High-yield Production Research Articles

A novel top-down approach for high yield production of graphene from natural graphite and its supercapacitor applications

The cost-effective and sustainable mass production of graphene is of utmost importance for its comprehensive application across various industries. Despite ball milling being the most efficacious method for obtaining high-quality graphene, industrial-scale production poses a significant challenge due to a lowered yield, heterogeneous media and reduced energy transfer efficiency. Furthermore, the consistent properties of graphene pose another major challenge when it comes to scaled-up processes. To address these challenges, we conducted a study aiming to produce high-quality graphene from natural graphite sourced from Indian ore through the utilisation of a horizontal planetary ball mill. Our analysis revealed that the graphene sheets produced via this method exhibit excellent structural integrity, rendering them highly beneficial for a wide range of applications. The diffraction pattern of the graphene sheets is dominated by the (002) plane, as shown by XRD data. The Raman spectrum indicates a compatible I2D/IG ratio, suggesting double- to triple-layer graphene sheets. The graphene sheets were seen to be layered and irregularly shaped in the SEM and TEM pictures, which is consistent with the sample's polycrystalline makeup. The findings demonstrate the large surface area and strong electrical conductivity of the synthesized graphene. Furthermore, the high specific capacitance value of 30F/g is shown by in-house graphene-based supercapacitors. Overall, the findings of this work show that manufacturing high-quality graphene from synthetic and natural graphite is feasible and effective when carried out on a horizontal planetary ball mill, and each batch of 200 g graphene requires $25 in production costs. This strategy promises to supply the rising demand for graphene by enabling more widespread, cost-effective manufacturing.

Target Development towards First Production of High-Molar- Activity 44gSc and 47Sc by Mass Separation at CERN-MEDICIS.

The radionuclides 43Sc, 44g/mSc, and 47Sc can be produced cost-effectively in sufficient yield for medical research and applications by irradiating natTi and natV target materials with protons. Maximizing the production yield of the therapeutic 47Sc in the highest cross section energy range of 24-70 MeV results in the co-production of long-lived, high-γ-ray-energy 46Sc and 48Sc contaminants if one does not use enriched target materials. Mass separation can be used to obtain high molar activity and isotopically pure Sc radionuclides from natural target materials; however, suitable operational conditions to obtain relevant activity released from irradiated natTi and natV have not yet been established at CERN-MEDICIS and ISOLDE. The objective of this work was to develop target units for the production, release, and purification of Sc radionuclides by mass separation as well as to investigate target materials for the mass separation that are compatible with high-yield Sc radionuclide production in the 9-70 MeV proton energy range. In this study, the in-target production yield obtained at MEDICIS with 1.4 GeV protons is compared with the production yield that can be reached with commercially available cyclotrons. The thick-target materials were irradiated at MEDICIS and comprised of metallic natTi, natV metallic foils, and natTiC pellets. The produced radionuclides were subsequently released, ionized, and extracted from various target and ion source units and mass separated. Mono-atomic Sc laser and molecule ionization with forced-electron-beam-induced arc-discharge ion sources were investigated. Sc radionuclide production in thick natTi and natV targets at MEDICIS is equivalent to low- to medium-energy cyclotron-irradiated targets at medically relevant yields, furthermore benefiting from the mass separation possibility. A two-step laser resonance ionization scheme was used to obtain mono-atomic Sc ion beams. Sc radionuclide release from irradiated target units most effectively could be promoted by volatile scandium fluoride formation. Thus, isotopically pure 44g/mSc, 46Sc, and 47Sc were obtained as mono-atomic and molecular ScF 2+ ion beams and collected for the first time at CERN-MEDICIS. Among all the investigated target materials, natTiC is the most suitable target material for Sc mass separation as molecular halide beams, due to high possible operating temperatures and sustained release.

Influence of FeSO4.7H2O, Indole-3-Butyric Acid and Different Nutrient Medium on In Vitro Sapling Propagation and Micrografting of Walnut (Juglans Regia L.)

Walnuts are considered a functional food and play a significant role worldwide in people's regular diets. The cultivation and trade of walnut rootstocks and saplings are crucial agricultural activities globally and in Türkiye. Rootstocks are used for grafting and propagating different walnut varieties, and they are essential for tree development, fruit yield, product quantity, and most importantly, their ability to adapt to various ecological conditions and resist diseases and pests. Fruit growing and production of fruit rootstocks of deep-rooted plants in horticulture is given high importance in many countries. However, poor rooting and slow-growing sapling prevent establishing high-yield clonal production. Especially walnut sapling propagation is more complicated and time-consuming than other woody plants. Our research aims to grow high-quality saplings by using biotechnological techniques. This study used modifications to the current in vitro basal medium and evaluated the effects on in vitro rooting and micrografting of walnut species. The optimal medium for shoots induction (2.93±0.90) of the plant was determined as 4 number medium (Murashige and Skoog (MS) nutrient medium with 5.4 µM NAA + 6.9 µM TDZ + 0.6 µM GA3 and solidified with 0.7% agar). In trials to develop sapling, the 10.33 and 6 rooted (2.5cm root length) shoots were obtained from MS nutrient medium with 14.7 and 19.6 µM of IBA and 348.42 µM of FeSO4. 7H2O, and two micrografted plants in MS medium with 19.6 µM of IBA and 348.42 µM of FeSO4.7H2O survived. After three months in the growth chamber, six saplings were ready for transfer to the field. Our findings suggested that FeSO4.7H2O and high dose IBA treatment in MS medium is efficient for in vitro rooting and obtaining in vitro micrografted saplings.

A citric acid cycle-deficient Escherichia coli as an efficient chassis for aerobic fermentations.

Tricarboxylic acid cycle (TCA cycle) plays an important role for aerobic growth of heterotrophic bacteria. Theoretically, eliminating TCA cycle would decrease carbon dissipation and facilitate chemicals biosynthesis. Here, we construct an E. coli strain without a functional TCA cycle that can serve as a versatile chassis for chemicals biosynthesis. We first use adaptive laboratory evolution to recover aerobic growth in minimal medium of TCA cycle-deficient E. coli. Inactivation of succinate dehydrogenase is a key event in the evolutionary trajectory. Supply of succinyl-CoA is identified as the growth limiting factor. By replacing endogenous succinyl-CoA dependent enzymes, we obtain an optimized TCA cycle-deficient E. coli strain. As a proof of concept, the strain is engineered for high-yield production of four separate products. This work enhances our understanding of the role of the TCA cycle in E. coli metabolism and demonstrates the advantages of using TCA cycle-deficient E. coli strain for biotechnological applications.

Biosynthesis of apigenin glucosides in engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25℃, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin- 4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37℃. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Imidazolium based Ionic liquid Anchored over Dendritic Mesoporous Silica Nanospheres possessing a Magnetic Core as an Efficient and Recyclable Base Catalyst

AbstractIn this study, Magnetic core‐shell dendritic mesoporous silica nanospheres immobilized with imidazolium‐based di‐cationic ionic liquid (Fe3O4@SiO2@DSiO2‐Im‐IL) possessing a large surface area and numerous active site for adsorption of various molecules to carry out organic transformations have been successfully developed. The synthesized nanocatalyst was found to be easily recyclable by applying external magnetic force. The structure, morphology, thermal stability, and magnetic behavior of the synthesized magnetic nanocatalyst were confirmed by different physiochemical techniques like FT‐IR, P‐XRD, FE‐SEM, TGA, VSM, BET, and TEM analysis. The efficiency of the developed heterogeneous nanocatalyst has been investigated for the green synthesis of numerous benzopyran derivatives by employing different aldehydes and active methylene compounds. The catalyst exhibits marvellous activity and efficiency in terms of high product yield, less catalyst use, and wide substrate scope even at room temperature. Finally, after the successful formation of the product, the catalyst was regenerated by utilizing an external magnetic field. The catalyst has been reused multiple times without any noteworthy loss in its activity which confirms the stability of the developed catalyst. The preparation of the catalyst is cost‐effective, and shows atom economy with the utilization of green solvent and without any waste production, which authenticates that it could be a greener pathway for the synthesis of the benzopyran derivatives.

Convergent Synthesis of Two Heterogeneous Fluxes from Glucose and Acetate for High-Yield Citramalate Production.

Biological production of citramalate has garnered attention due to its wide application for food additives and pharmaceuticals, although improvement of yield is known to be challenging. When glucose is used as the sole carbon source, carbon loss through decarboxylation steps for providing acetyl-CoA from pyruvate is inevitable. To avoid this, we engineered a strain to co-utilize glucose and cost-effective acetate while preventing carbon loss for enhancing citramalate production. The production pathway diverged to independently supply the precursors required for the synthesis of citramalate from glucose and acetate, respectively. Moreover, the phosphotransferase system was inactivated and the acetate assimilation pathway and the substrate ratio were optimized to enable the simultaneous and efficient utilization of both carbon sources. This yielded results (5.0 g/L, 0.87 mol/mol) surpassing the yield and titer of the control strain utilizing glucose as the sole carbon source in flask cultures, demonstrating an economically efficient strain redesign strategy for synthesizing various products.

Concentrated Solar Light Photoelectrochemical Water Splitting for Stable and High-Yield Hydrogen Production.

Photoelectrochemical water splitting is a promising technique for converting solar energy into low-cost and eco-friendly H2 fuel. However, the production rate of H2 is limited by the insufficient number of photogenerated charge carriers in the conventional photoelectrodes under 1 sun (100mWcm-2) light. Concentrated solar light irradiation can overcome the issue of low yield, but it leads to a new challenge of stability because the accelerated reaction alters the surface chemical composition of photoelectrodes. Here, it is demonstrated that loading Pt nanoparticles (NPs) on single crystalline GaN nanowires (NWs) grown on n+-p Si photoelectrode operates efficiently and stably under concentrated solar light. Although a large number of Pt NPs detach during the initial reaction due to H2 gas bubbling, some Pt NPs which have an epitaxial relation with GaN NWs remain stably anchored. In addition, the stability of the photoelectrode further improves by redepositing Pt NPs on the reacted Pt/GaN surface, which results in maintaining onset potential >0.5V versus reversible hydrogen electrode and photocurrent density >60mAcm-2 for over 1500 h. The heterointerface between Pt cocatalysts and single crystalline GaN nanostructures shows great potential in designing an efficient and stable photoelectrode for high-yield solar to H2 conversion.

Reduction in Interferon-Stimulated Genes Contributes to High-Yield Production of Influenza Virus in Suspension MDCK Cells.

Compared with the traditional vaccine produced in embryonated chicken eggs, cell-based manufacturing represented by the Madin-Darby canine kidney (MDCK) cell line has a larger production scale and reduces the risk of egg shortage in a pandemic. Establishing a culture system that enables high production of the influenza virus is a key issue in influenza vaccine production. Here, a serum-free suspension culture of MDCK (sMDCK) cells was obtained from adherent MDCK (aMDCK) cells by direct adaptation. Viral infection experiments showed that viral yields of influenza A/B virus in sMDCK cells were higher than in aMDCK cells. Transcriptome analysis revealed that numerous interferon-stimulated genes (ISGs) exhibited reduced expression in sMDCK cells. To further clarify the mechanism of high viral production in sMDCK cells, we demonstrated the antiviral role of RIG-I and IFIT3 in MDCK cells by knockdown and overexpression experiments. Furthermore, suppression of the JAK/STAT pathway enhances the viral accumulation in aMDCK cells instead of sMDCK cells, suggesting the reduction in the JAK/STAT pathway and ISGs promotes viral replication in sMDCK cells. Taken together, we elucidate the relationship between the host innate immune response and the high viral productive property of sMDCK cells, which helps optimize cell production processes and supports the production of cell-based influenza vaccines.

Green Synthesis of Bimetallic Nanoparticles and its Catalytic Activity of Multicomponent Reactions and Antibacterial Activity

AbstractThis present research work focuses on synthesizing Ag−Cu bimetallic nanoparticles utilizing an aqueous extract leaf of Excoecaria agallocha, employing a green synthesis route. The incorporation of silver (Ag) with copper (Cu) induces synergistic effects that enhance reaction kinetics and reduce the multicomponent reaction time. The catalysts synthesized through this environmentally friendly approach were thoroughly characterized using various analytical techniques, including HR‐TEM, XRD, SEM‐EDX, UV‐Vis Spectrophotometry, and FT‐IR. The UV‐visible spectrum displayed a peak at 257 nm, while the FT‐IR spectrum exhibited peaks at 574 cm−1, indicative of bimetallic peak reductions attributed to the influence of plant metabolites. HR‐TEM and XRD revealed the crystalline with (111), (200), (111), (200), and (220) plane values and shapes of BMNPs. Furthermore, the Ag−Cu bimetallic nanoparticles demonstrated outstanding catalytic performance with high product yield, along with notable reusability and recyclability and excellent antimicrobial activity when compared with commercial antibacterial chloramphenicol.

CAN Interceded Oxidative Coupling of β‐Dicarbonyl Compounds to 2‐Aryl/Heteroarylchromenes: A Regio‐ and Diastereoselective Synthesis of Tetrahydro‐benzofuro[3,2‐c]chromenones

AbstractA promising and useful method has been accomplished for regio‐ and diastereoselective synthesis of tetrahydro‐benzofuro[3,2‐c]chromenones through ceric(IV) ammonium nitrate (CAN) mediated oxidative coupling of cyclic/acyclic 1,3‐dicarbonyl compounds to 2‐aryl/heteroarylchromenes followed by a base assisted cyclization. The significant features of the method encircle easy accessibility of the starting materials, high air stability and low toxicity of oxidant CAN, high product yield, operationally simple and eco‐friendly reaction conditions. Trapping of a radical intermediate with radical scavenger TEMPO (detected by LC–MS) from the reaction mixture proved that the oxidative coupling is a radical mediated process.

High-yield recombinant adeno-associated viral vector production by multivariate optimization of bioprocess and transfection conditions.

Recombinant adeno-associated viral vectors (rAAVs) are one of the most used vehicles for gene therapy, with five rAAV therapeutics commercially approved by the FDA. To improve product yield, we optimized the suspension production process of rAAV8 vectors carrying a proprietary transgene using a commercially available transfection reagent, FectoVIR-AAV. Using a miniaturized automated 250 mL scale bioreactor system, we generated models of vector genome (vg) titer, capsid (cp) titer, and Vg:Cp percentage from two multivariate design of experiment studies, one centered around bioreactor operating parameters, and another based on the transfection conditions. Using the optimized process returned from these models, the vector genome titer from the bioreactor was improved to beyond 1 × 1012 vg/mL. Five critical parameters were identified that had large effects on the pre-purification vector quantity-the transfection pH, production pH, complexation time, viable cell density at transfection, and transfection reagent to DNA ratio. The optimized process was further assessed for its performance extending to six AAV serotypes, namely AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 carrying a transgene encoding for green fluorescent protein (GFP). Five of the six serotypes returned higher vector genome titers than the control condition. These data suggest that the choice of transfection reagent is a major factor in improving vector yield. The multivariate design of experiment approach is a powerful way to optimize production processes, and the optimized process from one AAV vector can to some extent be generalized to other serotypes and transgenes to accelerate development timelines of new programs.

How To Get Isocyanate?

Isocyanate, a pivotal chemical intermediate to synthesize polyurethane with widespread applications in household appliances, automobiles, and construction, is predominantly produced via the phosgene process, which currently holds a paramount status in industrial isocyanate production. Nonetheless, concerns arise from the toxicity of phosgene and the corrosiveness of hydrogen chloride, posing safety hazards. The synthesis of isocyanate using nonphosgene methods represents a promising avenue for future development. This article primarily focuses on the nonphosgene approach, which involves the formation of carbamate through the reaction of nitro-amino compounds with carbon monoxide, dimethyl carbonate, and urea, among other reagents, subsequently leading to the thermal decomposition of carbamate to get isocyanate. This paper emphasizes the progress in catalyst development during the carbamate decomposition process. Single-component metal catalysts, particularly zinc, exhibit advantages such as high activity, cost-effectiveness, and compatibility with a wide range of substrates. Composite catalysts enhance isocyanate yield by introducing a second component to adjust the active metal composition. The central research direction aims to optimize catalyst adaptation to reaction conditions, including temperature, pressure, time, and solvent, to achieve high raw material conversion and product yield.

Development of a drying method for proteins based on protein-hyaluronic acid precipitation

This study aims to develop a new method to dry proteins based on protein-hyaluronic acid (HA) precipitation and apply the precipitation-redissolution technique to develop highly concentrated protein formulations. Lysozyme was used as a model protein and HA with various molecular weights (MW) were investigated. Under low ionic strength, low-MW HA (e.g., MW: around 5 K) did not induce lysozyme precipitation. Conversely, high-MW HA (e.g., MW: approximately from 40 K to 1.5 M) precipitated more than 90 % of lysozyme. The dried lysozyme-HA precipitates had moisture levels between 4 % and 5 %. The lysozyme-HA precipitates could be redissolved using PBS (pH 7.4, ionic strength: ∼ 163 mM). The viscosity of the reconstituted solution was dependent on HA MW, e.g., 4 cP for HA40K, and 155 cP for HA1.5 M, suggesting low-MW HA might be a proper excipient for highly concentrated solution formulations for subcutaneous/intraocular injection and high-MW HA may fit for other applications. The tertiary structure of lysozyme after the precipitation-redissolution steps had no significant difference from that of the original lysozyme as confirmed by fluorescence spectroscopy. The denaturation temperature of lysozyme after the precipitation-redissolution steps and that of the original lysozyme were close, indicating they possessed similar thermal stability. The accelerated stability study revealed that lysozyme stored in the dry precipitates was more physically stable than that in the buffer solution. Overall, this new drying technique is suitable for drying proteins and exhibits several benefits such as minimum energy consumption, cost effectiveness, high production yield, and mild processing conditions. In addition, the precipitation-redissolution technique proposed in this study can potentially be used to develop highly concentrated formulations, especially for proteins experiencing poor stability in the liquid state.

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

Development of Integrated Vectors with Strong Constitutive Promoters for High-Yield Antibiotic Production in Mangrove-Derived Streptomyces.

It is important to improve the production of bioactive secondary products for drug development. The Escherichia coli-Streptomyces shuttle vector pSET152 and its derived vector pIB139 containing a strong constitutive promoter ermEp* are commonly used as integrative vectors in actinomycetes. Four new integrative vectors carrying the strong constitutive promoter kasOp*, hrdBp, SCO5768p, and SP44, respectively, were constructed and proven to be functional in different mangrove-derived Streptomyces host strains by using kanamycin resistance gene neo as a reporter. Some biosynthetic genes of elaiophylins, azalomycin Fs, and armeniaspirols were selected and inserted into these vectors to overexpress in their producers including Streptomyces sp. 219807, Streptomyces sp. 211726, and S. armeniacus DSM 43125, resulting in an approximately 1.1-1.4-fold enhancement of the antibiotic yields.

A sustainable process for electrochemical production of 2-Iodosylbenzoic acid

Herein, an environmentally friendly, scalable and highly efficient process for production of 2-iodosylbenzoic acid, a well-established hypervalent iodine oxidant is reported. It is based on anodic oxidation of 2-iodobenzoic acid in anhydrous AcONa-AcOH electrolyte followed by simple work-up of solid product. In particular, a benefit of the developed electrochemical process is absence of any chemical oxidant, application of environmentally benign solvent, high Faradaic efficiency and product yield. The process on the anode side combines anodic oxidation of reactant to the desired product with separation process, i.e. removal of Na+ cation from the anolyte through a cation-exchange membrane leading to drop of AcONa concentration. Consequently, the product of high purity can be obtained simply by filtering the anolyte solution followed by drying the filtration cake. Interestingly, no washing by organic solvents is required. Moreover, both anolyte filtrate and catholyte can be reused multiple times. Energy and material balance and cost and environmental footprint analysis showed benefits of the developed process and suggested clear strategy towards further reduction of the product cost and, in particular, significant decrease of a generated waste amount.

A Review on Organic Farming in India

Organic horticulture is a dynamic and sustainable approach to cultivating plants that prioritizes environmental health, biodiversity, and human well-being. This combination of high-yielding production technology has helped the country develop a food surplus as well as contributing to concerns of soil health, environmental pollution, pesticide toxicity, and sustainability of agricultural production. Alternatives for managing pests and diseases in organic farming mostly focus on preventive methods as opposed to curative therapies, which are founded on ecologically safer management techniques. The priority has been placed on maintaining the ecosystem’s health, allowing plants to become resistant to insect pests and illnesses.

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.