4-Octyl itaconate attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

Objectives: The aim of this study was to investigate the mechanism of itaconate's potential effect in diabetic kidney disease.Methods: Renal immune responsive gene 1 (IRG1) levels were measured in db/db mice and streptozotocin (STZ) + high-fat diet (HFD)-induced diabetic mice. Irg1 knockout mice were generated. db/db mice were treated with 4-octyl itaconate (4-OI, 50 mg/kg), a derivative of itaconate, for 4 weeks. Renal function and morphological changes were investigated. Ultrastructural alterations were determined by transmission electron microscopy.Results: Renal IRG1 levels were reduced in two diabetic models. STZ+HFD-treated Irg1 knockout mice exhibited aggravated renal tubular injury and worsened renal function. Treatment with 4-OI lowered urinary albumin-to-creatinine ratio and blood urea nitrogen levels, and restored renal histological changes in db/db mice. It improved mitochondrial damage, increased expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice. These were confirmed in vitro; 4-OI improved high glucose-induced abnormal mitochondrial morphology and TFAM expression in HK-2 cells, effects that were inhibited by PGC-1α silencing. Moreover, 4-OI reduced the number of apoptotic cells in the renal cortex of db/db mice. Further study showed that 4-OI increased renal Nrf2 expression and decreased oxidative stress levels in db/db mice. In HK-2 cells, 4-OI decreased high glucose-induced mitochondrial ROS production, which was reversed by Nrf2 silencing. Nrf2 depletion also inhibited 4-OI-mediated regulation of PGC-1α, TFAM, and mitochondrial apoptotic protein expressions.Conclusions: 4-OI attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

[Severe traumatic brain injury with damage to the posterior cranial fossa in a teenager]. / Tyazhelaya cherepno-mozgovaya travma s povrezhdeniem zadnei cherepnoi yamki u podrostka.

In the presented clinical observation of complex therapy of severe combined trauma: severe brain contusion, subarachnoid hemorrhage, closed fracture of the occipital bone, closed compression fracture of ThVI-ThVIII vertebral bodies, contusion of the lungs and kidneys, blunt abdominal trauma and closed fracture of both bones of the right leg in lower third with displacement) in a teenager after an accident, the need for dynamic introscopic examination of the patient is shown for timely detection of abnormalities in the state of brain structures and correction of treatment up to surgical intervention. The effectiveness of the inclusion of Cytoflavin in complex treatment regimens was noted in the form of positive dynamics of the clinical and introscopic picture. The results obtained may serve as a basis for further research.

Vanillin loaded-physically crosslinked PVA/chitosan/itaconic membranes for topical wound healing applications.

Vanillin loaded-physically crosslinked hydrogel membranes made of PVA/chitosan/itaconic acid (PVA-CS-IA) were prepared using freezing-thawing (F-T) cycle method. To ensure the entanglement of PVA-CS-IA chains, three F-T cycles were repeated. The polymeric chains entanglements were confirmed and characterized by different instrumental characterizations. Physicochemical properties for example, swelling ratio, mechanical characteristics, gel fraction percentage (GF%), hydrolytic degradation, and thermal stability of PVA-CS-IA membrane were discussed in detail. The findings showed that the swelling ratio, mechanical characteristics, and hydrolytic degradation of the crosslinked membranes enhanced with increasing CS-IA contents in membranes composition; however, GF% gradually declined with CS-IA content. Additionally, cell viability test using HFB-4 cell line and antimicrobial activity against Staphylococcus aureus and Escherichia coli were evaluated using MTT assay and the bacterium growth inhibition percentage method; respectively. Notably, with varying incubation durations and membrane concentrations, all examined constructed hydrogels showed significant cell survival percentages. The findings supported the notion that produced hydrogel membranes might be used in a professional setting as antibacterial dressings or biomaterials for quick wound healing rate.

Model-based investigation of the pH-dependent chromatographic separation of itaconic acid from aqueous solution using strongly hydrophobic adsorbents.

In this study, we propose a model for the simulation of the pH-dependent separation of dicarboxylic acids from aqueous solutions using strongly hydrophobic adsorbents. Building upon results of our previous study, where we experimentally investigated the pH-dependent adsorption behavior of the individual acid species of itaconic acid (IA) on a strongly hydrophobic adsorbent using in-line Raman spectroscopy, we utilize a transport-dispersive model as the basis for our simulation model. Instead of considering IA as a single component in our model, we simulated each acid species of IA individually. For this purpose, we expanded the transport-dispersive model with reaction terms in all aqueous phases. The reaction terms include all dissociation reactions of all involved components for each time step and spatial discretization. This model enables the time and spatial dependent simulation of the pH value in the chromatographic column and thus the time and spatial dependent knowledge of each acid species concentration. The consideration of activity coefficients due to high local ionic strength is achieved using the Truesdell-Jones (TdJ) model. The simulation model is successfully validated using experimental data from our previous study and used in a simulation study that demonstrates the potential of the model approach for analyzing associated separation tasks.

Tumor-associated macrophage-derived itaconic acid contributes to nasopharyngeal carcinoma progression by promoting immune escape via TET2.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of epithelial origin in head and neck with high incidence rate in South China, Southeast Asia and North Africa. The intervention of tumor-associated macrophages (Mφs) (TAMs)-mediated immunosuppression is a potential therapeutic strategy against tumor metastasis, but the exact mechanisms of TAM-mediated immunosuppression in nasopharyngeal carcinoma are unclear. Furthermore, how TAM affects the occurrence and development of nasopharyngeal carcinoma through metabolism is rarely involved. In this work, we revealed that NPC cells promoted M2-type Mφ polarization and elevated itaconic acid (ITA) release. Also, TAMs facilitated NPC cell proliferation, migration, and invasion through immune response gene 1 (IRG1)-catalyzed ITA production. Then, IRG1-mediated ITA production in TAMs repressed the killing of CD8+ T cells, induced M2-type polarization of TAMs, and reduced the phagocytosis of TAMs. Moreover, we demonstrated ITA played a tumor immunosuppressive role by binding and dampening ten-eleven translocation-2 (TET2) expression. Finally, we proved that ITA promotes NPC growth by facilitating immune escape in CD34+ hematopoietic stem cell humanized mice. In Conclusion, TAM-derived ITA facilitated NPC progression by enhancing immune escape through targeting TET2, highlighting that interfering with the metabolic pathway of ITA may be a potential strategy for NPC treatment.

Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages.

A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1ß in response to these innate activators. In contrast, the production of interferon (IFN)ß, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

The potential therapeutic role of itaconate and mesaconate on the detrimental effects of LPS-induced neuroinflammation in the brain.

Despite advances in antimicrobial and anti-inflammatory treatment, inflammation and its consequences remain a major challenge in the field of medicine. Inflammatory reactions can lead to life-threatening conditions such as septic shock, while chronic inflammation has the potential to worsen the condition of body tissues and ultimately lead to significant impairment of their functionality. Although the central nervous system has long been considered immune privileged to peripheral immune responses, recent research has shown that strong immune responses in the periphery also affect the brain, leading to reactive microglia, which belong to the innate immune system and reside in the brain, and neuroinflammation. The inflammatory response is primarily a protective mechanism to defend against pathogens and tissue damage. However, excessive and chronic inflammation can have negative effects on neuronal structure and function. Neuroinflammation underlies the pathogenesis of many neurological and neurodegenerative diseases and can accelerate their progression. Consequently, targeting inflammatory signaling pathways offers potential therapeutic strategies for various neuropathological conditions, particularly Parkinson's and Alzheimer's disease, by curbing inflammation. Here the blood-brain barrier is a major hurdle for potential therapeutic strategies, therefore it would be highly advantageous to foster and utilize brain innate anti-inflammatory mechanisms. The tricarboxylic acid cycle-derived metabolite itaconate is highly upregulated in activated macrophages and has been shown to act as an immunomodulator with anti-inflammatory and antimicrobial functions. Mesaconate, an isomer of itaconate, similarly reduces the inflammatory response in macrophages. Nevertheless, most studies have focused on its esterified forms and its peripheral effects, while its influence on the CNS remained largely unexplored. Therefore, this study investigated the immunomodulatory and therapeutic potential of endogenously synthesized itaconate and its isomer mesaconate in lipopolysaccharide (LPS)-induced neuroinflammatory processes. Our results show that both itaconate and mesaconate reduce LPS-induced neuroinflammation, as evidenced by lower levels of inflammatory mediators, reduced microglial reactivity and a rescue of synaptic plasticity, the cellular correlate of learning and memory processes in the brain. Overall, this study emphasizes that both itaconate and mesaconate have therapeutic potential for neuroinflammatory processes in the brain and are of remarkable importance due to their endogenous origin and production, which usually leads to high tolerance.

Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semipilot scale.

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD+-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.

Octenylsuccinylation strategy to construct high-amylose maize starch-myristic acid complexes with high thermal stability, complexation efficiency and anti-retrogradation ability.

Gelatinizing high-amylose maize starch (HAMSt) requires high temperatures to allow complexation with lipids, making it a challenging process. An octenylsuccinylation method was examined as a part of a strategy to decrease the gelatinization temperature of HAMSt, thereby promoting the complexation between HAMSt and myristic acid (MAc). Octenyl succinic anhydride (OSA) modification of HAMSt reduces the onset gelatinization temperature of HAMSt from 71.63 °C to 66.97 °C. Moreover, as the OSA concentration increased from 2 % to 11 %, the degree of substitution and molecular weights of the esterified HAMSt gradually increased from 0.0069 to 0.0184 and from 0.97 × 106 to 1.17 × 106 g/mol, respectively. Fourier transform infrared analysis indicated that the octenyl-succinate groups were grafted onto the HAMSt chains. The formation of HAMSt-MAc complexes improved the thermal stability of OSA-treated HAMSt (peak temperature increased by 0.11 °C-13.95 °C). Moreover, the diffraction intensity of the V-type peak of the 11 % sample was greater than that of other samples. Finally, the anti-retrogradation ability was in the order of OSA-HAMSt-MAc complexes > HAMSt-MAc complexes > HAMSt. Overall, our results indicate that octenylsuccinylation can be an effective strategy to promote the formation of OSA-HAMSt-MAc complexes and delay starch retrogradation.

[One-pot synthesis of itaconic acid by engineered Halomonas bluephagenesis TDZI-08].

Itaconic acid (IA) is one of the twelve high value-added platform compounds applied in various fields including coatings, adhesives, plastics, resins, and biofuels. In this study, we established a one-pot catalytic synthesis system for IA from citric acid based on the engineered salt-tolerant bacterial strain Halomonas bluephagenesis TDZI-08 after investigating factors that hindered the process and optimizing the carbon source, nitrogen source, inducer addition time, and surfactant dosage. The open, non-sterile, one-pot synthesis with TDZI-08 in a 5 L fermenter achieved the highest IA titer of 40.50 g/L, with a catalytic yield of 0.68 g IA/g citric acid during the catalytic stage and a total yield of 0.42 g IA/g (citric acid+gluconic acid). The one-pot synthesis system established in this study is simple and does not need sterilization or aseptic operations. The findings indicate the potential of H. bluephagenesis for industrial production of IA.

Divergent effects of itaconate isomers on Coxiella burnetii growth in macrophages and in axenic culture.

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.

Fabrication of injectable alginate hydrogels with sustained release of 4-octyl itaconate for articular anti-inflammatory.

BACKGROUND: Osteoarthritis (OA) is a chronic and degenerative joint disease that remains a great challenge in treatment due to the lack of effective therapies. 4-octyl itaconate (4-OI) is a novel and potent modulator of inflammation for the treatment of inflammatory disease. However, the clinical usage of 4-OI is limited due to its poor solubility and low bioavailability. As a promising drug delivery strategy, injectable hydrogels offers an effective approach to address these limitations of 4-OI. OBJECTIVE: The aim of the study was to verify that the composite 4-OI/SA hydrogels could achieve a controlled release of 4-OI and reduce damage to articular cartilage in the group of osteoarthritic rats treated with the system. METHODS: In this study, an injectable composite hydrogel containing sodium alginate (SA) and 4-octyl itaconate (4-OI) has been developed for continuous intra-articular administration in the treatment of OA. RESULTS: After intra-articular injection in arthritic rats, the as-prepared 4-OI/SA hydrogel containing of 62.5 µM 4-OI effectively significantly reduced the expression of TNF-α, IL-1ß, IL-6 and MMP3 in the ankle fluid. Most importantly, the as-prepared 4-OI/SA hydrogel system restored the morphological parameters of the ankle joints close to normal. CONCLUSION: 4-OI/SA hydrogel shows a good anti-inflammatory activity and reverse cartilage disruption, which provide a new strategy for the clinical treatment of OA.

Metabolic Messengers: itaconate.

The metabolite itaconate has emerged as an important immunoregulator with roles in antibacterial defence, inhibition of inflammation and, more recently, as an inhibitory factor in obesity. Itaconate is one of the most upregulated metabolites in inflammatory macrophages. It is produced owing to the disturbance of the tricarboxylic acid cycle and the diversion of aconitate to itaconate via the enzyme aconitate decarboxylase 1. In immunology, initial studies concentrated on the role of itaconate in inflammatory macrophages where it was shown to be inhibitory, but this has expanded as the impact of itaconate on other cell types is starting to emerge. This review focuses on itaconate as a key immunoregulatory metabolite and describes its diverse mechanisms of action and its many impacts on the immune and inflammatory responses and in cancer. We also examine the clinical relevance of this immunometabolite and its therapeutic potential for immune and inflammatory diseases.

The Aconitate Decarboxylase 1/Itaconate Pathway Modulates Immune Dysregulation and Associates with Cardiovascular Disease Markers and Disease Activity in Systemic Lupus Erythematosus.

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

The anti-inflammatory effects of itaconate and its derivatives in neurological disorders.

Almost 16 % of the global population is affected by neurological disorders, including neurodegenerative and cerebral neuroimmune diseases, triggered by acute or chronic inflammation. Neuroinflammation is recognized as a common pathogenic mechanism in a wide array of neurological conditions including Alzheimer's disease, Parkinson's disease, postoperative cognitive dysfunction, stroke, traumatic brain injury, and multiple sclerosis. Inflammatory process in the central nervous system (CNS) can lead to neuronal damage and neuronal apoptosis, consequently exacerbating these diseases. Itaconate, an immunomodulatory metabolite from the tricarboxylic acid cycle, suppresses neuroinflammation and modulates the CNS immune response. Emerging human studies suggest that itaconate levels in plasma and cerebrospinal fluid may serve as biomarkers associated with inflammatory responses in neurological disorders. Preclinical studies have shown that itaconate and its highly cell-permeable derivatives are promising candidates for preventing and treating neuroinflammation-related neurological disorders. The underlying mechanism may involve the regulation of immune cells in the CNS and neuroinflammation-related signaling pathways and molecules including Nrf2/KEAP1 signaling pathway, reactive oxygen species, and NLRP3 inflammasome. Here, we introduce the metabolism and function of itaconate and the synthesis and development of its derivatives. We summarize the potential impact and therapeutic potential of itaconate and its derivatives on brain immune cells and the associated signaling pathways and molecules, based on preclinical evidence via various neurological disorder models. We also discuss the challenges and potential solutions for clinical translation to promote further research on itaconate and its derivatives for neuroinflammation-related neurological disorders.

Enhancing methyl violet 2B pollutant removal from wastewater using Al-MOF encapsulated with poly (itaconic acid) grafted crosslinked chitosan composite sponge: Synthesis, characterization, DFT calculation, adsorption optimization via Box-Behnken Design.

In this research, aluminum metal-organic framework encapsulated with poly (itaconic acid) grafted crosslinked chitosan composite sponge (Al-MOF@PIC) was prepared. SEM, FTIR, XPS, XRD, and BET techniques were employed to thoroughly characterize the synthesized material and establish its structure and characteristics. The study discovered that the Al-MOF@PIC is an efficient way to remove dyes, which constitute a significant number of contaminants in industrial wastewater. Subsequently the adsorption of methyl violet 2B (MV-2B) dye, the surface area, pore size, and pore volume of the adsorbent decreased from 1860.68 m2/g, 1.62 nm, and 1.52 cc/g to 1426.45 m2/g, 1.11 nm, and 0.92 cc/g, individually. This modification suggested that a portion of the MV-2B dye had been removed by adsorption over the adsorbent's pores. The excellent adsorption capacity of the material was further confirmed by batch adsorption tests, which displayed a maximum adsorption capability of 646.76 mg/g for the elimination of MV-2B dye. The high adsorption energy of 26.8 kJ/mol designates that chemisorption is primarily responsible for MV-2B dye adsorption against the sponge adsorbent. The Al-MOF@PIC composite sponge demonstrated exceptional reusability over six cycles, demonstrating its strength and durability. The Al-MOF@PIC composite sponge successfully removes MV-2B from water by pore filling, π-π stacking, hydrogen bonding, and electrostatic interactions, which are the key mechanisms behind the adsorption of the dye pollutant. Its potential for practical applications is further demonstrated using Box Behnken-design (BBD) to optimize the adsorption consequences.

Direct Itaconate Production from Brown Macroalgae Using Engineered Vibrio sp. dhg.

Itaconate is a promising platform chemical with broad applicability, including the synthesis of poly(methyl methacrylate). Most studies on microbial itaconate production entail the use of crop-based feedstock, which imposes constraints due to its limited supply. Brown macroalgae have recently gained attention as next-generation biomass owing to their high biomass productivity and carbohydrate content and amenability to mass production. Therefore, the use of macroalgae for itaconate production warrants exploration. In this study, the direct production of itaconate from brown macroalgae was demonstrated using engineered Vibrio sp. dhg, which has emerged as an efficient platform host for brown macroalgal biorefineries. Specifically, to enhance production, cis-aconitate decarboxylase (Cad) from Aspergillus terreus was heterologously expressed and isocitrate dehydrogenase (icd) was deleted. Notably, the resulting strain, VIC, achieved itaconate titers of 2.5 and 1.5 g/L from a mixture of alginate and mannitol (10 g/L of each) and 40 g/L of raw Saccharina japonica (S. japonica), respectively. Overall, this study highlights the utility of brown macroalgae as feedstock, as well as that of Vibrio sp. dhg as a platform strain for improving itaconate bioproduction.

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.

Directed Evolution of Protein-Based Sensors for Anaerobic Biological Activation of Methane.

Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.