Design and engineering of E. coli metabolic sensor strains with a wide sensitivity range for glycerate.

Microbial biosensors are used to detect the presence of compounds provided externally or produced internally. The latter case is commonly constrained by the need to screen a large library of enzyme or pathway variants to identify those that can efficiently generate the desired compound. To address this limitation, we suggest the use of metabolic sensor strains which can grow only if the relevant compound is present and thus replace screening with direct selection. We used a computational platform to design metabolic sensor strains with varying dependencies on a specific compound. Our method systematically explores combinations of gene deletions and identifies how the growth requirement for a compound changes with the media composition. We demonstrate this approach by constructing a set of E. coli glycerate sensor strains. In each of these strains a different set of enzymes is disrupted such that central metabolism is effectively dissected into multiple segments, each requiring a dedicated carbon source. We find an almost perfect match between the predicted and experimental dependence on glycerate and show that the strains can be used to accurately detect glycerate concentrations across two orders of magnitude. Apart from demonstrating the potential application of metabolic sensor strains, our work reveals key phenomena in central metabolism, including spontaneous degradation of central metabolites and the importance of metabolic sinks for balancing small metabolic networks.

Introducing an Experimental Design Approach for Efficient Optimization of Chiral Derivatization Conditions for D- and L-Glyceric Acids.

A sensitive analytical method was developed for individual analyses of D- and L-glyceric acids by chiral derivatization - liquid chromatography-tandem mass spectrometry. To elucidate rapid and efficient optimization for derivatization we newly introduced a concept of design of experiments (DOE). The optimization of major 5 factors in the derivatization could be predicted with only 28 measurements. By applying DOE to optimization, the yields of desired derivatives increased five-fold against before optimization.

Separation of tartronic and glyceric acids by simulated moving bed chromatography.

The SMB unit developed by the Laboratory of Separation and Reaction Engineering (FlexSMB-LSRE®) was used to perform tartronic acid (TTA) and glyceric acid (GCA) separation and to validate the mathematical model in order to determine the optimum operating parameters of an industrial unit. The purity of the raffinate and extract streams in the experiments performed were 80% and 100%, respectively. The TTA and GCA productivities were 79 and 115 kg per liter of adsorbent per day, respectively and only 0.50 cubic meters of desorbent were required per kilogram of products. Under the optimum operating conditions, which were determined through an extensive simulation study based on the mathematical model developed to predict the performance of a real SMB unit, it was possible to achieve a productivity of 86 kg of TTA and 176 kg of GCA per cubic meter of adsorbent per day (considering the typical commercial purity value of 97% for both compounds) with an eluent consumption of 0.30 cubic meters per kilogram of products.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

BACKGROUND: Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. STUDY DESIGN AND METHODS: RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. RESULTS: Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. CONCLUSIONS: Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM.

Spectroscopic approaches to resolving ambiguities of hyper-polarized NMR signals from different reaction cascades.

The influx of exogenous substrates into cellular reaction cascades on the seconds time scale is directly observable by NMR spectroscopy when using nuclear spin polarization enhancement. Conventional NMR assignment spectra for the identification of reaction intermediates are not applicable in these experiments due to the non-equilibrium nature of the nuclear spin polarization enhancement. We show that ambiguities in the intracellular identification of transient reaction intermediates can be resolved by experimental schemes using site-specific isotope labelling, optimised referencing and response to external perturbations.

Microbial resolution of DL-glyceric acid for L-glyceric acid production with newly isolated bacterial strains.

To produce L-glyceric acid (L-GA) from DL-GA, microbial resolution was investigated using newly isolated bacterial strains capable of enantiospecific degradation of D-GA. Strains GA3R and GA72P, identified as Serratia and Pseudomonas species, respectively, exhausted D-GA within 72 h, resulting in production of L-GA with enantiomeric purity ≥89%.

Measurement of enolase activity in cell lysates.

Enolase (EC 4.2.1.11) is a cytosolic metalloenzyme responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate, the second to last step in glycolysis. In mammals, enolase is encoded by three homologous genes. These gene products not only possess distinct biochemical and immunological properties but also show different tissue distribution. Besides its glycolytic function, α-enolase plays a variety of roles in pathophysiological settings including oncogenesis, tumor progression, ischemia, and bacterial infection. The expression levels of α-enolase have been attributed diagnostic and prognostic value in a number of tumors. Furthermore, neuron-specific α-enolase is released into the cerebrospinal fluid as well as in the systemic circulation upon traumatic brain injury and ischemic episodes. Thus, the measurement of the enzymatic activity of enolase is relevant for diverse fields of investigation, including oncometabolism. Here, we described simple and rapid protocols to measure the activity of enolase in lysates from mammalian cells and tissues.

A conversation with Andrew Benson: reflections on the discovery of the Calvin-Benson cycle.

On June 26-27, 2012, one of us (BBB) made a video based on an interview conducted with Andrew A. Benson, Professor Emeritus, Scripps Institution of Oceanography, University of California, San Diego, CA, USA. The video was first shown in a seminar presented by BBB on July 27, 2012 at the Calvin Laboratory, University of California, Berkeley, to mark the departure of the Energy Biosciences Institute to a new building. Here we record the conversation taking place during the interview. The Brancraft Library on the Berkeley campus will house the video's transcript in its oral histories collection, and the video will be housed in its motion picture collection. The video and the transcript have also been posted on You Tube (http://youtu.be/GfQQJ2vR_xE).

Metabolomics of supragingival plaque and oral bacteria.

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

Liquid chromatography/tandem mass spectrometry of glycolytic intermediates: deconvolution of coeluting structural isomers based on unique product ion ratios.

A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample. Quantification is accomplished using isotopically labeled glucose, glucose-6-phosphate, and pyruvate as internal standards. In addition, a method to deconvolute peak areas of coeluting structural isomers based on unique product ion ratios has been developed to allow accurate quantification of the individual isomers 2-phosphoglycerate and 3-phosphoglycerate, as well as glucose-6-phosphate and fructose-6-phosphate. Intrasample precisions for glycolytic intermediate measurements in cell-free extracts using this method vary between 0.9% and 11.8%, averaging 3.5% (RSD). Calibration curves are linear over the range 0.1-100 microg/mL, and detection limits are estimated at 2-49 ng/mL. Spike recoveries in cell extracts vary from 53% to 127% averaging 91%. This method has the potential to demonstrate correlation of glycolytic intermediate flux to microbial production profiles toward acceleration of the bioprocess development cycle.

Differential chemical diagnosis of primary hyperoxaluria type II. Highly sensitive analysis of optical isomers of glyceric acid by GC/MS as diastereoisomeric derivatives.

We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.

Statistical total correlation spectroscopy: an exploratory approach for latent biomarker identification from metabolic 1H NMR data sets.

We describe here the implementation of the statistical total correlation spectroscopy (STOCSY) analysis method for aiding the identification of potential biomarker molecules in metabonomic studies based on NMR spectroscopic data. STOCSY takes advantage of the multicollinearity of the intensity variables in a set of spectra (in this case 1H NMR spectra) to generate a pseudo-two-dimensional NMR spectrum that displays the correlation among the intensities of the various peaks across the whole sample. This method is not limited to the usual connectivities that are deducible from more standard two-dimensional NMR spectroscopic methods, such as TOCSY. Moreover, two or more molecules involved in the same pathway can also present high intermolecular correlations because of biological covariance or can even be anticorrelated. This combination of STOCSY with supervised pattern recognition and particularly orthogonal projection on latent structure-discriminant analysis (O-PLS-DA) offers a new powerful framework for analysis of metabonomic data. In a first step O-PLS-DA extracts the part of NMR spectra related to discrimination. This information is then cross-combined with the STOCSY results to help identify the molecules responsible for the metabolic variation. To illustrate the applicability of the method, it has been applied to 1H NMR spectra of urine from a metabonomic study of a model of insulin resistance based on the administration of a carbohydrate diet to three different mice strains (C57BL/6Oxjr, BALB/cOxjr, and 129S6/SvEvOxjr) in which a series of metabolites of biological importance can be conclusively assigned and identified by use of the STOCSY approach.

Respiratory acclimation in Arabidopsis thaliana leaves at low temperature.

Acclimation of 25 degrees C-grown Arabidopsis thaliana at 5 degrees C resulted in a marked increase of leaf respiration in darkness (Rd) measured at 5 degrees C. Rd was particularly high in leaves developed at 5 degrees C. Leaf respiration (non-photorespiratory intracellular decarboxylation) in the light (Rl) also increased during cold acclimation, but less so than did Rd. The ratio Rd/Pt (Pt - true photosynthesis) was higher in more acclimated or cold-developed leaves, while the ratio Rl/Pt remained unchanged. In cold-acclimated leaves, Rl did not correlate with 3-phosphoglycerate and pyruvate nor with hexose phosphate pools in the cytosol. Rl in A. thaliana leaves was probably not limited by the substrate during cold acclimation. Under the conditions tested, Rd was more sensitive to low temperature stress than Rl.

A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes.

Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.

A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

Sampling tube device for monitoring intracellular metabolite dynamics.

Continuous sampling of microorganisms from a controlled bioreactor with rapid inactivation of metabolism and extraction of metabolites using precooled -40 degrees C perchloric acid solution (35%) was achieved with a sampling tube, thus fixing fast dynamic reactions at a certain position in the tube. After sampling was stopped (200 s) the tube was frozen at -80 degrees C and divided into identical parts and the extracted metabolites were analyzed enzymatically. A high resolution in time was achieved due to the axial dispersion of the metabolites in the sampling tube: The events of 1 s in the cells of the reactor were represented by 15 parts of the sampling tube. Axial dispersion was determined quantitatively with tracer measurements. The performance of the sampling tube device was evaluated with dynamic investigations on glucose-metabolism of Zymomonas mobilis. The dynamics of intracellular glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate concentrations were monitored after adding a glucose pulse to a glucose-limited steady-state culture.

High-performance liquid chromatographic assay for L-glyceric acid in body fluids. Application in primary hyperoxaluria type 2.

We describe a liquid chromatographic technique to determine L-glycerate in body fluids. The method is based on the derivatisation of the L-glycerate by incubation with lactate dehydrogenase and nicotinamide-adenine dinucleotide in the presence of phenylhydrazine. Oxidation of L-glycerate forms beta-hydroxypyruvate which is converted in turn into the related phenylhydrazone. The UV-absorbing derivative is determined using reversed-phase high performance liquid chromatography. The sensitivity was 5 mumol/l and 50 microliters of sample were required. The imprecision relative standard deviation was 4.5% and the recovery was 96.5 +/- 6.8% for L-glycerate in plasma. L-Glycerate concentrations in urine and plasma were less than 5 mumol/l in both normal individuals and patients with glycolic aciduria. In a patient with systemic oxalosis and normal plasma glycolate, plasma L-glyceric acid was 887 mumol/l.

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver.

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.

Thermal synthesis and hydrolysis of polyglyceric acid.

Polyglyceric acid was synthesized by thermal condensation of glyceric acid at 80 degrees in the presence and absence of two mole percent of sulfuric acid catalyst. The acid catalyst accelerated the polymerization over 100-fold and made possible the synthesis of insoluble polymers of both L- and DL-glyceric acid by heating for less than 1 day. Racemization of L-glyceric acid yielded less than 1% D-glyceric acid in condensations carried out at 80 degrees C with catalyst for 1 day and without catalyst for 12 days. The condensation of L-glyceric acid yielded an insoluble polymer much more readily than condensation of DL-glyceric acid. Studies of the hydrolysis of poly-DL-glyceric acid revealed that it was considerably more stable under mild acidic conditions compared to neutral pH. The relationship of this study to the origin of life is discussed.