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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.

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.

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 17S-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.

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.

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.

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.

BackgroundNudibranchs 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.ResultsWe 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.ConclusionsAltogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

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.

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.

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.

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.

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 Cα-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.

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.

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.

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.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved. Here, we apply a biosynthetic approach to explore the composite structures accessible in one type of melanin, eumelanin. Using a combination of solid-state NMR, dynamic nuclear polarization, and electron microscopy, we reveal how a variety of monomers are enzymatically polymerized into their corresponding eumelanin pigments. We demonstrate how this approach can be used to unite structure with an understanding of enzymatic activity, substrate scope, and the regulation of nanostructural features. Overall, this data reveals how intermediate metabolites of the Raper-Mason metabolic pathway contribute to polymerization, allowing us to revisit the original proposal of how eumelanin is biosynthesized.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces.

Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4'-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium toward favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables 1 week of stability to palmitoyl-AcpP from Escherichia coli. 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.

Contrast-enhanced ultrasound (CEUS) offers the exciting prospect of retaining the ease of ultrasound imaging while enhancing imaging clarity, diagnostic specificity, and theranostic capability. To advance the capabilities of CEUS, the synthesis and understanding of new ultrasound contrast agents (UCAs) is a necessity. Many UCAs are nano- or micro-scale materials composed of a perfluorocarbon (PFC) and stabilizer that synergistically induce an ultrasound response that is both information-rich and easily differentiated from natural tissue. In this work, we probe the extent to which CEUS is modulated through variation in a PFC stabilized with fluorine-modified polydopamine nanoparticles (PDA NPs). The high level of synthetic tunability in this system allows us to study signal as a function of particle aggregation and PFC volatility in a systematic manner. Separation of aggregated and non-aggregated nanoparticles lead to a fundamentally different signal response, and for this system, PFC volatility has little effect on CEUS intensity despite a range of over 50 °C in boiling point. To further explore the imaging tunability and multimodality, Fe3+-chelation was employed to generate an enhanced photoacoustic (PA) signal in addition to the US signal. In vitro and in vivo results demonstrate that PFC-loaded PDA NPs show stronger PA signal than the non-PFC ones, indicating that the PA signal can be used for in situ differentiation between PFC-loading levels. In sum, these data evince the rich role synthetic chemistry can play in guiding new directions of development for UCAs.

At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo via a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur with other heteroatoms results in significant structural changes, thus suggesting more rigorous selections of isosteric substitutes is needed.

We describe the design, synthesis, and antitumor activity of an 18 carbon α,ω-dicarboxylic acid monoconjugated via an ester linkage to paclitaxel (PTX). This 1,18-octadecanedioic acid-PTX (ODDA-PTX) prodrug readily forms a noncovalent complex with human serum albumin (HSA). Preservation of the terminal carboxylic acid moiety on ODDA-PTX enables binding to HSA in the same manner as native long-chain fatty acids (LCFAs), within hydrophobic pockets, maintaining favorable electrostatic contacts between the ω-carboxylate of ODDA-PTX and positively charged amino acid residues of the protein. This carrier strategy for small molecule drugs is based on naturally evolved interactions between LCFAs and HSA, demonstrated here for PTX. ODDA-PTX shows differentiated pharmacokinetics, higher maximum tolerated doses and increased efficacy in vivo in multiple subcutaneous murine xenograft models of human cancer, as compared to two FDA-approved clinical formulations, Cremophor EL-formulated paclitaxel (crPTX) and Abraxane (nanoparticle albumin-bound (nab)-paclitaxel).

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and β-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d-amino acids through the use of auxiliary epimerization domains, converting l-amino acids into d-amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.

The discovery of peptide substrates for enzymes with exclusive, selective activities is a central goal in chemical biology. In this paper, we develop a hybrid computational and biochemical method to rapidly optimize peptides for specific, orthogonal biochemical functions. The method is an iterative machine learning process by which experimental data is deposited into a mathematical algorithm that selects potential peptide substrates to be tested experimentally. Once tested, the algorithm uses the experimental data to refine future selections. This process is repeated until a suitable set of de novo peptide substrates are discovered. We employed this technology to discover orthogonal peptide substrates for 4'-phosphopantetheinyl transferase, an enzyme class that covalently modifies proteins. In this manner, we have demonstrated that machine learning can be leveraged to guide peptide optimization for specific biochemical functions not immediately accessible by biological screening techniques, such as phage display and random mutagenesis.

The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism.

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

Ocean-derived, airborne microbes play important roles in Earth's climate system and human health, yet little is known about factors controlling their transfer from the ocean to the atmosphere. Here, we study microbiomes of isolated sea spray aerosol (SSA) collected in a unique ocean-atmosphere facility and demonstrate taxon-specific aerosolization of bacteria and viruses. These trends are conserved within taxonomic orders and classes, and temporal variation in aerosolization is similarly shared by related taxa. We observe enhanced transfer into SSA of Actinobacteria, certain Gammaproteobacteria, and lipid-enveloped viruses; conversely, Flavobacteriia, some Alphaproteobacteria, and Caudovirales are generally under-represented in SSA. Viruses do not transfer to SSA as efficiently as bacteria. The enrichment of mycolic acid-coated Corynebacteriales and lipid-enveloped viruses (inferred from genomic comparisons) suggests that hydrophobic properties increase transport to the sea surface and SSA. Our results identify taxa relevant to atmospheric processes and a framework to further elucidate aerosolization mechanisms influencing microbial and viral transport pathways.

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.

Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. In splicing reporter assays and pre-clinical patient-derived AML models, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.