Rapid evaluation of PHD2 inhibitory activity of natural products based on capillary electrophoresis online stacking strategy.

Prolyl hydroxylase domain 2 (PHD2) is an important enzyme in the human body that perceives changes in oxygen concentration and regulates response in hypoxic environments. Evaluation of PHD2 inhibitory activity of natural products is crucial for drug development of hypoxia related diseases. At present, the detection of low concentration of α-ketoglutaric acid (the substrate of PHD2 enzymatic reaction) requires derivatization reactions or sample pretreatment, which undoubtedly increases the workload of PHD2 inhibitory activity evaluation. In this paper, a direct detection approach of α-ketoglutaric acid was established by using the online stacking strategy of capillary electrophoresis to evaluate the PHD2 inhibitory activity of natural products. Under optimized conditions, detection of a single sample can be achieved within 2 min. By calculation, the intraday precision RSD of the apparent electrophoretic mobility and peak areas of α-ketoglutaric acid are 0.92 % and 0.79 %, respectively, and the interday RSD were 1.27 % and 0.96 % respectively. The recoveries of the present approach were 97.9-105.2 %, and the LOQ and LOD were 2.0 µM and 5.0 µM, respectively. Furthermore, this approach was applied for the evaluation of inhibitory activity of PHD2 for 13 natural products, and PHD2 inhibitory activity of salvianolic acid A was firstly reported. The present work not only realizes evaluation of PHD2 inhibitory activity through direct detection of α-ketoglutaric acid, but also provides technical support for the discovery of potential drug molecules in hypoxia related diseases.

A Stereoselective Strigolactone Biosynthesis Catalyzed by a 2-Oxoglutarate-Dependent Dioxygenase in Sorghum.

Seeds of root parasitic plants, Striga, Orobanche and Phelipanche spp., are induced to germinate by strigolactones (SLs) exudated from host roots. In Striga-resistant cultivars of Sorghum bicolor, the loss-of-function of the Low Germination Stimulant 1 (LGS1) gene changes the major SL from 5-deoxystrigol (5DS) to orobanchol, which has an opposite C-ring stereochemistry. The biosynthetic pathway of 5DS catalyzed by LGS1 has not been fully elucidated. Since other unknown regulators, in addition to LGS1 encoding a sulfotransferase, appear to be necessary for the stereoselective biosynthesis of 5DS, we examined Sobic.005G213500 (Sb3500), encoding a 2-oxoglutarate-dependent dioxygenase, as a candidate regulator, which is co-expressed with LGS1 and located 5'-upstream of LGS1 in the sorghum genome. When LGS1 was expressed with known SL biosynthetic enzyme genes including the cytochrome P450 SbMAX1a in Nicotiana benthamiana leaves, 5DS and its diastereomer 4-deoxyorobanchol (4DO) were produced in approximately equal amounts, while the production of 5DS was significantly larger than that of 4DO when Sb3500 was also co-expressed. We also confirmed the stereoselective 5DS production in an in vitro feeding experiment using synthetic chemicals with recombinant proteins expressed in Escherichia coli and yeast. This finding demonstrates that Sb3500 is a stereoselective regulator in the conversion of the SL precursor carlactone to 5DS, catalyzed by LGS1 and SbMAX1a, providing a detailed understanding of how different SLs are produced to combat parasitic weed infestations.

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.

The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method. The assay uses a self-assembled monolayer presenting a hydrazide group that covalently captures the αKG product of IDH1, where it can then be detected by MALDI-TOF mass spectrometry. Co-capture of an isotopically-labeled αKG internal standard allows the αKG concentration to be quantitated. The assay was used to analyze a series of standard αKG solutions and produced minimal error in measured αKG concentration values. The suitability of the assay for high-throughput analysis was evaluated in a 384-sample biochemical IDH1 screen. Cells expressing IDH1 were lysed and the lysate was applied to the monolayer to capture αKG, which was then quantitated using the SAMDI-MS assay. Cells in which IDH1 expression was reduced by small-interfering RNA exhibited a corresponding decrease in αKG concentration as measured by the assay. Application of the assay toward the high-throughput screening of IDH1 inhibitors or knockdown agents may facilitate the discovery of treatments for GBM.

A genetic toolkit for the analysis of metabolic changes in Drosophila provides new insights into metabolic responses to stress and malignant transformation.

Regulation of the energetic metabolism occurs fundamentally at the cellular level, so analytical strategies must aim to attain single cell resolution to fully embrace its inherent complexity. We have developed methods to utilize a toolset of metabolic FRET sensors for assessing lactate, pyruvate and 2-oxoglutarate levels of Drosophila tissues in vivo by imaging techniques. We show here how the energetic metabolism is altered by hypoxia: While some larval tissues respond to low oxygen levels by executing a metabolic switch towards lactic fermentation, the fat body and salivary glands do not alter their energetic metabolism. Analysis of tumor metabolism revealed that depending on the genetic background, some tumors undergo a lactogenic switch typical of the Warburg effect, while other tumors do not. This toolset allows for developmental and physiologic studies in genetically manipulated Drosophila individuals in vivo.

Production of pyruvic acid from glycerol by Yarrowia lipolytica.

The aim of the study was to screen Yarrowia lipolytica strains for keto acid production and determine optimal conditions for pyruvic acid biosynthesis from glycerol by the best producer. The analyzed parameters were thiamine concentration, medium pH, stirring speed, and substrate concentration. The screening was performed in flask cultures, whereas pyruvic acid production was carried out in 5-L stirred-tank reactor with 2 L of working volume. In total, 24 Y. lipolytica strains were compared for their abilities to produce pyruvic and α-ketoglutaric acids. The total concentration of both acids ranged from 0.1 to 15.03 g/L. Ten strains were selected for keto acid biosynthesis in bioreactor. The Y. lipolytica SKO 6 strain was identified as the best producer of pyruvic acid. In the selected conditions (thiamine concentration 1.5 µg/L, pH 4.0, stirring speed 800 rpm, 150 g/L of glycerol), the strain Y. lipolytica SKO 6 produced 99.3 g/L of pyruvic acid, with process yield of 0.63 g/g and volumetric production rate of 1.18 g/L/h. Higher titer of pyruvic acid was obtained during fed-batch culture with 200 g/L of glycerol, reaching 125.8 g/L from pure glycerol (yield 0.68 g/g) and 124.4 g/L from crude glycerol (yield 0.62 g/g). Results obtained for the strain Y. lipolytica SKO 6 proved the suitability of microbial production of pyruvic acid at industrial scale.

Fast, affordable and eco-friendly enzyme kinetic method for the assay of α-ketoglutaric acid in medical product and sports supplements.

A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d-4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l-glutamic acid using d-phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration. The method showed good linearity (r2 = 0.9994) over an AKG concentration range of 20-160 µM. The limits of detection and quantitation were 4.09 and 13.62 µM respectively. It was simple, inexpensive, accurate and precise, as well as repeatable, and was not interfered with by excipients in the samples. Regarding the environmental friendliness, the method was free from the use of organic solvents or hazardous reagents and required no chemical pre-treatment of samples. The proposed method gave assay results tested in real samples in agreement with the HPLC method and commercial assay kits, therefore being suitable for routine analysis of AKG in quality control laboratories.

Citric Acid Metabolism in Resistant Hypertension: Underlying Mechanisms and Metabolic Prediction of Treatment Response.

Resistant hypertension (RH) affects 9% to 12% of hypertensive adults. Prolonged exposure to suboptimal blood pressure control results in end-organ damage and cardiovascular risk. Spironolactone is the most effective drug for treatment, but not all patients respond and side effects are not negligible. Little is known on the mechanisms responsible for RH. We aimed to identify metabolic alterations in urine. In addition, a potential capacity of metabolites to predict response to spironolactone was investigated. Urine was collected from 29 patients with RH and from a group of 13 subjects with pseudo-RH. For patients, samples were collected before and after spironolactone administration and were classified in responders (n=19) and nonresponders (n=10). Nuclear magnetic resonance was applied to identify altered metabolites and pathways. Metabolites were confirmed by liquid chromatography-mass spectrometry. Citric acid cycle was the pathway most significantly altered (P<0.0001). Metabolic concentrations were quantified and ranged from ng/mL malate to µg/mL citrate. Citrate and oxaloacetate increased in RH versus pseudoresistant. Together with α-ketoglutarate and malate, they were able to discriminate between responders and nonresponders, being the 4 metabolites increased in nonresponders. Combined as a prediction panel, they showed receiver operating characteristiccurve with area under the curve of 0.96. We show that citric acid cycle and deregulation of reactive oxygen species homeostasis control continue its activation after hypertension was developed. A metabolic panel showing alteration before spironolactone treatment and predicting future response of patients is shown. These molecular indicators will contribute optimizing the rate of control of RH patients with spironolactone.

Discovery of a novel calcium-sensitive fluorescent probe for α-ketoglutarate.

α-Ketoglutarate (α-KG), a pivotal metabolite in energy metabolism, has been implicated in nonalcoholic fatty liver disease (NAFLD) and several cancers. It is recently proposed that plasma α-KG is a surrogate biomarker of NAFLD. Here, we report the development of a novel "turn-on" chemosensor for α-KG that contains a coumarin moiety as a fluorophore. Using benzothiazole-coumarin (BTC) as inspiration, we designed a probe for calcium ion recognition that possesses a unique fluorophore compared with previously reported probes for α-KG measurement. This chemosensor is based on the specific Schiff base reaction and the calcium ion recognition property of the widely used calcium indicator BTC. The probe was synthesized, and a series of parallel experiments were conducted to optimize the chemical recognition process. Compared to the initial weak fluorescence, a remarkable 7.6-fold enhancement in fluorescence intensity (I/I0 at 495 nm) was observed for the conditions in which the probe (1 µmol/L), α-KG (50 µmol/L), and Ca2+ (100 µmol/L) were incubated at 30 °C in EtOH. The probe displayed good selectivity for α-KG even in an environment with an abundance of amino acids and other interfering species such as glutaric acid. We determined that the quantitative detection range of α-KG in EtOH was between 5 and 50 µmol/L. Finally, probe in serum loaded with α-KG (10 mmol/L) showed a 7.4-fold fluorescence enhancement. In summary, a novel probe for detecting the biomarker α-KG through a typical Schiff base reaction has been discovered. With further optimization, this probe may be a good alternative for detecting the physiological metabolite α-KG.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Oncometabolites d- and l-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions.

Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.

Co-expression of L-glutamate oxidase and catalase in Escherichia coli to produce α-ketoglutaric acid by whole-cell biocatalyst.

OBJECTIVES: To improve the production of α-ketoglutaric acid (α-KG) from L-glutamate by whole-cell biocatalysis. RESULTS: A novel and highly active L-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg-1 at pH 6.0, 35 oC. The apparent Km and Vmax values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg-1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H2O2, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l-1 with a conversion rate from L-glutamate of 98.5% after 12 h. CONCLUSIONS: An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

Comparative genomics analysis of a series of Yarrowia lipolytica WSH-Z06 mutants with varied capacity for α-ketoglutarate production.

Yarrowia lipolytica is one of the most intensively investigated α-ketoglutaric acid (α-KG) producers, and metabolic engineering has proven effective for enhancing production. However, regulation of α-KG metabolism remains poorly understood. Genetic engineering of new strains is accompanied by potential safety concerns in some countries and regions. A series of mutants with varied capacity for α-KG production were obtained using random mutagenesis of Y. lipolytica WSH-Z06. Comparative genomics analysis was implemented to identify genes candidates associated with α-KG production. Manipulation of genes regulating mitochondrial biogenesis and energy metabolism could improve α-KG production, while genes involved in regulating transformation between keto acids and amino acids may decrease production. One gene associated with cell cycle control well represented in all mutants, whereas this gene involved in cell concentration do not appear to influence α-KG production. The results shed light on α-KG production in eukaryotic cells, and pave the way for a high-throughput screening and random mutagenesis method for enhancing α-KG production.

Dietary alpha-ketoglutarate increases cold tolerance in Drosophila melanogaster and enhances protein pool and antioxidant defense in sex-specific manner.

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5-10mM) recovered faster from chill coma (0°C for 15min or 3h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2h recovery from continuous cold exposure (-1°C for 3-31h) (LTi50) was higher for flies reared on 10mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

α-ketoglutarate is associated with delayed wound healing in diabetes.

AIM: A high level of matrix metalloproteinase 9 (MMP-9) is a predictor of poor wound healing in diabetic foot ulcers. In skin keratinocytes, site-specific DNA demethylation plays an important role in MMP-9 expression. Ten-eleven translocation enzyme 2 (TET2) protein, one member of TET family, could rely on α-ketoglutarate (α-KG) as cosubstrate to exhibit catalytic activity of DNA demethylation. Here, we aimed to explore the changes of α-KG and its relationship with MMP-9 and TET2 during diabetic wound healing. METHODS: Seventy-one cases of patients with diabetic foot ulcers and 53 cases of nondiabetic ulcers were enrolled. Serum, urine and wound fluids were collected for measurement of α-KG levels and MMP-9 expression. Skin tissues were collected for the measurement of TET2 and MMP-9 expression. Clinical parameters were collected, and transcutaneous oxygen pressure (TcPO2) levels of feet were detected. RESULTS: The levels of α-KG, TET2 and MMP-9 were significantly increased in diabetic wound compared with nondiabetic wound (P = 0·010, 0·016 and 0·025). There was a significant correlation between a low TcPO2 and a high α-KG level of wound fluids (r = -0·395, P = 0·002). Further analysis showed that α-KG concentration had a positive correlation with both haemoglobin A1c (HbA1C) and 2 h postprandial blood glucose (PBG) (r = 0·393, P = 0·005; r = 0·320, P = 0·025, respectively). CONCLUSIONS: The levels of α-KG, TET2 and MMP-9 were significantly increased in diabetic wound compared with nondiabetic wound. Elevated α-KG was related to local hypoxia ischaemia status and systematic poor glycaemic control.

HPLC determination of α-ketoglutaramate [5-amino-2,5-dioxopentanoate] in biological samples.

α-Ketoglutaramate is an important glutamine metabolite in mammals, plants, and many bacteria. It is also a nicotine metabolite in certain bacteria. Previously published methods for the determination of α-ketoglutaramate in biological samples have considerable drawbacks. Here, we describe a relatively simple high-performance liquid chromatography (HPLC)-based method for measurement of α-ketoglutaramate in plasma and deproteinized tissues that overcomes these drawbacks. Concentrations of α-ketoglutaramate in normal rat liver, kidney, brain, and plasma were found to be approximately 216, 13, 6, and 19 µM, respectively. The HPLC method should be useful for studying the role of α-ketoglutaramate in eukaryotic glutamine metabolism and in bacterial nicotine metabolism.

Metabolic phenotyping of an adoptive transfer mouse model of experimental colitis and impact of dietary fish oil intake.

Inflammatory bowel diseases are acute and chronic disabling inflammatory disorders with multiple complex etiologies that are not well-defined. Chronic intestinal inflammation has been linked to an energy-deficient state of gut epithelium with alterations in oxidative metabolism. Plasma-, urine-, stool-, and liver-specific metabonomic analyses are reported in a naïve T cell adoptive transfer (AT) experimental model of colitis, which evaluated the impact of long-chain n-3 polyunsaturated fatty acid (PUFA)-enriched diet. Metabolic profiles of AT animals and their controls under chow diet or fish oil supplementation were compared to describe the (i) consequences of inflammatory processes and (ii) the differential impact of n-3 fatty acids. Inflammation was associated with higher glycoprotein levels (related to acute-phase response) and remodeling of PUFAs. Low triglyceride levels and enhanced PUFA levels in the liver suggest activation of lipolytic pathways that could lead to the observed increase of phospholipids in the liver (including plasmalogens and sphingomyelins). In parallel, the increase in stool excretion of most amino acids may indicate a protein-losing enteropathy. Fecal content of glutamine was lower in AT mice, a feature exacerbated under fish oil intervention that may reflect a functional relationship between intestinal inflammatory status and glutamine metabolism. The decrease in Krebs cycle intermediates in urine (succinate, α-ketoglutarate) also suggests a reduction in the glutaminolytic pathway at a systemic level. Our data indicate that inflammatory status is related to this overall loss of energy homeostasis.

A rapid, one step preparation for measuring selected free plus SO2-bound wine carbonyls by HPLC-DAD/MS.

Carbonyl compounds are produced during fermentation and chemical oxidation during wine making and aging, and they are important to wine flavor and color stability. Since wine also contains these compounds as α-hydroxysulfonates as a result of their reaction with sulfur dioxide, an alkaline pre-treatment requiring oxygen exclusion has been used to release these bound carbonyls for analysis. By modifying the method to hydrolyze the hydroxysulfonates with heating and acid in the presence of 2,4-dinitrophenylhydrazine (DNPH), the carbonyl compounds are simultaneously and quickly released and derivatized, resulting in a simpler and more rapid method. In addition, the method avoids air exclusion complications during hydrolysis by the addition of sulfur dioxide. The method was optimized for temperature, reaction time, and the concentrations of DNPH, sulfur dioxide and acid. The hydrazones were shown to be stable for 10 h, adequate time for chromatographic analysis by HPLC-DAD/MS. This method is demonstrated for 2-ketoglutaric acid, pyruvic acid, acetoin and acetaldehyde, wine carbonyls of very different reactivities, and it offers good specificity, high recovery and low limits of detection. This new rapid, simple method is demonstrated for the measurement of carbonyl compounds in a range of wines of different ages and grape varieties.

Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the α-KG utilization pathway.

We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 µM min(-1) to 56.7 µM min(-1). Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.

A single fluorescent protein-based sensor for in vivo 2-oxogluatarate detection in cell.

2-Oxoglutarate (2OG) is an important currency stands at the crossroad between carbon and nitrogen metabolism. Recent research found that 2OG acts as a signal in the regulation of nitrogen metabolism in prokaryote. While in eukaryotic cells, 2OG is also attractive since tricarboxylic acid cycle (TCA cycle) in tumor cells was found to undergo metabolic alterations such as the Warburg effect. A method of tracing this key metabolite 2OG at the cellular level is highly desirable. In order to visualize and monitor 2-oxoglutarate metabolism in single living cells, we developed a novel sensor by inserting the functional 2OG-binding domain GAF of the NifA protein into YFP. This sensor was found to be highly specific to 2OG. Following binding of 2OG, fluorescence intensity of the sensor increased with increasing 2OG concentration and reached a 1.5-fold maximum fluorescence signal change (F/F0-1), kinetics of fluorescence signal upon 2OG association with sensor was fast, the dynamic response range of the mOGsor sensors was 100 µM-100 mM. Dissociation between sensor and 2OG was verified both in vitro and in vivo. This sensor reported cellular 2OG dynamics in E. coli cells in real time upon different nutrition challenges and manifested the differences in 2OG pool accumulation and consumption rate.