Electrochemical Synthesis of Itaconic Acid Derivatives via Chemodivergent Single and Double Carboxylation of Allenes with CO2.

Leveraging electrochemistry, a new synthesis of non-natural derivatives of itaconic acid is proposed by utilizing carbon dioxide (CO2) as a valuable C1 synthon. An electrochemical cross-electrophile coupling between allenoates and CO2 was targeted, allowing for the synthesis of both mono- and di-carboxylation products in a catalyst- and additive-free environment (yields up to 87 %, 30 examples). Elaboration of the model mono-carboxylation product, and detailed cyclovoltammetric, as well as mechanistic analyses complete the present investigation.

Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch.

Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.

Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12 g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Metabolic engineering of Shewanella oneidensis to produce glutamate and itaconic acid.

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products. KEY POINTS: •Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production. •Deletion of ackA/pta redirects carbon flux towards TCA cycle. •Heterologous expression of cadA and acnB enables itaconic acid production.

Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation.

BACKGROUND: In the inflammatory milieu of diabetic chronic wounds, macrophages undergo substantial metabolic reprogramming and play a pivotal role in orchestrating immune responses. Itaconic acid, primarily synthesized by inflammatory macrophages as a byproduct in the tricarboxylic acid cycle, has recently gained increasing attention as an immunomodulator. This study aims to assess the immunomodulatory capacity of an itaconic acid derivative, 4-Octyl itaconate (OI), which was covalently conjugated to electrospun nanofibers and investigated through in vitro studies and a full-thickness wound model of diabetic mice. RESULTS: OI was feasibly conjugated onto chitosan (CS), which was then grafted to electrospun polycaprolactone/gelatin (PG) nanofibers to obtain P/G-CS-OI membranes. The P/G-CS-OI membrane exhibited good mechanical strength, compliance, and biocompatibility. In addition, the sustained OI release endowed the nanofiber membrane with great antioxidative and anti-inflammatory activities as revealed in in vitro and in vivo studies. Specifically, the P/G-CS-OI membrane activated nuclear factor-erythroid-2-related factor 2 (NRF2) by alkylating Kelch-like ECH-associated protein 1 (KEAP1). This antioxidative response modulates macrophage polarization, leading to mitigated inflammatory responses, enhanced angiogenesis, and recovered re-epithelization, finally contributing to improved healing of mouse diabetic wounds. CONCLUSIONS: The P/G-CS-OI nanofiber membrane shows good capacity in macrophage modulation and might be promising for diabetic chronic wound treatment.

Engineering of itaconic acid pathway via co-localization of CadA and AcnA in recombinant Escherichia coli.

Itaconic acid is an excellent polymeric precursor with a wide range of industrial applications. The efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. However, limitation in the itaconic acid precursor supply was revealed by finding out the key intermediate of the tricarboxylic acid in the itaconic acid pathway. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool to increase itaconic acid productivity are required. In this study, we introduce a synthetic protein scaffold system between CadA and AcnA to physically combine the two enzymes. Through the introduction of a synthetic protein scaffold, 2.1 g L-1 of itaconic acid was produced at pH 7 and 37 °C. By fermentation, 20.1 g L-1 for 48 h of itaconic acid was produced with a yield of 0.34 g g-1 glycerol. These results suggest that carbon flux was successfully increased itaconic acid productivity.

Safety Assessment of Vinylpyrrolidone Polymers as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 30 vinylpyrrolidone polymers as used in cosmetic products; most of these ingredients have the reported cosmetic function of film former in common. The Panel reviewed data relevant to the safety of these ingredients, and determined that 27 vinylpyrrolidone polymers are safe in cosmetics in the present practices of use and concentration described in the safety assessment. The Panel also concluded that the available data are insufficient to make a determination that 3 vinylpyrrolidone polymers (all urethanes) are safe under the intended conditions of use in cosmetic formulations.

Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

BACKGROUND: Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. RESULTS: In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. CONCLUSIONS: This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

Microbiome-metabolomics analysis reveals abatement effects of itaconic acid on odorous compound production in Arbor Acre broilers.

BACKGROUND: Public complaints concerning odor emissions from intensive livestock and poultry farms continue to grow, as nauseous odorous compounds have adverse impacts on the environment and human health. Itaconic acid is a metabolite from the citric acid cycle of the host and shows volatile odor-reducing effects during animal production operations. However, the specific role of itaconic acid in decreasing intestinal odorous compound production remains unclear. A total of 360 one-day-old chicks were randomly divided into 6 treatment groups: control group (basal diet) and itaconic acid groups (basal diet + 2, 4, 6, 8 and 10 g/kg itaconic acid). The feeding experiment lasted for 42 d. RESULTS: Dietary itaconic acid supplementation linearly and quadratically decreased (P < 0.05) the cecal concentrations of indole and skatole but did not affect (P > 0.05) those of lactic, acetic, propionic and butyric acids. The cecal microbial shift was significant in response to 6 g/kg itaconic acid supplementation, in that the abundances of Firmicutes, Ruminococcus and Clostridium were increased (P < 0.05), while those of Bacteroidetes, Escherichia-Shigella and Bacteroides were decreased (P < 0.05), indicative of increased microbial richness and diversity. Furthermore, a total of 35 significantly (P < 0.05) modified metabolites were obtained by metabolomic analysis. Itaconic acid decreased (P < 0.05) the levels of nicotinic acid, nicotinamide, glucose-6-phosphate, fumatic acid and malic acid and increased (P < 0.05) 5-methoxytroptomine, dodecanoic acid and stearic acid, which are connected with the glycolytic pathway, citrate acid cycle and tryptophan metabolism. Correlation analysis indicated significant correlations between the altered cecal microbiota and metabolites; Firmicutes, Ruminococcus and Clostridium were shown to be negatively correlated with indole and skatole production, while Bacteroidetes, Escherichia-Shigella and Bacteroides were positively correlated with indole and skatole production. CONCLUSIONS: Itaconic acid decreased cecal indole and skatole levels and altered the microbiome and metabolome in favor of odorous compound reduction. These findings provide new insight into the role of itaconic acid and expand its application potential in broilers.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates the detection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4 °C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 µM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences.

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high - 120 g/L - glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Construction of cell factory through combinatorial metabolic engineering for efficient production of itaconic acid.

BACKGROUND: Itaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid. RESULTS: This study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g(DCW) among heterologous hosts at 48 h. CONCLUSIONS: The excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.

Synergistic effects on itaconic acid production in engineered Aspergillus niger expressing the two distinct biosynthesis clusters from Aspergillus terreus and Ustilago maydis.

BACKGROUND: Itaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus fermentation are limited in terms of process efficiency and productivity. To construct more efficient IA production strains, A. niger was used as a chassis for engineering IA production by assembling the key components of IA biosynthesis pathways from both A. terreus and Ustilago maydis. RESULTS: Recombinant A. niger S1596 overexpressing the A. terreus IA biosynthesis genes cadA, mttA, mfsA produced IA of 4.32 g/L, while A. niger S2120 overexpressing the U. maydis IA gene cluster adi1, tad1, mtt1, itp1 achieved IA of 3.02 g/L. Integration of the two IA production pathways led to the construction of A. niger S2083 with IA titers of 5.58 g/L. Increasing cadA copy number in strain S2083 created strain S2209 with titers of 7.99 g/L and deleting ictA to block IA degradation in S2209 created strain S2288 with IA titers of 8.70 g/L. Overexpressing acoA to enhance the supply of IA precursor in strain S2288 generated strain S2444 with IA titers of 9.08 g/L in shake flask. CONCLUSION: Recombinant A. niger overexpressing the U. maydis IA biosynthesis pathway was capable of IA accumulation. Combined expression of the two IA biosynthesis pathways from A. terreus and U. maydis in A. niger resulted in much higher IA titers. Furthermore, increasing cadA copy number, deleting ictA to block IA degradation and overexpressing acoA to enhance IA precursor supply all showed beneficial effects on IA accumulation.

Safranin-O cationic dye removal from wastewater using carboxymethyl cellulose-grafted-poly(acrylic acid-co-itaconic acid) nanocomposite hydrogel.

Copolymer of acrylic acid (AA) and itaconic acid (IA) grafted onto sodium carboxymethyl cellulose hydrogel (CMC-g-poly (AA-co-IA)) was successfully synthesized as an adsorbent to remove safranin-O from wastewater. The swelling and removal efficiencies of CMC-g-poly (AA-co-IA) were enhanced by increasing IA/AA molar ratio as well as by incorporation of montmorillonite clay nano-sheets (MMT). The surface area of MMT, CMC-g-poly (AA-co-IA), and CMC-g-poly (AA-co-IA) samples was 15.632, 0.61452, and 0.66584 m2/g, respectively, indicating the effectiveness of MMT nano-sheets in improving hydrogel surface area. The maximum removal efficiency of CMC-g-poly (AA-co-IA)/MMT under optimum conditions i.e., pH of 8, initial concentration of 10 mg/L, adsorbent dose of 2 g/L, and contact time of 40 min was ascertained 99.78% using a response surface methodology-central composite design (RSM-CCD). Pseudo-second-order and Langmuir models giving the maximum monolayer adsorption capacity of 18.5185 mg/g and 19.1205 mg/g for CMC-g-poly (AA-co-IA) and CMC-g-poly (AA-co-IA)/MMT samples, respectively are the best-fitted models for kinetic and equilibrium data. Thermodynamically, safranin-O decontamination was spontaneous, exothermic, and entropy decreasing. Moreover, ad (de)sorption behavior study showed that CMC-g-poly (AA-co-IA)/MMT performance was not changed after multiple recovery steps. Therefore, CMC-g-poly (AA-co-IA)/MMT was considered as a highly potential adsorbent for safranin-O removal from wastewater.

Itaconic acid hybrids as potential anticancer agents.

In this paper, we report the synthesis of novel hybrids 2-14 based on itaconic acid and fluoroaniline, pyridine, indole and quinoline scaffolds. Itaconic acid is a naturally occurring compound with a Michael acceptor moiety, a key structural feature in several anticancer and antiviral drugs, responsible for the covalent binding of a drug to the cysteine residue of a specific protein. Aromatic parts of the hybrids also come from the substances reported as anticancer or antiviral agents. The synthetic route employed to access the amido-ester hybrids 2-13 used monomethyl itaconate or monomethyl itaconyl chloride and corresponding amines as the starting materials. Dimers 14 and 15 with two aminoindole or mefloquine moieties were prepared from itaconic acid and corresponding amino derivative, using standard coupling conditions (HATU/DIEA). All hybrids exerted anticancer effects in vitro against almost all the tumour cell lines that were evaluated (MCF-7, HCT 116, H460, LN-229, Capan-1, DND-41, HL-60, K-562, Z-138). Solid tumour cells were, in general, more responsive than the haematological cancer cells. The MCF-7 breast adenocarcinoma cell line appeared the most sensitive. Amido-ester 12 with chloroquine core and mefloquine homodimer 15 showed the highest activity with GI50 values between 0.7 and 8.6 µM. In addition, compound 15 also exerted antiviral activity against Zika virus and Coxsackievirus B4 in low micromolar concentrations.

Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis.

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.

Degradation of Polymer-Drug Conjugate Nanoparticles Based on Lactic and Itaconic Acid.

Tuberculosis (TB) is still a significant threat to human health. A promising solution is engineering nanoparticulate drug carriers to deliver anti-TB molecules. Itaconic acid (ITA) potentially has anti-TB activity; however, its incorporation in nanoparticles (NP) is challenging. Here we show an approach for preparing polymer-ITA conjugate NPs and a methodology for investigating the NP degradation and ITA release mechanism. The conjugate was synthesized by the two-directional growing of polylactic acid (PLA) chains, followed by capping their extremities with ITA. The poly(lactate)-itaconate PLA-ITA was then used to formulate NPs. The degradation and drug release processes of the polymer conjugate NPs were studied qualitatively and quantitatively. The molecular structures of released species were characterized by using liquid NMR spectroscopy and mass spectrometry. We discovered a complex NP hydrolysis process forming diverse oligomers, as well as monomeric lactic acid (LA) and drug ITA. The slow degradation process led to a low release of free drugs, although raising the pH from 5.3 to 7.4 induced a slight increase in the amounts of released products. TEM images showed that bulk erosion is likely to play the primary role in the degradation of PLA-ITA NPs. The overall results and methodology can be of interest for understanding the mechanisms of NP degradation and drug release of this new polymer-drug conjugate system.