The relationships between the metabolic fluxes and 13C-labeled isotopomer distribution for the flux analysis of the main metabolic pathways

It has been known that ¹³C-labeling technique is quite useful in estimating the metabolic fluxes. Although the program-based flux analysis is powerful, it is not easy to be confident with the result obtained without experiences and exhaustive trial and errors based on statistical analysis for the confidence intervals in practice. It is, therefore, quite important to grasp the relationship between the fluxes and the ¹³C-labeled isotopomer distribution to get deeper insight into the metabolic flux analysis. In the present research, it was shown explicitly how the isotopomer distribution changes with respect to the fluxes in relation to the labeling patterns of the substrate, where either labeled glucose, acetate, or pyruvate was used as a carbon source. Some of the analytical expressions were derived based on the matrix representation, and they were utilized for analysis. It was shown that the isotopomer pattern does not necessarily change uniformly with respect to fluxes, but changes in a complicated way in particular for the case of using pyruvate as a carbon source where some isotopomers do not necessarily change monotonically. It was shown to be quite important to grasp how the isotopomer pattern changes with respect to fluxes and the labeling pattern of the substrate for flux determination and the experimental design. It was also shown that the mixture of [1-¹³C] acetate and [2-¹³C] acetate should not be used from the information index point of view. Some of the experimental data were evaluated from the present approach. It was also shown that the isotopomer distribution is less sensitive to the bidirectional fluxes in the reversible pathway.     

13C NMR spectroscopy of Methanobacterium thermoautotrophicum. Carbon fluxes and primary metabolic pathways

The flux of 13C-labeled carbons from the soluble metabolite 2,3-cyclopyrophosphoglycerate (CPP), a novel compound found in high concentrations exclusively in methanobacteria and methanobrevibacter, into carbohydrate-containing material has been deduced by solid-state 13C NMR spectroscopy which strongly argues for a role in gluconeogenesis for this unique metabolite. The turnover rates, but not the steady-state levels, of CPP labeled by 13CO2 or [13C]acetate depend dramatically on cell growth conditions. When the demand for carbohydrate synthesis is reduced (i.e. in stationary phase), the rates of CPP biosynthesis and degradation decrease 10-fold, and the disaccharide alpha, alpha-trehalose accumulates. Valinomycin, a metabolic inhibitor of Methanobacterium thermoautotrophicum growth, does not affect steady-state levels of CPP, but does decrease 13C uptake into the CPP pool. The effects of these different conditions on CPP labeling suggest stringent regulation of CPP linked to cellular metabolism. Labeling of CPP by [6-(13)C]glucose, which does not serve as an energy or carbon source for this organism, provides strong evidence that glucose is cleaved by the reverse of the gluconeogenesis pathway. This metabolic pathway linking glucose with triose phosphate type precursors and an analysis of the 13C NMR spectrum of CPP labeled by incubating cells with [U-13C]glucose have established that in vivo phosphoenolpyruvate synthetase must be reversible.     

NMR structure analysis of uniformly 13C-labeled carbohydrates

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of ¹³C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly ¹³C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-¹³C)-sucrose, 342 Da] and one compound of medium molecular weight (¹³C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The ¹³C resonances are traced using ¹³C–¹³C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the ¹³C resonances, the ¹H chemical shifts are derived in a straightforward manner using one-bond ¹H–¹³C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the ¹ J _CC splitting of the ¹³C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either ¹³C or ¹H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T₂ relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the ¹H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the ¹³C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with ¹⁵N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and ¹³C-detected (H)CACO spectra.     

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.     

Synthesis and NMR spectra of 13C-labeled coenzyme A esters

The synthesis of coenzyme A thioesters of 13C-labeled acetate, propionate, succinate, and methyl malonate is described. The average yields were 94%. The 13C-NMR spectra were determined to provide a reference for the resonance positions of these metabolites. The synthesis of coenzyme thioesters of small-molecular-weight acids labeled with 13C has not been described previously, nor have the resonance positions been previously reported.     

产琥珀酸放线杆菌的原生质体制备与再生

基因组重组技术是一项重要的菌种改造技术,原生质体制备和再生是进行基因组重组的前提和基础。目前少有关于产琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC2650原生质体研究的报道。为了优化该菌的原生质体制备和再生条件,及利用基因组重组技术构建优良菌种提供参考,研究了甘氨酸预处理,菌龄,酶浓度,作用时间,温度对产琥珀酸放线杆菌原生质体制备和再生的影响,并考察了不同渗透压稳定剂对其再生的影响。结果表明,菌体在添加了0.6mg/ml甘氨酸的TSB培养基中培养5h后收集,用SMM稀释到OD660=1.0,用0.025mg/ml溶菌酶在37℃下酶解45min制备原生质体,将原生质体涂布于含0.3mol/L蔗糖的再生培养基中,再生率最大,达到40.9%。确定了产琥珀酸放线杆菌原生质体制备和再生的最佳条件,所用的原生质体制备的方法对琥珀酸的产生没有影响,这为进一步开展该菌的原生质体诱变及基因组重组等研究奠定了基础。     

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Combining metabolic flux analysis tools and 13C NMR to estimate intracellular fluxes of cultured astrocytes

In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase.     

13C and 1H NMR study of cellulose metabolism by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.     

Effect of shear on morphology,viability and metabolic activity of succinic acid-producing Actinobacillus succinogenes biofilms

Two custom-designed bioreactors were used to evaluate the effect of shear on biofilms of a succinic acid producer, Actinobacillus succinogenes. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. The smooth, low porosity biofilms obtained under high shear conditions had an average cell viability of 79% compared to 57% at the lowest shear used. The maximum cell-based succinic acid productivity for high shear biofilm was 2.4 g g⁻¹DCW h⁻¹ compared to the 0.8 g g⁻¹DCW h⁻¹ of the low shear biofilm. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed higher cell metabolic activities for high shear developed biofilm compared to biofilm developed at low shear conditions. Results clearly indicated that high shear biofilm cultivation has beneficial morphological, viability, and cell-based productivity characteristics.

     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

外源添加代谢中间体对产琥珀酸放线杆菌厌氧发酵制备丁二酸的影响

考察了外源添加中间代谢产物对菌体生长及发酵产酸的影响,结果表明添加0.5g/L磷酸烯醇式丙酮酸(PEP)时丁二酸产量最高。围绕产琥珀酸放线杆菌NJ113厌氧发酵产丁二酸的代谢网络进行代谢通量分析,发现添加PEP后己糖磷酸途径(HMP)与糖酵解途径(EMP)的通量比由39.4∶60.3提高至76.8∶22.6,解决了丁二酸合成过程中还原力不足的矛盾,导致PEP生成草酰乙酸的通量提高了23.8%,丁二酸代谢通量从99.8mmol/(gDCW·h)增至124.4mmol/(gDCW·h),而副产物乙酸及甲酸的代谢通量分别降低了22.9%、15.4%;关键酶活分析结果表明,添加0.5g/LPEP后PEP羧化激酶比酶活达到1910U/mg,与对照相比提高了74.7%,而丙酮酸激酶的比酶活降低了67.5%。最终丁二酸浓度为29.1g/L,收率达到76.2%,比未添加PEP时提高了11.0%。     

琥珀酸放线杆菌发酵培养基的优化

对琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC1593发酵产生琥珀酸培养基的主要成分,及其含量进行优化。通过单因素试验,得出发酵培养基中葡萄糖、酵母膏和玉米浆的含量对产生琥珀酸有显著影响;采用响应面法(RSM),得出多元二次回归方程拟合的三种因素与琥珀酸含量间的函数关系,并根据优化结果与实验,CGMCC1593产琥珀酸达到41.69g/L。     

In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways

Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.     

13C-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO3 and H2 concentrations

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO(2) and H(2) concentrations by producing more succinate and by producing less formate, acetate, and ethanol. To determine how intermediary fluxes in A. succinogenes respond to CO(2) and H(2) perturbations, (13)C-metabolic flux analysis was performed in batch cultures at two different NaHCO(3) concentrations, with and without H(2), using a substrate mixture of [1-(13)C]glucose, [U-(13)C]glucose, and unlabeled NaHCO(3). The resulting amino acid, organic acid, and glycogen isotopomers were analyzed by gas chromatography-mass spectrometry and NMR. In all conditions, exchange flux was observed through malic enzyme and/or oxaloacetate decarboxylase. The presence of an exchange flux between oxaloacetate, malate, and pyruvate indicates that, in addition to phosphoenolpyruvate, oxaloacetate, and malate, pyruvate is a fourth node for flux distribution between succinate and alternative fermentation products. High NaHCO(3) concentrations decreased the amount of flux shunted by C(4)-decarboxylating activities from the succinate-producing C(4) pathway to the formate-, acetate-, and ethanol-producing C(3) pathway. In addition, pyruvate carboxylating flux increased in response to high NaHCO(3) concentrations. C(3)-pathway dehydrogenase fluxes increased or decreased appropriately in response to the different redox demands imposed by the different NaHCO(3) and H(2) concentrations. Overall, these metabolic flux changes allowed A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These results are discussed with respect to A. succinogenes' physiology and to metabolic engineering strategies to increase the flux to succinate.     

Helix-helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy

The rates at which a peptide hexamer and a peptide octamer interconvert between left- and right-handed helical forms in CD2Cl2 solution have been characterized by 13C dynamic NMR (DNMR) spectroscopy. The peptide esters studied are Fmoc-(Aib)n-OtBu (n = 6 and 8), where Fmoc is 9-fluorenylmethyoxycarbonyl and Aib is the strongly helix-forming residue alpha-aminoisobutyric acid. Because the Aib residue is itself achiral, homooligomers of this residue form a 50/50 mixture of enantiomeric 3(10)-helices in solution. It has been demonstrated (R.-P. Hummel, C. Toniolo, and G. Jung, Angewandte Chemie International Edition, 1987, Vol. 26, pp. 1150-1152) that oligomers of Aib interconvert on the millisecond timescale. We have performed lineshape analysis of 13C-NMR spectra collected for our peptides enriched with 13C at a single residue. Rate constants for the octamer range from 6 s(-1) at 196 K to about 56,500 s(-1) at 320 K. At all temperatures, the hexamer interconverts about three times faster than the octamer. Eyring plots of the data reveal experimentally indistinguishable DeltaH++ values for the hexamer and octamer of 37.8 +/- 0.6 and 37.6 +/- 0.4 kJ mol(-1) respectively. The difference in the rates of interconversion is dictated by entropic factors. The hexamer and octamer exhibit negative DeltaS++ values of -29.0(-1) +/- 2.5 and -37.3 +/- 1.7 J K(-1) mol(-1), respectively. A mechanism for the helix-helix interconversion is proposed. and calculated DeltaG++ values are compared to the estimate for a decamer undergoing a helix-helix interconversion.     

Enhancement of succinic acid production by osmotic-tolerant mutant strain of Actinobacillus succinogenes

Fermentation and succinic acid production by Actinobacillus succinogenes YZ0819 was inhibited by high NaCl. To enhance the resistance of this strain to osmotic stress, an NaCl-tolerant mutant strain of A. succinogenes (CH050) was screened and selected through a continuous culture using survival in 0.7 M NaCl as the selection criterion. Using Na₂CO₃ as the pH regulator and glucose as the carbon source in batch fermentation, the isolated osmo-resistant stain, A. succinogenes CH050, produced up to 66 g/l succinic acid with a yield of 73.37% (w/w). The concentration of succinic acid and mass yield were increased by 37.5 and 4.37%, respectively, compared to the parent strain. The dry cell weight reached 10.1 g/l, which is 37% higher than that of the parent strain. The high tolerance of A. succinogenes CH050 to osmotic stress increased improved the succinic acid production from batch fermentation.