Enhancing glucaric acid production from myo-inositol in Escherichia coli by eliminating cell-to-cell variation.

Glucaric acid (GA) is a value-added chemical and can be used to manufacture food additives, anticancer drugs, and polymers. The non-genetic cell-to-cell variations in GA biosynthesis are naturally inherent, indicating the presence of both high- and low-performance cells in culture. Low-performance cells can lead to nutrient waste and inefficient production. Furthermore, myo-inositol oxygenase (MIOX) is a key rate-limiting enzyme with the problem of low stability and activity in GA production. Therefore, eliminating cell-to-cell variations and increasing MIOX stability can select high-performance cells and improve GA production. In this study, an in vivo GA bioselector was constructed based on GA biosensor and tetracycline efflux pump protein TetA to continuously select GA-efficient production strains. Additionally, the upper limit of the GA biosensor was improved to 40 g/L based on ribosome-binding site optimization, achieving efficient enrichment of GA high-performance cells. A small ubiquitin-like modifier (SUMO) enhanced MIOX stability and activity. Overall, we used the GA bioselector and SUMO-MIOX fusion in fed-batch GA production and achieved a 5.52-g/L titer in Escherichia coli, which was 17-fold higher than that of the original strain.IMPORTANCEGlucaric acid is a non-toxic valuable product that was mainly synthesized by chemical methods. Due to the problems of non-selectivity, inefficiency, and environmental pollution, GA biosynthesis has attracted significant attention. The non-genetic cell-to-cell variations and MIOX stability were both critical factors for GA production. In addition, the high detection limit of the GA biosensor was a key condition for performing high-throughput screening of GA-efficient production strains. To increase GA titer, this work eliminated the cell-to-cell variations by GA bioselector constructed based on GA biosensor and TetA, and improved the stability and activity of MIOX in the GA biosynthetic pathway through fusing the SUMO to MIOX. Finally, these approaches improved the GA production by 17-fold to 5.52 g/L at 65 h. This study represents a significant step toward the industrial application of GA biosynthetic pathways in E. coli.

Study on the osmotic response and function of myo-inositol oxygenase in euryhaline fish nile tilapia (Oreochromis niloticus).

To understand the role of myo-inositol oxygenase (miox) in the osmotic regulation of Nile tilapia, its expression was analyzed in various tissues. The results showed that the expression of miox gene was highest in the kidney, followed by the liver, and was significantly upregulated in the kidney and liver under 1 h hyperosmotic stress. The relative luminescence efficiency of the miox gene transcription starting site (-4,617 to +312 bp) under hyperosmotic stress was measured. Two fragments (-1,640/-1,619 and -620/-599) could induce the luminescence activity. Moreover, the -1,640/-1,619 and -620/-599 responded to hyperosmotic stress and high-glucose stimulation by base mutation, suggesting that osmotic and carbohydrate response elements may exist in this region. Finally, the salinity tolerance of Nile tilapia was significantly reduced after the knocking down of miox gene. The accumulation of myo-inositol was affected, and the expression of enzymes in glucose metabolism was significantly reduced after the miox gene was knocked down. Furthermore, hyperosmotic stress can cause oxidative stress, and MIOX may help maintain the cell redox balance under hyperosmotic stress. In summary, MIOX is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.NEW & NOTEWORTHY Myo-inositol oxygenase (MIOX) is the rate-limiting enzyme that catalyzes the first step of MI metabolism and determines MI content in aquatic animals. To understand the role of miox in the osmotic regulation of Nile tilapia, we analyzed its expression in different tissues and its function under hyperosmotic stress. This study showed that miox is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.

Biosensor development for single-cell detection of glucuronate.

Recent work in biosensors has shown promise to enable high throughput searches through large genetic libraries. However, just as physiological limitations and lack of in-depth mechanistic knowledge can prevent us from achieving high titers in microbial systems; similar roadblocks can appear in the application of biosensors. Here, we characterized a previously developed transcription-factor (ExuR) based galacturonate biosensor for its other cognate ligand, glucuronate. Though we saw an ideal response to glucuronate from the biosensor in controlled and ideal experimental circumstances, these results began to deviate from a well-behaved system when we explored the application of the sensor to different MIOX homologs. Through modifications to circuit architecture and culture conditions, we were able to decrease this variation and use these more optimal conditions to apply the biosensor for the separation of two closely related MIOX homologs. ONE-SENTENCE SUMMARY: In this work, a transcription-factor biosensor was investigated for its potential to screen a library of myo -inositol oxygenase variants while seeking to mitigate the impact the production pathway appeared to have on the biosensor.

Genome-wide identification of the myo-inositol oxygenase gene family in alfalfa (Medicago sativa L.) and expression analysis under abiotic stress.

Myo-inositol oxygenase (MIOX), a pivotal enzyme in the myo-inositol oxygenation pathway, catalyzes the cleavage of myo-inositol to UDP-glucuronic acid and plays a major role in plant adaptation to abiotic stress factors. However, studies pertaining to the MIOX gene family in alfalfa (Medicago sativa L.) are lacking. Therefore, this study characterized ten MsMIOX genes in the alfalfa genome. These genes were divisible into two classes distributed over three chromosomes and produced 12 pairs of fragment repeats and one pair of tandem repeats. Physicochemical properties, subcellular location, protein structure, conserved motifs, and gene structure pertinent to these MsMIOX genes were analyzed. Construction of a phylogenetic tree revealed that similar gene structures and conserved motifs were present in the same MsMIOX groups. Analysis of cis-acting elements revealed the presence of stress- and hormone-induced expression elements in the promoter regions of the MsMIOX genes. qRT-PCR analysis revealed that MsMIOX genes could be induced by various abiotic stress factors, such as salt, saline-alkali, drought, and cold. Under such conditions, MIOX activity in alfalfa was significantly increased. Heterologous MsMIOX2 expression in yeast enhanced salt, saline-alkali, drought, and cold tolerance. Overexpression of MsMIOX2 in the hairy roots of alfalfa decreased O2- and H2O2 content and enhanced the abiotic stress tolerance. This study offers comprehensive perspectives on the functional features of the MsMIOX family and provides a candidate gene for improving the abiotic stress tolerance of alfalfa.

Wheat derived glucuronokinase as a potential target for regulating ascorbic acid and phytic acid content with increased root length under drought and ABA stresses in Arabidopsis thaliana.

Glucuronokinase (GlcAK) converts glucuronic acid into glucuronic acid-1-phosphate, which is then converted into UDP-glucuronic acid (UDP-GlcA) via myo-inositol oxygenase (MIOX) pathway. UDP-GlcA acts as a precursor in the synthesis of nucleotide-sugar moieties forming cell wall biomass. GlcAK being present at the bifurcation point between UDP-GlcA and ascorbic acid (AsA) biosyntheses, makes it necessary to study its role in plants. In this study, the three homoeologs of GlcAK gene from hexaploid wheat were overexpressed in Arabidopsis thaliana. The GlcAK overexpressing transgenic lines showed decreased contents of AsA and phytic acid (PA) as compared to control plants. Root length and seed germination analyses under abiotic stress (drought and abscisic acid) conditions revealed enhanced root length in transgenic lines as compared to control plants. These results indicate that the MIOX pathway might be contributing towards AsA biosynthesis as evident by the decreased AsA content in the GlcAK overexpressing transgenic Arabidopsis thaliana plants. Findings of the present study will enhance the understanding of the involvement of GlcAK gene in MIOX pathway and subsequent physiological effects in plants.

Inositol in Disease and Development: Roles of Catabolism via myo-Inositol Oxygenase in Drosophila melanogaster.

Inositol depletion has been associated with diabetes and related complications. Increased inositol catabolism, via myo-inositol oxygenase (MIOX), has been implicated in decreased renal function. This study demonstrates that the fruit fly Drosophila melanogaster catabolizes myo-inositol via MIOX. The levels of mRNA encoding MIOX and MIOX specific activity are increased when fruit flies are grown on a diet with inositol as the sole sugar. Inositol as the sole dietary sugar can support D. melanogaster survival, indicating that there is sufficient catabolism for basic energy requirements, allowing for adaptation to various environments. The elimination of MIOX activity, via a piggyBac WH-element inserted into the MIOX gene, results in developmental defects including pupal lethality and pharate flies without proboscises. In contrast, RNAi strains with reduced levels of mRNA encoding MIOX and reduced MIOX specific activity develop to become phenotypically wild-type-appearing adult flies. myo-Inositol levels in larval tissues are highest in the strain with this most extreme loss of myo-inositol catabolism. Larval tissues from the RNAi strains have inositol levels higher than wild-type larval tissues but lower levels than the piggyBac WH-element insertion strain. myo-Inositol supplementation of the diet further increases the myo-inositol levels in the larval tissues of all the strains, without any noticeable effects on development. Obesity and blood (hemolymph) glucose, two hallmarks of diabetes, were reduced in the RNAi strains and further reduced in the piggyBac WH-element insertion strain. Collectively, these data suggest that moderately increased myo-inositol levels do not cause developmental defects and directly correspond to reduced larval obesity and blood (hemolymph) glucose.

[Engineering Saccharomyces cerevisiae for efficient production of glucaric acid].

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.

Modulation of gentamicin-induced acute kidney injury by myo-inositol oxygenase via the ROS/ALOX-12/12-HETE/GPR31 signaling pathway.

In this investigation, a potentially novel signaling pathway in gentamicin-induced acute kidney injury-worsened by overexpression of proximal tubular enzyme, myo-inositol oxygenase (MIOX)-was elucidated. WT, MIOX-transgenic (MIOX-Tg), and MIOX-KO mice were used. Gentamicin was administered to induce tubular injury. MIOX-Tg mice had severe tubular lesions associated with increased serum creatinine and proteinuria. Lesions were relatively mild, with no rise in serum creatinine and no albuminuria in MIOX-KO mice. Transfection of HK-2 cells with MIOX-pcDNA led to increased gentamicin-induced reactive oxygen species (ROS). Marked increase of ROS-mediated lipid hydroperoxidation was noted in MIOX-Tg mice, as assessed by 4-HNE staining. This was associated with increased expression of arachidonate 12-lipoxygenase (ALOX-12) and generation of 12-hydroxyeicosatetraenoic acid (12-HETE). In addition, notable monocyte/macrophage influx, upregulation of NF-κB and inflammatory cytokines, and apoptosis was observed in MIOX-Tg mice. Treatment of cells with ALOX-12 siRNA abolished gentamicin-mediated induction of cytokines and 12-HETE generation. HETE-12 treatment promoted this effect, along with upregulation of various signaling kinases and activation of GPCR31. Similarly, treatment of cells or mice with the ALOX-12 inhibitor ML355 attenuated inflammatory response, kinase signaling cascade, and albuminuria. Collectively, these studies highlight a potentially novel mechanism (i.e., the ROS/ALOX-12/12-HETE/GPR31 signaling axis) relevant to gentamicin-induced nephrotoxicity modulated by MIOX.

Potential of engineering the myo-inositol oxidation pathway to increase stress resilience in plants.

Myo-inositol is one of the most abundant form of inositol. The myo-inositol (MI) serves as substrate to diverse biosynthesis pathways and hence it is conserved across life forms. The biosynthesis of MI is well studied in animals. Beyond biosynthesis pathway, implications of MI pathway and enzymes hold potential implications in plant physiology and crop improvement. Myo-inositol oxygenase (MIOX) enzyme catabolize MI into D-glucuronic acid (D-GlcUA). The MIOX enzyme family is well studied across few plants. More recently, the MI associated pathway's crosstalk with other important biosynthesis and stress responsive pathways in plants has drawn attention. The overall outcome from different plant species studied so far are very suggestive that MI derivatives and associated pathways could open new directions to explore stress responsive novel metabolic networks. There are evidences for upregulation of MI metabolic pathway genes, specially MIOX under different stress condition. We also found MIOX genes getting differentially expressed according to developmental and stress signals in Arabidopsis and wheat. In this review we try to highlight the missing links and put forward a tailored view over myo-inositol oxidation pathway and MIOX proteins.

Myo-inositol oxygenase overexpression exacerbates cadmium-induced kidney injury via oxidant stress and necroptosis.

Conceivably, like other forms of acute kidney injury, cadmium-induced renal injury may also be associated with oxidative stress and various forms of cell death, including necroptosis, a form of regulated necrosis-associated cell death. Myo-inositol oxygenase (MIOX), an enzyme localized in renal proximal tubules, regulates oxidative stress and programmed cell death in various forms of renal injuries. Herein, the role and potential mechanism(s) by which MIOX potentiates cadmium-induced renal tubular damage were investigated. Overexpression of MIOX exacerbated cadmium-induced cell death and proximal tubular injury in mice, whereas MIOX gene disruption attenuated cellular damage in vitro and in vivo. Furthermore, necroptosis was observed in the renal tubular compartment, and, more importantly, it was corroborated by inhibitor experiments with necrostatin-1 (Nec-1). Coadministration of Nec-1 dampened including receptor-interacting protein kinase (RIP)1/RIP3/mixed-lineage kinase domain-like signaling, which is relevant to the process of necroptosis. Interestingly, the necroptosis induced by cadmium in tubules was modulated by MIOX expression profile. Also, the increased reactive oxygen species generation and NADPH consumption were accelerated by MIOX overexpression, and they were mitigated by Nec-1 administration. These findings suggest that MIOX-potentiated redox injury and necroptosis are intricately involved in the pathogenesis of cadmium-induced nephropathy, and this may yield novel potential therapeutic targets for amelioration of cadmium-induced kidney injury.NEW & NOTEWORTHY This is a seminal article documenting the role of myo-inositol oxygenase (MIOX), a renal proximal tubule-specific enzyme, in the exacerbation of cadmium-induced acute kidney injury by perturbing redox balance and inducing necroptosis. MIOX gene disruption or administration of necrostatin-1 (a necroptosis inhibitor) diminished cadmium-induced renal damage, in both in vitro and in vivo systems, suggesting a therapeutic potential of MIOX to attenuate necroptosis and relevant signaling pathways in cadmium-induced renal injury.

Ferroptosis-related gene model to predict overall survival of papillary thyroid carcinoma.

BACKGROUND: Ferroptosis is a form of programmed cell death that is closely associated with the development of various tumors. However, the correlation between ferroptosis and papillary thyroid carcinoma (PTC) is unclear. This study was performed to investigate the expression and prognostic value of ferroptosis-related genes (FRG) in PTC. METHODS: mRNA expression profiles and corresponding clinical data of patients with PTC were analyzed to identify factors affecting prognosis. Independent risk factors were used to establish a predictive receiver operating characteristic model. Single-sample gene set enrichment analysis (ssGSEA) was used to evaluate the correlation between ferroptosis and immune cells. RESULTS: Most genes related to FRG (78.8%) were differentially expressed between the tumor and adjacent normal tissues. In univariate Cox regression analysis, 12 differentially expressed genes were associated with prognostic survival. We constructed a prognostic model of eight FRG, including DPP4, GPX4, GSS, ISCU, MIOX, PGD, TF, and TFRC, and divided patients into two groups: high and low risk. The high-risk group exhibited a significantly reduced overall survival rate. In multivariate Cox regression analysis, the risk score was used as an independent prognostic factor. ssGSEA showed that immune cell types and their expression in the high- and low-risk groups were significant. CONCLUSION: This study constructed a prognostic model of ferroptosis-related genes and determined its usefulness as an independent prognostic factor, providing a reference for the treatment and prognosis of patients with PTC.

Identification of a glycolysis-related gene signature associated with clinical outcome for patients with lung squamous cell carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC), one of the main types of lung cancer, has caused a huge social burden. There has been no significant progress in its therapy in recent years, Resulting in a poor prognosis. This study aims to develop a glycolysis-related gene signature to predict patients' survival with LUSC and explore new therapeutic targets. METHODS: We obtained the mRNA expression and clinical information of 550 patients with LUSC from the Cancer Genome Atlas (TCGA) database. Glycolysis genes were identified by Gene Set Enrichment Analysis (GSEA). The glycolysis-related gene signature was established using the Cox regression analysis. RESULTS: We developed five glycolysis-related genes signature (HKDC1, AGL, ALDH7A1, SLC16A3, and MIOX) to calculate each patient's risk score. According to the risk score, patients were divided into high- and low-risk groups and exhibited significant differences in overall survival (OS) between the two groups. The ROC curves showed that the AUC was 0.707 for the training cohort and 0.651 for the validation cohort. Additionally, the risk score was confirmed as an independent risk factor for LUSC patients by Cox regression analysis. CONCLUSION: We built a gene signature to clarify the connection between glycolysis and LUSC. This model performs well in evaluating patients' survival with LUSC and provides new biomarkers for targeted therapy.

Effect of magnesium ions on glucaric acid production in the engineered Saccharomyces cerevisiae.

Glucaric acid has been successfully produced in Escherichia coli and fungus. Here, we first analyzed the effects of different metal ions on glucaric acid production in the engineered Saccharomyces cerevisiae Bga-3 strain harboring the glucaric acid synthesis pathway. We found that magnesium ions could promote the growth rate of yeast cells, and thus, increase the glucaric acid production by elevating the glucose and myo-inositol utilization of Bga-3 strain. RNA-Seq transcriptome analysis results showed that the upregulation of genes involved in the gluconeogenesis pathway, as well as the downregulation of genes associated with the glycolysis pathway and pentose phosphate pathway in response to MgCl2 were all benefit for the enhancement of the glucose-6-phosphate flux, which was the precursor for myo-inositol and glucaric acid. In addition, we found that MgCl2 could also increase the activity of MIOX4, which was also crucial for glucaric acid synthesis. At last, a final glucaric acid titer of 10.6 g/L, the highest reported titer, was achieved in the fed-batch fermentation using a 5-L bioreactor by adding 100 mM MgCl2. Our findings will provide a new way of promoting the production of other chemicals in the engineered yeast cells.

Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae.

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ±â€¯0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.

Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana.

The production of wheat is severely affected by abiotic stresses such as cold, drought, salinity, and high temperature. Although constitutive promoters are frequently used to regulate the expression of alien genes, these may lead to undesirable side-effects in transgenic plants. Therefore, identification and characterization of an inducible promoter that can express transgene only when exposed to stresses are of great importance in the genetic engineering of crop plants. Previous studies have indicated the abiotic stress-responsive behavior of myo-inositol oxygenase (MIOX) gene in different plants. Here, we isolated the MIOX gene promoter from wheat (TaMIOX). The in-silico analysis revealed the presence of various abiotic stress-responsive cis-elements in the promoter region. The TaMIOX promoter was fused with the UidA reporter gene and transformed into Arabidopsis thaliana. The T3 single-copy homozygous lines were analyzed for GUS activity using histochemical and fluorometric assays. Transcript expression of TaMIOX::UidA was significantly up-regulated by heat (five fold), cold (seven fold), and drought (five fold) stresses as compared to transgenic plants grown without stress-induced conditions. The CaMV35S::UidA plants showed very high GUS activity even in normal conditions. In contrast, the TaMIOX::UidA plants showed prominent GUS activity only in stress treatments (cold, heat, and drought), which suggests the inducible behavior of the TaMIOX promoter. The substrate myo-inositol feeding assay of TaMIOX::UidA plants showed lesser GUS activity as compared to plants treated in abiotic stress conditions. Results support that the TaMIOX promoter could be used as a potential candidate for conditional expression of the transgene in abiotic stress conditions.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation.

BACKGROUND: Ascorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear. RESULTS: In this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity. CONCLUSIONS: These results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

Myo-inositol Oxygenase (MIOX) Overexpression Drives the Progression of Renal Tubulointerstitial Injury in Diabetes.

Conceivably, upregulation of myo-inositol oxygenase (MIOX) is associated with altered cellular redox. Its promoter includes oxidant-response elements, and we also discovered binding sites for XBP1, a transcription factor of endoplasmic reticulum (ER) stress response. Previous studies indicate that MIOX's upregulation in acute tubular injury is mediated by oxidant and ER stress. Here, we investigated whether hyperglycemia leads to accentuation of oxidant and ER stress while these boost each other's activities, thereby augmenting tubulointerstitial injury/fibrosis. We generated MIOX-overexpressing transgenic (MIOX-TG) and MIOX knockout (MIOX-KO) mice. A diabetic state was induced by streptozotocin administration. Also, MIOX-KO were crossbred with Ins2 Akita to generate Ins2 Akita/KO mice. MIOX-TG mice had worsening renal functions with kidneys having increased oxidant/ER stress, as reflected by DCF/dihydroethidium staining, perturbed NAD-to-NADH and glutathione-to-glutathione disulfide ratios, increased NOX4 expression, apoptosis and its executionary molecules, accentuation of TGF-ß signaling, Smads and XBP1 nuclear translocation, expression of GRP78 and XBP1 (ER stress markers), and accelerated tubulointerstitial fibrosis. These changes were not seen in MIOX-KO mice. Interestingly, such changes were remarkably reduced in Ins2 Akita/KO mice and, likewise, in vitro experiments with XBP1 siRNA. These findings suggest that MIOX expression accentuates, while its deficiency shields kidneys from, tubulointerstitial injury by dampening oxidant and ER stress, which mutually enhance each other's activity.

Myo-inositol oxygenase expression profile modulates pathogenic ferroptosis in the renal proximal tubule.

Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which were alleviated by ferrostatin-1, a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, WT mice, MIOX-overexpressing transgenic (MIOX-Tg) mice, and MIOX-KO mice were subjected to cisplatin treatment. In comparison with cisplatin-treated WT mice, cisplatin-treated MIOX-Tg mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of cisplatin-induced AKI, is modulated by the expression profile of MIOX.

Production of d-glucuronic acid from myo-inositol using Escherichia coli whole-cell biocatalyst overexpressing a novel myo-inositol oxygenase from Thermothelomyces thermophile.

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.

Myo-inositol oxygenase accentuates renal tubular injury initiated by endoplasmic reticulum stress.

Besides oxidant stress, endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of various metabolic disorders affecting the kidney. These two forms of stresses are not mutually exclusive to each other and may operate by a feedback loop in worsening the cellular injury. To attest to this contention, studies were performed to assess whether in such a setting, there is worsening of tubulointerstitial injury. We employed tunicamycin as a model of ER stress and used tubular cells and mice overexpressing myo-inositol oxygenase (MIOX), an enzyme involved in glycolytic events with excessive generation of ROS. Concomitant treatment of tunicamycin and transfection of cells with MIOX-pcDNA led to a marked generation of ROS, which was reduced by MIOX-siRNA. Likewise, an accentuated expression of ER stress sensors, GRP78, XBP1, and CHOP, was observed, which was reduced with MIOX-siRNA. These sensors were markedly elevated in MIOX-TG mice compared with WT treated with tunicamycin. This was accompanied with marked deterioration of tubular morphology, along with impairment of renal functions. Interestingly, minimal damage and elevation of ER stressors was observed in MIOX-KO mice. Downstream events that were more adversely affected in MIOX-TG mice included accentuated expression of proapoptogenic proteins, proinflammatory cytokines, and extracellular matrix constituents, although expression of these molecules was unaffected in MIOX-KO mice. Also, their tunicamycin-induced accentuated expression in tubular cells was notably reduced with MIOX-siRNA. These studies suggest that the biology of MIOX-induced oxidant stress and tunicamycin-induced ER stress are interlinked, and both of the events may feed into each other to amplify the tubulointerstitial injury.