[Metabolic engineering of Escherichia coli for the production of cadaverine].

Cadaverine is a fundamental C5 building block in the production of polyamides. Due to the limited regeneration efficiency of intracellular pyridoxal 5'-phosphate (PLP), the current fermentation-based production of cadaverine exhibits low efficiency. In this study, we developed an Escherichia coli strain L01 by introducing lysine decarboxylase (lysine decarboxylase, LDC, a key enzyme in the synthesis of cadaverine) into a lysine-producing strain E. coli LY-4, achieving a cadaverine tier of 1.07 g/L in shake flask fermentation. Subsequently, a dual metabolic pathway enhancement strategy was proposed to synergistically strengthen both endogenous and exogenous PLP synthesis modules, thereby improving intracellular PLP synthesis. The optimized strain L11 achieved a cadaverine titer of 9.23 g/L in shake flask fermentation. Finally, the fermentation process for cadaverine production by strain L11 was optimized in a 5 L fermenter. After 48 h of fed-batch fermentation, the engineered strain L11 achieved the cadaverine titer, yield, and productivity of 54.43 g/L, 0.22 g/g, and 1.13 g/(L·h), respectively. This study provides a theoretical and technical foundation for establishing microbial cell factories for bioamine production.

[Clinical and genetic features of children with 3-methylcrotonyl-coenzyme A carboxylase deficiency: an analysis of six cases]. / 3-ç”²åŸºå·´è±†é °è¾ é ¶Aç¾§åŒ–é ¶ç¼ºä¹ç—‡6ä¾‹æ‚£å„¿çš„ä¸´åºŠç‰¹å¾åŠé—ä¼ å­¦åˆ†æž.

OBJECTIVES: To investigate the clinical and genetic features of children with 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD). METHODS: A retrospective analysis was conducted on the clinical manifestations and genetic testing results of six children with MCCD who attended Children's Hospital Affiliated to Zhengzhou University from January 2018 to October 2023. RESULTS: Among the six children with MCCD, there were 4 boys and 2 girls, with a mean age of 7 days at the time of attending the hospital and 45 days at the time of confirmed diagnosis. Of all children, one had abnormal urine odor and five had no clinical symptoms. All six children had increases in blood 3-hydroxyisovaleryl carnitine and urinary 3-hydroxyisovaleric acid and 3-methylcrotonoylglycine, and five of them had a reduction in free carnitine. A total of six mutations were identified in the MCCC1 gene, i.e., c.1630del(p.R544Dfs*2), c.269A>G(p.D90G), c.1609T>A(p.F537I), c.639+2T>A, c.761+1G>T, and c.1331G>A(p.R444H), and three mutations were identified in the MCCC2 gene, i.e., c.838G>T(p.D280Y), c.592C>T(p.Q198*,366), and c.1342G>A(p.G448A). Among these mutations, c.269A>G(p.D90G) and c.1609T>A(p.F537I) had not been previously reported in the literature. There was one case of maternal MCCD, and the child carried a heterozygous mutation from her mother. Five children with a reduction in free carnitine were given supplementation of L-carnitine, and free carnitine was restored to the normal level at the last follow-up visit. CONCLUSIONS: This study identifies two new mutations, c.269A>G(p.D90G) and c.1609T>A(p.F537I), thereby expanding the mutation spectrum of the MCCC1 gene. A combination of blood amino acid and acylcarnitine profiles, urine organic acid analysis, and genetic testing can facilitate early diagnosis and treatment of MCCD, and provide essential data for genetic counseling.

ACOD1 mediates Staphylococcus aureus-induced inflammatory response via the TLR4/NF-κB signaling pathway.

Staphylococcus aureus (SA) is a common Gram-positive bacterium that activates inflammatory cells, expressing various cytokines and inducing an inflammatory response. Recent research revealed aconitate decarboxylase 1 (ACOD1) as a regulator of the immune response through various metabolic pathways, playing a dual role in the inflammatory response. However, the mechanism by which ACOD1 participates in the regulation of SA-induced inflammatory responses in macrophages remains unknown. Therefore, this study aims to investigate the function and underlying regulatory mechanisms of ACOD1 in SA-induced inflammatory response. This study reveals that SA induced a macrophage inflammatory response and upregulated ACOD1 expression. ACOD1 knockdown significantly inhibited SA-induced macrophage inflammatory response, attenuated SA-induced nuclear envelope wrinkling, and plasma membrane rupture, and suppressed the TLR4/NF-κB signaling pathway. Furthermore, ACOD1 knockdown reduced the inflammatory response and alleviated lung tissue injury and cellular damage, leading to decreased bacterial loads in the lungs of SA-infected mice. Collectively, these findings demonstrate that SA induces an inflammatory response in macrophages and increases ACOD1 expression. ACOD1 enhances SA-induced inflammatory responses via the TLR4/NF-κB signaling pathway. Our findings highlight the significant role of ACOD1 in mediating the inflammatory response in SA-infected macrophages and elucidate its molecular mechanism in regulating the SA-induced inflammatory response.

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation. ONE-SENTENCE SUMMARY: Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

Clinical, biochemical and genetic characteristics and long-term follow-up of five patients with malonyl-CoA decarboxylase deficiency.

BACKGROUND: Malonyl-CoA decarboxylase (MLYCD) deficiency, also known as malonic aciduria (MAD), is a rare autosomal recessive inherited metabolic defect. In this study, we aimed to investigate the clinical and molecular features of five patients with MAD in order to increase clinicians' awareness of the disease. METHODS: Sanger sequencing was used to detect and genetically analyze the MLYCD variations in the preexisting patients and their parents. RESULTS: Five patients with MAD (5 months to 9.6 years old; two males and three females) rarely exhibited metabolic decompensation episodes or seizures. All patients exhibited varying degrees of developmental delay and hypotonia. Our study expands the spectrum of variants of the MLYCD gene. MLYCD gene variations were detected in all five patients, and five new variants were identified: c.60delG (p.Arg21Glyfs*52), c.928C > T (p.Arg310*), c.1293G > T (p.Trp431Cys), c.721T > C (p.Ser241Pro), and Exons 4-5 deletion. Additionally, there is no correlation between various genotypes and phenotypes. CONCLUSION: A high-medium-chain triglyceride and low-long-chain triglyceride diet supplemented with L-carnitine was effective in most patients and may improve cardiomyopathy and muscle weakness. Newborn screening may aid in the early diagnosis, treatment, and prognosis of this rare disorder.

A monoallelic UXS1 variant associated with short-limbed short stature.

BACKGROUND: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more. METHODS: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics. RESULTS: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father. CONCLUSION: This is the first report linking UXS1 to short-limbed short stature in humans.

Metabolic and enzymatic engineering approach for the production of 2-phenylethanol in engineered Escherichia coli.

2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.

Fine-Tuning Pyridoxal 5'-Phosphate Synthesis in Escherichia coli for Cadaverine Production in Minimal Culture Media.

Cadaverine is a critical C5 monomer for the production of polyamides. Pyridoxal 5'-phosphate (PLP), as a crucial cofactor for the key enzyme lysine decarboxylase in the cadaverine biosynthesis pathway, has seen a persistent shortage, leading to limitations in cadaverine production. To address this issue, a dual-pathway strategy was implemented, synergistically enhancing both endogenous and heterologous PLP synthesis modules and resulting in improved PLP synthesis. Subsequently, a growth-stage-dependent molecular switch was introduced to balance the precursor competition between PLP synthesis and cell growth. Additionally, a PLP sensor-based negative feedback circuit was constructed by integrating a newly identified PLP-responsive promoter PygjH and an arabinose-regulated system, dynamically regulating the expression of the PLP synthetic genes and preventing excessive intracellular PLP accumulation. The optimal strain, L18, cultivated in the minimal medium AM1, demonstrated cadaverine production with a titer, yield, and productivity of 64.03 g/L, 0.23 g/g glucose, and 1.33 g/L/h, respectively. This represents the highest titer reported to date in engineered Escherichia coli by fed-batch fermentation in a minimal medium.

Novel biallelic PISD missense variants cause spondyloepimetaphyseal dysplasia with disproportionate short stature and fragmented mitochondrial morphology.

Biallelic variants in PISD cause a phenotypic spectrum ranging from short stature with spondyloepimetaphyseal dysplasia (SEMD) to a multisystem disorder affecting eyes, ears, bones, and brain. PISD encodes the mitochondrial-localized enzyme phosphatidylserine decarboxylase. The PISD precursor is self-cleaved to generate a heteromeric mature enzyme that converts phosphatidylserine to the phospholipid phosphatidylethanolamine. We describe a 17-year-old male patient, born to unrelated healthy parents, with disproportionate short stature and SEMD, featuring platyspondyly, prominent epiphyses, and metaphyseal dysplasia. Trio genome sequencing revealed compound heterozygous PISD variants c.569C>T; p.(Ser190Leu) and c.799C>T; p.(His267Tyr) in the patient. Investigation of fibroblasts showed similar levels of the PISD precursor protein in both patient and control cells. However, patient cells had a significantly higher proportion of fragmented mitochondria compared to control cells cultured under basal condition and after treatment with 2-deoxyglucose that represses glycolysis and stimulates respiration. Structural data from the PISD orthologue in Escherichia coli suggest that the amino acid substitutions Ser190Leu and His267Tyr likely impair PISD's autoprocessing activity and/or phosphatidylethanolamine biosynthesis. Based on the data, we propose that the novel PISD p.(Ser190Leu) and p.(His267Tyr) variants likely act as hypomorphs and underlie the pure skeletal phenotype in the patient.

Itaconate protects ferroptotic neurons by alkylating GPx4 post stroke.

Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4's enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.

Taurine Alleviates Experimental Colitis by Enhancing Intestinal Barrier Function and Inhibiting Inflammatory Response through TLR4/NF-κB Signaling.

Taurine (Tau) is a semiessential amino acid in mammals with preventive and therapeutic effects on several intestinal disorders. However, the exact function of taurine in ulcerative colitis (UC) is still largely unclear. In this study, we used two taurine-deficient mouse models (CSAD-/- and TauT-/- mice) to explore the influence of taurine on the progression of UC in both dextran sulfate sodium (DSS)-induced colitis and LPS-stimulated Caco-2 cells. We found that cysteine sulfinic acid decarboxylase (CSAD) and taurine transporter (TauT) expressions and taurine levels were markedly reduced in colonic tissues of mice treated with DSS. The CSAD and TauT knockouts exacerbated DSS-induced clinical symptoms and pathological damage and aggravated the intestinal barrier dysfunction and the colonic mucosal inflammatory response. Conversely, taurine pretreatment enhanced the intestinal barrier functions by increasing goblet cells and upregulating tight junction protein expression. Importantly, taurine bound with TLR4 and inhibited the TLR4/NF-κB pathway, ultimately reducing proinflammatory factors (TNF-α and IL-6) and oxidative stress. Our findings highlight the essential role of taurine in maintaining the intestinal barrier integrity and inhibiting intestinal inflammation, indicating that taurine is a promising supplement for colitis treatment.

Acod1/itaconate activates Nrf2 in pulmonary microvascular endothelial cells to protect against the obesity-induced pulmonary microvascular endotheliopathy.

BACKGROUND: Obesity is the main risk factor leading to the development of various respiratory diseases, such as asthma and pulmonary hypertension. Pulmonary microvascular endothelial cells (PMVECs) play a significant role in the development of lung diseases. Aconitate decarboxylase 1 (Acod1) mediates the production of itaconate, and Acod1/itaconate axis has been reported to play a protective role in multiple diseases. However, the roles of Acod1/itaconate axis in the PMVECs of obese mice are still unclear. METHODS: mRNA-seq was performed to identify the differentially expressed genes (DEGs) between high-fat diet (HFD)-induced PMVECs and chow-fed PMVECs in mice (|log2 fold change| ≥ 1, p ≤ 0.05). Free fatty acid (FFA) was used to induce cell injury, inflammation and mitochondrial oxidative stress in mouse PMVECs after transfection with the Acod1 overexpressed plasmid or 4-Octyl Itaconate (4-OI) administration. In addition, we investigated whether the nuclear factor erythroid 2-like 2 (Nrf2) pathway was involved in the effects of Acod1/itaconate in FFA-induced PMVECs. RESULTS: Down-regulated Acod1 was identified in HFD mouse PMVECs by mRNA-seq. Acod1 expression was also reduced in FFA-treated PMVECs. Acod1 overexpression inhibited cell injury, inflammation and mitochondrial oxidative stress induced by FFA in mouse PMVECs. 4-OI administration showed the consistent results in FFA-treated mouse PMVECs. Moreover, silencing Nrf2 reversed the effects of Acod1 overexpression and 4-OI administration in FFA-treated PMVECs, indicating that Nrf2 activation was required for the protective effects of Acod1/itaconate. CONCLUSION: Our results demonstrated that Acod1/Itaconate axis might protect mouse PMVECs from FFA-induced injury, inflammation and mitochondrial oxidative stress via activating Nrf2 pathway. It was meaningful for the treatment of obesity-caused pulmonary microvascular endotheliopathy.

IRG1/itaconate alleviates acute liver injury in septic mice by suppressing NLRP3 expression and its mediated macrophage pyroptosis via regulation of the Nrf2 pathway.

Sepsis, a systemic inflammatory response triggered by infection, has a considerably high mortality rate. However, effective prevention and intervention measures against sepsis remain insufficient. Therefore, this study aimed to investigate the mechanisms underlying the protective properties of immune response gene-1 (IRG1) and 4-Octyl itaconate (OI) during acute liver damage in mice with sepsis. A sepsis mouse model was established to compare wild-type and IRG1-/- groups. The impact of IRG1/Itaconate on pro- and anti-inflammatory cytokines was evaluated using J774A.1 cells. IRG1/Itaconate substantially reduced pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines. It reduced pathological damage to liver tissues, preserved normal liver function, decreased the release of reactive oxygen species (ROS) and LDH, and enhanced the GSH/GSSG ratio. Moreover, IRG1 and itaconic acid activated the Nrf2 signaling pathway, regulating the expression of its downstream antioxidative stress-related proteins. Additionally, they inhibited the activity of NLRP3 inflammatory vesicles to suppress the expression of macrophage-associated pyroptosis signaling molecules. Our findings demonstrate that IRG1/OI inhibits NLRP3 inflammatory vesicle activation and macrophage pyroptosis by modulating the Nrf2 signaling pathway, thereby attenuating acute liver injury in mice with sepsis. These findings could facilitate the clinical application of IRG1/Itaconate to prevent sepsis-induced acute liver injury.

Sedentary behavior in mice induces metabolic inflexibility by suppressing skeletal muscle pyruvate metabolism.

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in the absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.

Rationally Engineering pH Adaptation of Acid-Induced Arginine Decarboxylase from Escherichia coli to Alkaline Environments to Efficiently Biosynthesize Putrescine.

Acid-induced arginine decarboxylase AdiA is a typical homo-oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole-cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0-9.0), and its enzymatic activity is 35-fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L-1 with a conversion of 0.94 mol·mol-1 in whole-cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH-adaptation engineering of other oligomeric decarboxylases.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

ACOD1, rather than itaconate, facilitates p62-mediated activation of Nrf2 in microglia post spinal cord contusion.

BACKGROUND: Spinal cord injury (SCI)-induced neuroinflammation and oxidative stress (OS) are crucial events causing neurological dysfunction. Aconitate decarboxylase 1 (ACOD1) and its metabolite itaconate (Ita) inhibit inflammation and OS by promoting alkylation of Keap1 to induce Nrf2 expression; however, it is unclear whether there is another pathway regulating their effects in inflammation-activated microglia after SCI. METHODS: Adult male C57BL/6 ACOD1-/- mice and their wild-type (WT) littermates were subjected to a moderate thoracic spinal cord contusion. The degree of neuroinflammation and OS in the injured spinal cord were assessed using qPCR, western blot, flow cytometry, immunofluorescence, and trans-well assay. We then employed immunoprecipitation-western blot, chromatin immunoprecipitation (ChIP)-PCR, dual-luciferase assay, and immunofluorescence-confocal imaging to examine the molecular mechanisms of ACOD1. Finally, the locomotor function was evaluated with the Basso Mouse Scale and footprint assay. RESULTS: Both in vitro and in vivo, microglia with transcriptional blockage of ACOD1 exhibited more severe levels of neuroinflammation and OS, in which the expression of p62/Keap1/Nrf2 was down-regulated. Furthermore, silencing ACOD1 exacerbated neurological dysfunction in SCI mice. Administration of exogenous Ita or 4-octyl itaconate reduced p62 phosphorylation. Besides, ACOD1 was capable of interacting with phosphorylated p62 to enhance Nrf2 activation, which in turn further promoted transcription of ACOD1. CONCLUSIONS: Here, we identified an unreported ACOD1-p62-Nrf2-ACOD1 feedback loop exerting anti-inflammatory and anti-OS in inflammatory microglia, and demonstrated the neuroprotective role of ACOD1 after SCI, which was different from that of endogenous and exogenous Ita. The present study extends the functions of ACOD1 and uncovers marked property differences between endogenous and exogenous Ita. KEY POINTS: ACOD1 attenuated neuroinflammation and oxidative stress after spinal cord injury. ACOD1, not itaconate, interacted with p-p62 to facilitate Nrf2 expression and nuclear translocation. Nrf2 was capable of promoting ACOD1 transcription in microglia.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Recent Advances of Oxalate Decarboxylase: Biochemical Characteristics, Catalysis Mechanisms, and Gene Expression and Regulation.

Oxalate decarboxylase (OXDC) is a typical Mn2+/Mn3+ dependent metal enzyme and splits oxalate to formate and CO2 without any organic cofactors. Fungi and bacteria are the main organisms expressing the OXDC gene, but with a significantly different mechanism of gene expression and regulation. Many articles reported its potential applications in the clinical treatment of hyperoxaluria, low-oxalate food processing, degradation of oxalate salt deposits, oxalate acid diagnostics, biocontrol, biodemulsifier, and electrochemical oxidation. However, some questions still remain to be clarified about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II)/Mn(III), the nature of dioxygen involved in the catalytic mechanism, and how OXDC acquires Mn(II) /Mn(III). This review mainly summarizes its biochemical and structure characteristics, gene expression and regulation, and catalysis mechanism. We also deep-mined oxalate decarboxylase gene data from National Center for Biotechnology Information to give some insights to explore new OXDC with diverse biochemical properties.