Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum . Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain and other neurotoxic symptoms in vertebrates. Here we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

Until today, the range and the time to fill up the energy storage of an electric vehicle takes much longer than that of vehicles with combustion engine. For the user, these deficits are associated with a loss of comfort, especially during long-distance journeys. One possible approach to significantly reducing charging times is to provide the option of thermal power transfer in addition to electrical power during vehicle fast charging in order to dissipate heat during the fast charging process and thus further shorten the charging process. This approach is selected in the publicly funded research project CoolEV.Despite these and comparable efforts, charging time will not be comparable to the time required for refuelling in the foreseeable future. The question arises as to what extent shortening the charging time can contribute to increasing convenience. The possibility of completing the charging process like a short "pit stop" in the drive-through is seen as a decisive gain in convenience. Closely related to this is the question of how long vehicle users maximally remain at the vehicle during charging in order to then immediately continue their journey. As a result, a statement can be generated as to whether charging stations in a drive-through layout are target-oriented and under which conditions they are to be preferred to those with parking bays.In order to investigate the questions, an architectural model of an ideal loading park with a drive-through layout is first created. This model is then imported into a VR environment and animated allowing test persons to virtually experience and evaluate the charging process. Subject tests are conducted and user behavior as a function of charging time is captured and documented by video. The results are compared and discussed with the subjective impressions of the test persons, which are collected with questionnaires.

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

Microbes that can recycle one-carbon (C-1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+ H-2, or CO+CO2+H-2). We detect the prioritization of proteome allocation for C-1 fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant in vivo for CO oxidation, H-2 metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. IMPORTANCE Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C-1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate in silico reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.

Significance Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.

The R-type voltage-gated Ca 2+ (Ca v ) channels Ca v 2.3, widely expressed in neuronal and neuroendocrine cells, represent potential drug targets for pain, seizures, epilepsy, and Parkinson’s disease. Despite their physiological importance, there have lacked selective small-molecule inhibitors targeting these channels. High-resolution structures may aid rational drug design. Here, we report the cryo-EM structure of human Ca v 2.3 in complex with α2δ−1 and β3 subunits at an overall resolution of 3.1 Å. The structure is nearly identical to that of Ca v 2.2, with VSD II in the down state and the other three VSDs up. A phosphatidylinositol 4,5-bisphosphate (PIP2) molecule binds to the interface of VSD II and the tightly closed pore domain. We also determined the cryo-EM structure of a Ca v 2.3 mutant in which a Ca v 2-unique cytosolic helix in repeat II (designated the CH2 II helix) is deleted. This mutant, named ΔCH2, still reserves a down VSD II , but PIP2 is invisible and the juxtamembrane region on the cytosolic side is barely discernible. Our structural and electrophysiological characterizations of the wild type and ΔCH2 Ca v 2.3 show that the CH2 II helix stabilizes the inactivated conformation of the channel by tightening the cytosolic juxtamembrane segments, while CH2 II helix is not necessary for locking the down state of VSD II .

High levels of anthropogenic CO2 emissions are driving the warming of global climate. If this pattern of increasing emissions does not change, it will cause further climate change with severe consequences for the human population. On top of this, the increasing accumulation of solid waste within the linear economy model is threatening global biosustainability. The magnitude of these challenges requires several approaches to capture and utilize waste carbon and establish a circular economy. Microbial gas fermentation presents an exciting opportunity to capture carbon oxides from gaseous and solid waste streams with high feedstock flexibility and selectivity. Here we discuss available microbial systems and review in detail the metabolism of both anaerobic acetogens and aerobic hydrogenotrophs and their ability to utilize C1 waste feedstocks. More specifically, we provide an overview of the systems-level understanding of metabolism, key metabolic pathways, scale-up opportunities and commercial successes, and the most recent technological advances in strain and process engineering. Finally, we also discuss in detail the gaps and opportunities to advance the understanding of these autotrophic biocatalysts for the efficient and economically viable production of bioproducts from recycled carbon.

Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

The peripheral effects of ω-conotoxins, selective blockers of N-type voltage-gated calcium channels (CaV2.2), have not been characterised across different clinically relevant pain models. This study examines the effects of locally administered ω-conotoxin MVIIA, GVIA, and CVIF on mechanical and thermal paw withdrawal threshold (PWT) in postsurgical pain (PSP), cisplatin-induced neuropathy (CisIPN), and oxaliplatin-induced neuropathy (OIPN) rodent models. Intraplantar injection of 300, 100 and 30 nM MVIIA significantly (p < 0.0001, p < 0.0001, and p < 0.05, respectively) alleviated mechanical allodynia of mice in PSP model compared to vehicle control group. Similarly, intraplantar injection of 300, 100, and 30 nM MVIIA (p < 0.0001, p < 0.01, and p < 0.05, respectively), and 300 nM and 100 nM GVIA (p < 0.0001 and p < 0.05, respectively) significantly increased mechanical thresholds of mice in OIPN model. The ED50 of GVIA and MVIIA in OIPN was found to be 1.8 pmol/paw and 0.8 pmol/paw, respectively. However, none of the ω-conotoxins were effective in a mouse model of CisIPN. The intraplantar administration of 300 nM GVIA, MVIIA, and CVIF did not cause any locomotor side effects. The intraplantar administration of MVIIA can alleviate incision-induced mechanical allodynia, and GVIA and MVIIA effectively reduce OIPN associated mechanical pain, without locomotor side effects, in rodent models. In contrast, CVIF was inactive in these pain models, suggesting it is unable to block a subset of N-type voltage-gated calcium channels associated with nociceptors in the skin.

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that NaV channel subtypes NaV1.7-NaV1.9 are involved in nociception. More recently, NaV1.1, NaV1.3 and NaV1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived NaV inhibitors into smaller, more "drug-like" molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. We designed a series of small, stable and novel NaV probes displaying NaV subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to NaV subtype modulation.

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

Vincristine, oxaliplatin, and cisplatin are commonly prescribed chemotherapeutic agents for the treatment of many tumors. However, a main side effect is chemotherapy-induced peripheral neuropathy (CIPN), which may lead to changes in chemotherapeutic treatment. Although symptoms associated with CIPN are recapitulated by mouse models, there is limited knowledge of how these drugs affect the expression of genes in sensory neurons. The present study carried out a transcriptomic analysis of dorsal root ganglia following vincristine, oxaliplatin, and cisplatin treatment with a view to gain insight into the comparative pathophysiological mechanisms of CIPN. RNA-Seq revealed 368, 295, and 256 differential expressed genes induced by treatment with vincristine, oxaliplatin, and cisplatin, respectively, and only 5 shared genes were dysregulated in all 3 groups. Cell type enrichment analysis and gene set enrichment analysis showed predominant effects on genes associated with the immune system after treatment with vincristine, while oxaliplatin treatment affected mainly neuronal genes. Treatment with cisplatin resulted in a mixed gene expression signature. Perspective These results provide insight into the recruitment of immune responses to dorsal root ganglia and indicate enhanced neuroinflammatory processes following administration of vincristine, oxaliplatin, and cisplatin. These gene expression signatures may provide insight into novel drug targets for treatment of CIPN.

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (NaVs) play a critical role in this process. In particular, the NaV subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of NaV1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.

Vincristine is an antineoplastic substance that is part of many chemotherapy regimens, used especially for the treatment of a variety of pediatric cancers including leukemias and brain tumors. Unfortunately, many vincristine-treated patients develop peripheral neuropathy, a side effect characterized by sensory, motoric, and autonomic symptoms. The sensory symptoms include pain, in particular hypersensitivity to light touch, as well as loss of sensory discrimination to detect vibration and touch. The symptoms of vincristine-induced neuropathy are only poorly controlled by currently available analgesics and therefore often necessitate dose reductions or even cessation of treatment. The aim of this study was to identify new therapeutic targets for the treatment of vincristine-induced peripheral neuropathy (VIPN) by combining behavioral experiments, histology, and pharmacology after vincristine treatment. Local intraplantar injection of vincristine into the hind paw caused dose- and time-dependent mechanical hypersensitivity that developed into mechanical hyposensitivity at high doses, and lead to a pronounced, dose-dependent infiltration of immune cells at the site of injection. Importantly, administration of minocycline effectively prevented the development of mechanical hypersensitivity and infiltration of immune cells in mouse models of vincristine induce peripheral neuropathy (VIPN) based on intraperitoneal or intraplantar administration of vincristine. Similarly, Toll-like receptor 4 knockout mice showed diminished vincristine-induced mechanical hypersensitivity and immune cell infiltration, while treatment with the anti-inflammatory meloxicam had no effect. These results provide evidence for the involvement of Toll-like receptor 4 in the development of VIPN and suggest that minocycline and/or direct Toll-like receptor 4 antagonists may be an effective preventative treatment for patients receiving vincristine.