Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport

d-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce d-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in d-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941 bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in d-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85 g/L of d-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli.     

Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.     

高效合成葡萄糖二酸酿酒酵母工程菌的构建

葡萄糖二酸是天然存在的一种重要二元酸,其在医疗保健和化工工业等领域具有很高的实际应用价值,因此被称为"最具价值的生物炼制产品之一".以酿酒酵母(Saccharomyces cerevisiae)为底盘微生物,文中考察了过量表达肌醇转运蛋白Itr1、融合表达肌醇加氧酶和葡萄糖醛酸脱氢酶以及弱化表达葡萄糖6-磷酸脱氢酶基因...     

Production of glucaric acid from myo-inositol in engineered Pichia pastoris

A potential myo-inositol oxygenase (ppMIOX) was identified as a functional enzyme and a glucaric acid synthetic pathway was firstly constructed in Pichia pastoris. Coexpression of the native ppMIOX and the urinate dehydrogenase (Udh) from Pseudomonas putida KT2440 led to obvious accumulation of glucaric acid (90.46 ± 0.04 mg/L) from myo-inositol whereas no glucaric acid was detected from glucose. In comparison, coexpression of the heterologous mouse MIOX (mMIOX) and Udh resulted in higher titers of glucaric acid from glucose and myo-inositol, 107.19 ± 11.91 mg/L and 785.4 ± 1.41 mg/L, respectively. By applying a fusion expression strategy with flexible peptides, the mMIOX specific activity and the glucaric acid concentration were significantly increased. Using glucose and myo-inositol as carbon substrates, the production of glucaric acid was substantially enhanced to 6.61 ± 0.30 g/L in fed-batch cultures. To the best of our knowledge, this is the highest reported value to date.     

myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience: A NEW MECHANISM RELEVANT TO THE PATHOGENESIS OF DIABETIC NEPHROPATHY*

Diabetic nephropathy (DN) is characterized by perturbations in metabolic/cellular signaling pathways with generation of reactive oxygen species (ROS). The ROS are regarded as a common denominator of various pathways, and they inflict injury on renal glomerular cells. Recent studies indicate that tubular pathobiology also plays a role in the progression of DN. However, the mechanism(s) for how high (25 mm) glucose (HG) ambience induces tubular damage remains enigmatic. myo-Inositol oxygenase (MIOX) is a tubular enzyme that catabolizes myo-inositol to d-glucuronate via the glucuronate-xylulose (G-X) pathway. In this study, we demonstrated that G-X pathway enzymes are expressed in the kidney, and MIOX expression/bioactivity was up-regulated under HG ambience in LLC-PK1 cells, a tubular cell line. We further investigated whether MIOX overexpression leads to accentuation of tubulo-interstitial injury, as gauged by some of the parameters relevant to the progression of DN. Under HG ambience, MIOX overexpression accentuated redox imbalance, perturbed NAD⁺/NADH ratios, increased ROS generation, depleted reduced glutathione, reduced GSH/GSSG ratio, and enhanced adaptive changes in the profile of the antioxidant defense system. These changes were also accompanied by mitochondrial dysfunctions, DNA damage and induction of apoptosis, accentuated activity of profibrogenic cytokine, and expression of fibronectin, the latter two being the major hallmarks of DN. These perturbations were largely blocked by various ROS inhibitors (Mito Q, diphenyleneiodonium chloride, and N-acetylcysteine) and MIOX/NOX4 siRNA. In conclusion, this study highlights a novel mechanism where MIOX under HG ambience exacerbates renal injury during the progression of diabetic nephropathy following the generation of excessive ROS via an unexplored G-X pathway.     

Down-regulation of the <Emphasis Type="Italic">myo</Emphasis>-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis

The enzyme myo-inositol oxygenase (MIOX; E.C. 1.13.99.1) catalyzes the ring-opening four-electron oxidation of myo-inositol into glucuronic acid, which is subsequently activated to UDP-glucuronic acid (UDP-GlcA) and serves as a precursor for plant cell wall polysaccharides. Starting from single T-DNA insertion lines in different MIOX-genes a quadruple knockdown (miox1/2/4/5-mutant) was obtained by crossing, which exhibits greater than 90% down-regulation of all four functional MIOX genes. Miox1/2/4/5-mutant shows no visible phenotype and produces viable pollen. The alternative pathway to UDP-glucuronic acid via UDP-glucose is upregulated in the miox1/2/4/5-mutant as a compensatory mechanism. Miox1/2/4/5-mutant is impaired in the utilization of myo-inositol for seedling growth. The incorporation of myo-inositol derived sugars into cell walls is strongly (>90%) inhibited. Instead, myo-inositol and metabolites produced from myo-inositol such as galactinol accumulate in the miox1/2/4/5-mutant. The increase in galactinol and raffinose family oligosaccharides does not enhance stress tolerance. The ascorbic acid levels are the same in mutant and wild type plants.     

Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat

The myo-inositol level of plasma was determined during pre- and postnatal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly 10-fold. Immediately after birth, the myo-inositol level decreased but was maintained at values 3–4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 mM.The biosynthesis of myo-inositol from glucose 6-phosphate is catalyzed by glucose 6-phosphate:l-myo-inositol-1-phosphate cyclase and l-myo-inositol-1-phosphate phosphatase. The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that the fetal liver accounted for over 48% of the total carcass cyclase and 26% of the total carcass phosphatase activity. Developmental changes correlated well with the pattern of myo-inositol in fetal rat plasma. Similarly, the enzymes of the myo-inositol biosynthetic pathway increased in rat mammary gland in close agreement with the myo-inositol content of milk and diminished to prelactation activities within 24 hr after the onset of involution.The myo-inositol level of colostrum and milk of five human subjects was highest (2.8 mM) before birth and decreased to 40% of that level 5 days postpartum, where it remained for at least 3 weeks. Even after 7 months of lactation, the milk of one subject contained 3–4-fold more myo-inositol than all commercial infant formulas analyzed.     

Manipulation of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid biosynthesis pathways in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>: elevated GDP-mannose pyrophosphorylase activity enhances <Emphasis Type="SmallCaps">l</Emphasis>-ascorbate levels in red fruit

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff–Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.     

Enzymatic assay of d-glucuronate using uronate dehydrogenase

d-Glucuronate is a key metabolite in the process of detoxification of xenobiotics and in a recently constructed synthetic pathway to produce d-glucaric acid, a “top value-added chemical” from biomass. A simple and specific assay of d-glucuronate would be useful for studying these processes, but existing assays are either time-consuming or nonspecific. Using uronate dehydrogenase cloned from Agrobacterium tumefaciens, we developed an assay for d-glucuronate with a detection limit of 5 μM. This method was shown to be more suitable for a system with many interfering compounds than previous methods and was also applied to assays for myo-inositol oxygenase activity.     

Dietary phytase and myo-inositol supplementation are associated with distinct plasma metabolome profile in broiler chickens

Phytase enzyme is used as a dietary supplement in broiler nutrition to improve phosphorous bioavailability. Phytase deliberates phosphate groups from phytic acid and produces myo-inositol after total dephosphorylation. Myo-inositol is a bioactive compound having beneficial modulatory effects on metabolism in humans. However, it is not well understood if and how phytic acid degradation products, particularly myo-inositol, can modulate metabolism in broiler chicken. The purpose of this study was to investigate effects of dietary supplements of phytase and myo-inositol on the blood plasma metabolome profile of broiler chickens. Broilers were provided a nutrient-adequate control diet or the same diet supplemented with either 3.5 g myo-inositol or 500, 1500 or 3000 units of phytase, per kilogram of feed (grower diet). Broilers were group-housed in floor pens (eight pens per diet) and provided one of the treatment diets for 22 days. Then, blood was collected from one bird per pen, resulting in eight replicated measurements per diet. A targeted metabolomics approach was applied to the heparin plasma. Body weight of the birds was not significantly affected by the treatments. Plasma myo-inositol concentrations were significantly increased by myo-inositol supplementation and phytase supplementation at 500 and 1500 units/kg. Metabolites generally affected by phytase supplementation belonged to the groups of acyl-carnitines, phosphatidylcholines, sphingomyelins, lysophosphatidylcholine, biogenic amines and amino acids. Compared to the control diet, phytase supplements had significantly higher plasma concentrations of kynurenine and creatinine, but lower concentrations of histamine and cis-4-hydroxyproline. Myo-inositol supplementation significantly increased plasma concentrations of dopamine and serotonine. While some metabolites were similarly affected by myo-inositol and phytase supplementation, others were distinctly differently affected. We conclude that myo-inositol, either as a directly added supplement or indirectly released from phytate upon phytase supplementation, can affect specific metabolic pathways. Additional effects found on phytase supplementation may be related to intermediary phytate degradation products. Results are indicative for innovative hypothesis to be tested in future experiments, for instance, with regard to relationships between phytase or myo-inositol supplements and bird immunity or behaviour.     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

UDP-glucose: Indoleacetic acid glucosyl transferase and indoleacetyl-glucose: Myo-inositol indoleacetyl transferase

We have demonstrated the in vitro enzymatic synthesis of an ester of indole-3-acetic acid (IAA) and glucose and of IAA and myo-inositol by the following reaction sequence: lt]o| li]1) IAA + UDPG ? IAA-glucose +UDP li]2) IAA-glucose +myo-inositol → IAA-itmyo-inositol +glucose The enzymes were partially purified from extracts of immature kernels of Zea mays sweet corn and the two activities separated on a Sephadex G-150 column. Products were characterized, primarily, by comparison of their 70 eV mass spectra with those of authentic synthetic standards. To our knowledge this is the first example of enzymatically catalyzed acylation by a 1-O-acylsugar.     

Myo-inositol oxygenase from oat seedlings

Summary Enzyme preparations from oat seedlings showing the activity ofmyo-inositol oxygenase (E.C.1.13.99.1) have been described previously. In contrast tomyo-inositol oxygenase preparations from other sources, e.g. rat kidney or yeast, the oat enzyme seemed to exhibit a somewhat less stringent activity, acting on other inositols and inositol methyl ethers as well as onmyo-inositol.By purification of the enzyme present in the extract from oat seedlings with the help of an affinity gel specific for enzymes acting onmyo-inositol a homogeneous enzyme preparation was obtained, which shows the same strict specificity as themyo-inositol oxygenase from other sources. It has a molecular weight of 62,000 and tends to aggregate to oligomers (up to tetramers) under physiological pH-values; in more alkaline media dissociation to monomers is observed. The action on the other inositols and inositol methyl ethers is apparently due to one or more other enzymes, which are also adsorbed on the affinity gel, but can be separated from themyo-inositol oxygenase by elution with increasing concentrations ofmyo-inositol.Dedicated to Professor Karl KRATZL on the occasion of his 60th birthday.     

The enzymes involved in the synthesis of phytic acid in Lemna gibba (Studies on the biosynthesis of cyclitols,XL.)

Summary The biosynthesis of phytic acid is known to be catalyzed by enzymes causing a stepwise phosphorylation of myo-inositol or 1l-myo-inositol 1-phosphate with adenosine triphosphate as phosphate donor. The kinases responsible for these phosphorylations in Lemna gibba were purified by affinity chromatography on a Sepharose gel carrying myo-inositol 2-phosphate at the binding site. Three fractions with enzymatic activity could be identified; in the first one, we find myo-inositol kinase (EC 2.7.1.64) phosphorylating myo-inositol to 1l-myo-inositol 1-phosphate; the second one brings about the phosphorylation of myo-inositol trisphosphate to phytic acid; the third one phosphorylates myo-inositol 1-phosphate to a myo-inositol trisphosphate. An enzyme oxidizing 1l-myo-inositol 1-phosphate to an uronic acid derivative is found in the first two fractions. In the presence of ATP, Mg²⁺ Mn²⁺, and the second and the third enzyme fractions in an appropriate mixture, 1l-myo-inositol 1-phosphate can be phosphorylated to phytic acid. The structure of the trisphosphate acting as an intermediate is not yet known.     

In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.     

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.     

The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis

The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-bisphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4].In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively, in the deficient cell, whereas those of aldolase and citrate synthase decreased by 65 and 27%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities.The thermostability of phosphofructokinase (at 53°C) increased, while that of aldolase (at 48°C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52°C) was also unchanged.