids, polyketides, and non-ribosomal peptides, producing metabolites with important pharmaceutical, environmental, and industrial properties. Recent findings demonstrate that these pathways rely on selective communication mechanisms involving protein-protein interactions (PPIs) that guide enzyme reactivity and timing. While rational design of these PPIs could enable pathway design and modification, this goal remains a challenge due to the complex nature of protein interfaces. Computational methods offer an encouraging avenue, though many score functions fail to predict experimental observables, leading to low success rates. Here, we improve upon the Rosetta score function, leveraging experimental data through iterative rounds of computational prediction and mutagenesis, to design a hybrid fatty acid-non-ribosomal peptide initiation pathway. By increasing the weight of the electrostatic score term, the computational protocol proved to be more predictive, requiring fewer rounds of iteration to identify mutants with high in vitro activity. This allowed efficient design of new PPIs between a non-ribosomal peptide synthetase adenylation domain, PltF, and a fatty acid synthase acyl carrier protein, AcpP, as validated by activity and structural studies. This method provides a promising platform for customized pathway design, establishing a standard for carrier-protein-dependent pathway engineering through PPI optimization.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva., Results: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously., Conclusions: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract., (© 2023. The Author(s).)

Highly functionalized skeletons of macrolide natural products gain access to rare spatial arrangements of atoms, where changes in stereochemistry can have a profound impact on the structure and function. Spliceosome modulators present a unique consensus motif, with the majority targeting a key interface within the SF3B spliceosome complex. Our recent preparative-scale synthetic campaign of 17 S -FD-895 provided unique access to stereochemical analogues of this complex macrolide. Here, we report on the preparation and systematic activity evaluation of multiple FD-895 analogues. These studies examine the effects of modifications at specific stereocenters within the molecule and highlight future directions for medicinal chemical optimization of spliceosome modulators.

Neuroimmune pathways regulate brain function to influence complex behavior and play a role in several neuropsychiatric diseases, including alcohol use disorder (AUD). In particular, the interleukin-1 (IL-1) system has emerged as a key regulator of the brain's response to ethanol (alcohol). Here we investigated the mechanisms underlying ethanol-induced neuroadaptation of IL-1β signaling at GABAergic synapses in the prelimbic region of the medial prefrontal cortex (mPFC), an area responsible for integrating contextual information to mediate conflicting motivational drives. We exposed C57BL/6J male mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and conducted ex vivo electrophysiology and molecular analyses. We found that the IL-1 system regulates basal mPFC function through its actions at inhibitory synapses on prelimbic layer 2/3 pyramidal neurons. IL-1β can selectively recruit either neuroprotective (PI3K/Akt) or pro-inflammatory (MyD88/p38 MAPK) mechanisms to produce opposing synaptic effects. In ethanol naïve conditions, there was a strong PI3K/Akt bias leading to a disinhibition of pyramidal neurons. Ethanol dependence produced opposite IL-1 effects - enhanced local inhibition via a switch in IL-1β signaling to the canonical pro-inflammatory MyD88 pathway. Ethanol dependence also increased cellular IL-1β in the mPFC, while decreasing expression of downstream effectors (Akt, p38 MAPK). Thus, IL-1β may represent a key neural substrate in ethanol-induced cortical dysfunction. As the IL-1 receptor antagonist (kineret) is already FDA-approved for other diseases, this work underscores the high therapeutic potential of IL-1 signaling/neuroimmune-based treatments for AUD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Herein, we investigate synthetic routes to a close mimic of natural pheomelanin. Three different oxidative polymerization routes were attempted to generate synthetic pheomelanin, each giving rise to structurally dissimilar materials. Among them, the route employing 5-cysteinyl-dihydroxyphenylalanine (5-CD) as a monomer was verified as a close analogue of extracted pheomelanin from humans and birds. The resulting biomimetic and natural pheomelanins were compared via various techniques, including solid-state Nuclear Magnetic Resonance (ssNMR) and Electron Paramagnetic Resonance (EPR). This synthetic pheomelanin closely mimics the structure of natural pheomelanin as determined by parallel characterization of pheomelanin extracted from multiple biological sources. With a good synthetic biomimetic material in hand, we describe cation-π interactions as an important driving force for pheomelanogenesis, further advancing our fundamental understanding of this important biological pigment., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy., Competing Interests: Declaration of interests M.D.B. is a co-founder of Aspera Biomedicines. C.H.M.J. is a co-founder of Aspera Biomedicines and Impact Biomedicines and has received royalties for intellectual property licensed by Forty Seven Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation., Competing Interests: Declaration of interests C.H.M.J. is a co-founder of Aspera Biomedicines and Impact Biomedicines and receives royalties from Forty Seven Inc. M.D.B. is a co-founder of Aspera Biomedicines. C.H.M.J., L.A.C., M.D.B., L.B., P.K.M., C.N.M., R.H.D., J.J.L.C., T.W., I.v.d.W., P.W., and W.M. are named on patents related to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Human carbonic anhydrase II (hCAII) is a metalloenzyme essential to critical physiological processes in the body. hCA inhibitors are used clinically for the treatment of indications ranging from glaucoma to epilepsy. Targeted protein degraders have emerged as a promising means of inducing the degradation of disease-implicated proteins by using the endogenous quality control mechanisms of a cell. Here, a series of heterobifunctional degrader candidates targeting hCAII were developed from a simple aryl sulfonamide fragment. Degrader candidates were functionalized to produce either cereblon E3 ubiquitin ligase (CRBN) recruiting proteolysis targeting chimeras (PROTACs) or adamantyl-based hydrophobic tags (HyTs). Screens in HEK293 cells identified two PROTAC small-molecule degraders of hCA. Optimization of linker length and composition yielded a degrader with sub-nanomolar potency and sustained depletion of hCAII over prolonged treatments. Mechanistic studies suggest that this optimized degrader depletes hCAII through the same mechanism as previously reported CRBN-recruiting heterobifunctional degraders.

Acyl-homoserine lactone synthases make specific AHL quorum sensing signals to aid virulence in Gram-negative bacteria. Here, we use solution NMR spectroscopy to demonstrate that the carrier protein-enzyme interface accurately reveals substrate recognition mechanisms in two quorum signal synthases.

Accumulation of plastics in the Earth's oceans is causing widespread disruption to marine ecosystems. To help mitigate the environmental burden caused by non-degradable plastics, we have previously developed a commercially relevant polyurethane (PU) foam derived from renewable biological materials that can be depolymerized into its constituent monomers and consumed by microorganisms in soil or compost. Here we demonstrate that these same PU foams can be biodegraded by marine microorganisms in the ocean and by isolated marine microorganisms in an ex situ seawater environment. Using Fourier-transform infrared (FTIR) spectroscopy, we tracked molecular changes imparted by microbial breakdown of the PU polymers; and utilized scanning electron microscopy (SEM) to demonstrate the loss of physical structure associated with colonization of microorganisms on the PU foams. We subsequently enriched, isolated, and identified individual microorganisms, from six marine sites around San Diego, CA, that are capable of depolymerizing, metabolizing, and accumulating biomass using these PU foams as a sole carbon source. Analysis using SEM, FTIR, and gas chromatography-mass spectrometry (GCMS) confirmed that these microorganisms depolymerized the PU into its constitutive diols, diacids, and other PU fragments. SEM and FTIR results from isolated organismal biodegradation experiments exactly matched those from ex situ and ocean biodegradation samples, suggesting that these PU foam would undergo biodegradation in a natural ocean environment by enzymatic depolymerization of the PU foams and eventual uptake of the degradation products into biomass by marine microorganisms, should these foams unintentionally end up in the marine environment, as many plastics do., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.D.B., R.S.P., and S.P.M. are founders of, and hold an equity position in, Algenesis Inc. N.R.G, M.T., D.Z., L.J., and R.S., are employees and shareholders in Algenesis Inc., a company that could benefit from this research., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Non-ribosomal peptides play a critical role in the clinic as therapeutic agents. To access more chemically diverse therapeutics, non-ribosomal peptide synthetases (NRPSs) have been targeted for engineering through combinatorial biosynthesis; however, this has been met with limited success in part due to the lack of proper protein-protein interactions between non-cognate proteins. Herein, we report our use of chemical biology to enable X-ray crystallography, molecular dynamics (MD) simulations, and biochemical studies to elucidate binding specificities between peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically, we determined X-ray crystal structures of a type II PCP crosslinked to its cognate A domain, PigG and PigI, and of PigG crosslinked to a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures possess large protein-protein interfaces that predominantly feature hydrophobic interactions, with specific electrostatic interactions that orient the substrate for active site delivery. MD simulations of the PCP-A domain complexes and unbound PCP structures provide a dynamical evaluation of the transient interactions formed at PCP-A domain interfaces, which confirm the previously hypothesized role of a PCP loop as a crucial recognition element. Finally, we demonstrate that the interfacial interactions at the PCP loop 1 region can be modified to control PCP binding specificity through gain-of-function mutations. This work suggests that loop conformational preferences and dynamism account for improved shape complementary in the PCP-A domain interactions. Ultimately, these studies show how crystallographic, biochemical, and computational methods can be used to rationally re-engineer NRPSs for non-cognate interactions.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.

To realize the commercialization of sustainable materials, new polymers must be generated and systematically evaluated for material characteristics and end-of-life treatment. Polyester polyols made from renewable monomers have found limited adoption in thermoplastic polyurethane (TPU) applications, and their broad adoption in manufacturing may be possible with a more detailed understanding of their structure and properties. To this end, we prepared a series of bio-based crystalline and amorphous polyester polyols utilizing azelaic acid and varying branched or non-branched diols. The prepared polyols showed viscosities in the range of 504-781 cP at 70 °C, with resulting TPUs that displayed excellent thermal and mechanical properties. TPUs prepared from crystalline azelate polyester polyol exhibited excellent mechanical properties compared to TPUs prepared from amorphous polyols. These were used to demonstrate prototype products, such as watch bands and cup-shaped forms. Importantly, the prepared TPUs had up to 85% bio-carbon content. Studies such as these will be important for the development of renewable materials that display mechanical properties suitable for commercially viable, sustainable products.

Although melanin is one of the most ubiquitous polymers in living systems, our understanding of its molecular structure, biosynthesis and biophysical properties has been limited to only a small number of organisms other than humans. This is in part due to the difficulty associated with isolating pure melanin. While purification methods exist, they typically involve harsh treatments with strong acid/base conditions combined with elevated temperatures that can lead to the polymer backbone degradation. To be successful, a viable isolation method must deliver a selective, yet complete degradation of non-melanin biopolymers as well as remove small molecule metabolites that are not integrative to the melanin backbone. Here, we demonstrate the use of chemoenzymatic processing guided by fluorescent probes for the purification and isolation of native mammalian melanin without significant induction of chemical degradation. This multi-step purification-tracking methodology enables quantitative isolation of pure melanin from mammalian tissue for spectroscopic characterization., (© 2022 Wiley-VCH GmbH.)

Flow chemistry offers a solution for replacing batch methods in chemical preparation where intermediates or products may pose toxicity or instability hazards. Ozonolysis offers an ideal opportunity for flow chemistry solutions, but multiple barriers to entry exist for use of these methods, including equipment cost and performance optimization. To address these challenges, we developed a programmable DIY syringe pump system to use for a continuous flow multireactor process using 3D-printed parts, off-the-shelf stepper motors, and an Arduino microcontroller. Reaction kinetics of ozonide formation informed the use of an integrated batch-flow approach, where ozone addition to an olefin was timed to coincide with fluid movement of a single-syringe pump, followed by downstream Pinnick oxidation and reductive quench in flow. The system was demonstrated by continuous preparation of azelaic acid from ozonolysis of palmitoleic acid, a process limited to low production volumes via batch chemistry. High total production of azelaic acid with 80% yield was obtained from an algae oil sourced unsaturated fatty acid: a product with important applications in medicine, cosmetics, and polymers. This low-cost, scalable approach offers the potential for rapid prototyping and distributed chemical production., Competing Interests: The authors declare the following competing financial interest(s): M.D.B. is a founder and advisor to Algenesis Materials, a biotechnology company interested in developing renewable materials., (© 2022 The Authors. Published by American Chemical Society.)

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli , unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.

The β-ketoacyl-acyl carrier protein synthase, or ketosynthase (KS), catalyses carbon-carbon bond formation in fatty acid and polyketide biosynthesis via a decarboxylative Claisen-like condensation. In prokaryotes, standalone elongating KSs interact with the acyl carrier protein (ACP) which shuttles substrates to each partner enzyme in the elongation cycle for catalysis. Despite ongoing research for more than 50 years since KS was first identified in E. coli , the complex mechanism of KSs continues to be unravelled, including recent understanding of gating motifs, KS-ACP interactions, substrate recognition and delivery, and roles in unsaturated fatty acid biosynthesis. In this review, we summarize the latest studies, primarily conducted through structural biology and molecular probe design, that shed light on the emerging enzymology of standalone elongating KSs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Alterations in RNA splicing are associated with different malignancies, including leukemia, lymphoma, and solid tumors. The RNA splicing modulators such as FD-895 and pladienolide B have been investigated in different malignancies to target/modulate spliceosome for therapeutic purpose. Different cell lines were screened using an RNA splicing modulator to test in vitro cytotoxicity and the ability to modulate RNA splicing capability via induction of intron retention (using RT-PCR and qPCR). The Cignal Finder Reporter Array evaluated [pathways affected by the splice modulators in HeLa cells. Further, the candidates associated with the pathways were validated at protein level using western blot assay, and gene-gene interaction studies were carried out using GeneMANIA. We show that FD-895 and pladienolide B induces higher apoptosis levels than conventional chemotherapy in different solid tumors. In addition, both agents modulate Wnt signaling pathways and mRNA splicing. Specifically, FD-895 and pladienolide B significantly downregulates Wnt signaling pathway-associated transcripts (GSK3β and LRP5) and both transcript and proteins including LEF1, CCND1, LRP6, and pLRP6 at the transcript, total protein, and protein phosphorylation's levels. These results indicate FD-895 and pladienolide B inhibit Wnt signaling by decreasing LRP6 phosphorylation and modulating mRNA splicing through induction of intron retention in solid tumors.

Non-ribosomal peptide synthetases (NRPSs) are attractive targets for biosynthetic pathway engineering due to their modular architecture and the therapeutic relevance of their products. With catalysis mediated by specific protein-protein interactions formed between the peptidyl carrier protein (PCP) and its partner enzymes, NRPS enzymology and control remains fertile ground for discovery. This review focuses on the recent efforts within structural biology by compiling high-resolution structural data that shed light into the various protein-protein interfaces formed between the PCP and its partner enzymes, including the phosphopantetheinyl transferase (PPTase), adenylation (A) domain, condensation (C) domain, thioesterase (TE) domain and other tailoring enzymes within the synthetase. Integrating our understanding of how the PCP recognizes partner proteins with the potential to use directed evolution and combinatorial biosynthetic methods will enhance future efforts in discovery and production of new bioactive compounds., Competing Interests: We have no conflict of interest to declare., (© 2022 The Authors.)

Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the C α -H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Melanin, a widespread pigment found in many taxa, is widely recognized for its high refractive index, ultraviolet (UV) protection, radical quenching ability, metal binding, and many other unique properties. The aforementioned characteristic traits make melanin a potential candidate for biomedical, separation, structural coloration, and space applications. However, the commercially available natural (sepia) and synthetic melanin are very expensive, limiting their use in various applications. Additionally, eumelanin has been the primary focus in most of these studies. In the present study, we demonstrate that melanin can be extracted from the pathogenic black knot fungus Apiosporina morbosa with a yield of ∼10% using the acid-base extraction method. The extracted melanin shows irregular morphology. Chemical characterization using X-ray photoelectron spectroscopy, infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy reveals that the melanin derived from black knots is the less explored nitrogen-free allomelanin. Additionally, the extracted melanin shows broadband UV absorption typical of other types of melanin. Because of the wide availability and low cost of black knots and the invasive nature of the fungus, black knots can serve as an alternative green source for obtaining allomelanin at a low cost, which could stimulate its use as an UV light absorber and antioxidant in cosmetics and packaging industries., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Lipoic acid is an essential cofactor produced in all organisms by diverting octanoic acid derived as an intermediate of type II fatty acid biosynthesis. In bacteria, octanoic acid is transferred from the acyl carrier protein (ACP) to the lipoylated target protein by the octanoyltransferase LipB. LipB has a well-documented substrate selectivity, indicating a mechanism of octanoic acid recognition. The present study reveals the precise protein-protein interactions (PPIs) responsible for this selectivity in Escherichia coli through a combination of solution-state protein NMR titration with high-resolution docking of the experimentally examined substrates. We examine the structural changes of substrate-bound ACP and determine the precise geometry of the LipB interface. Thermodynamic effects from varying substrates were observed by NMR, and steric occlusion of docked models indicates how LipB interprets proper substrate identity via allosteric binding. This study provides a model for elucidating how substrate identity is transferred through the ACP structure to regulate activity in octanoyl transferases., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

To combat the rise in antibiotic resistance, new targets must be identified and probes against them developed. Protein-protein interactions (PPI) of bacterial type II fatty acid biosynthesis (FAS-II) represent an untapped, yet rich area for new antibiotic discovery. Here, we present a computational and in vitro workflow for the discovery of new inhibitors of PPI in Escherichia coli FAS-II. As part of this study, we identified suramin, an existing treatment for African sleeping sickness, to effectively block the interaction of E. coli dehydratase FabA and the acyl carrier protein EcACP, with an IC 50 = 85 μΜ. This finding validates a workflow that combines in silico screening with in vitro PPI assays to identify probes appropriate for further optimization., (© 2021 John Wiley & Sons A/S.)

A current bottleneck in the development of proteolysis targeting chimeras (PROTACs) is the empirical nature of linker length structure-activity relationships (SARs). A multidisciplinary approach to alleviate the bottleneck is detailed here. First, we examine four published synthetic approaches that have been developed to increase synthetic throughput. We then discuss advances in structural biology and computational chemistry that have led to successful rational PROTAC design efforts and give promise to de novo linker design in silico . Lastly, we present a model generated from a curated list of linker SARs studies normalized to reflect how linear linker length affects the observed degradation potency (DC 50 ).

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro . As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a noncanonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

Using Nile Red and BODIPY 493/503 dye-staining and fluorescence microscopy, twenty cyanobacterial strains, including ten commercially available strains and ten environmental isolates from estuaries, freshwater ponds, and lagoons, were screened for the accumulation of ecologically important and potentially biotechnologically significant carbon storage granules such as polyhydroxyalkanoates (PHA). Dye-staining granules were observed in six strains. Three Synechocystis, spp. strains WHSYN, LSNM, and CGF-1, and a Phormidium-like sp. CGFILA were isolated from environmental sources and found to produce granules of polyhydroxyalkanoate (PHA) according to PHA synthase gene (phaC) PCR screening and 1 H NMR analyses. The environmental isolate, Nodularia sp. Las Olas and commercially available Phormidium cf. iriguum CCALA 759 displayed granules but screened negative for PHA according to phaC PCR and 1 H NMR analyses. Partial polyhydroxyalkanoate synthase subunit C (phaC) and 16S rRNA gene sequences obtained from the PHA-accumulating strains and analyzed alongside publicly available phaC, phaE, 16S rRNA, and 23S rRNA data help in understanding the distribution and evolutionary history of PHA biosynthesis within the phylum Cyanobacteria. The data show that the presence of phaC is highly conserved within the genus Synechocystis, and present in at least one isolate of Phormidium. Maximum likelihood analyses and cophylogenetic modeling of PHA synthase gene sequences provide evidence of a recent horizontal gene transfer event between distant genera of cyanobacteria related to Pleurocapsa sp. PCC 7327 and Phormidium-like sp. CGFILA. These findings will help guide additional screening for PHA producers, and may explain why some Phormidium species produce PHAs, while others do not., (© 2021 Phycological Society of America.)

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

Due to the depletion of fossil fuels, higher oil prices, and greenhouse gas emissions, the scientific community has been conducting an ongoing search for viable renewable alternatives to petroleum-based products, with the anticipation of increased adaptation in the coming years. New academic and industrial developments have encouraged the utilization of renewable resources for the development of ecofriendly and sustainable materials, and here, we focus on those advances that impact polyurethane (PU) materials. Vegetable oils, algae oils, and polysaccharides are included among the major renewable resources that have supported the development of sustainable PU precursors to date. Renewable feedstocks such as algae have the benefit of requiring only sunshine, carbon dioxide, and trace minerals to generate a sustainable biomass source, offering an improved carbon footprint to lessen environmental impacts. Incorporation of renewable content into commercially viable polymer materials, particularly PUs, has increasing and realistic potential. Biobased polyols can currently be purchased, and the potential to expand into new monomers offers exciting possibilities for new product development. This Review highlights the latest developments in PU chemistry from renewable raw materials, as well as the various biological precursors being employed in the synthesis of thermoset and thermoplastic PUs. We also provide an overview of literature reports that focus on biobased polyols and isocyanates, the two major precursors to PUs.

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products., Competing Interests: The authors declare no competing interest.

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.