定量稳定性同位素探针技术及其在微生物生态学研究中的应用

定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率。qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高速等密度梯度离心将被同位素标记的重链核酸与未被标记的轻链核酸进行分离,并对所有组分微生物类群进行绝对定量和测序分析,基于GC含量和未标记处理DNA密度曲线量化参与吸收转化的DNA同位素丰度。本文重点阐述qSIP的技术原理、数据分析流程及其在微生物生态学研究中的应用进展,并对该技术存在的问题进行了分析和展望。     

Quantitative approaches for analysing fluxes through plant metabolic networks using NMR and stable isotope labelling

The quantitative analysis of metabolic networks is a prerequisite for understanding the integration and regulation of plant metabolism and for devising rational approaches for manipulating resource allocation in plants. The analysis of steady state stable isotope labelling experiments using nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful method for determining these fluxes in micro-organisms and its application to heterotrophic plant metabolism is increasing. After an introductory discussion of the well known role of stable isotopes in pathway delineation, the review considers their application to metabolic flux analysis in plants. These applications are divided into two groups – small scale analyses of fluxes through particular pathways and large scale analyses of multiple fluxes through metabolic networks – and the problems caused by the complexity of intermediary metabolism in plants are discussed. It is concluded that metabolic flux analysis provides a powerful method for defining the metabolic phenotype of wild type, mutant and transgenic plants and that its development should be pursued.     

Strategies for metabolic flux analysis in plants using isotope labelling

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.     

How stable isotopes may help to elucidate primary nitrogen metabolism and its interaction with (photo)respiration in C3 leaves

Intense efforts are currently devoted to elucidate the metabolic networks of plants, in which nitrogen assimilation is of particular importance because it is strongly related to plant growth. In addition, at the leaf level, primary nitrogen metabolism interacts with photosynthesis, day respiration, and photorespiration, simply because nitrogen assimilation needs energy, reductant, and carbon skeletons which are provided by these processes. While some recent studies have focused on metabolomics and genomics of plant leaves, the actual metabolic fluxes associated with nitrogen metabolism operating in leaves are not very well known. In the present paper, it is emphasized that (12)C/(13)C and (14)N/(15)N stable isotopes have proved to be useful tools to investigate such metabolic fluxes and isotopic data are reviewed in the light of some recent advances in this area. Although the potential of stable isotopes remains high, it is somewhat limited by our knowledge of some isotope effects associated with enzymatic reactions. Therefore, this paper should be viewed as a call for more fundamental studies on isotope effects by plant enzymes.     

Metabolic dynamics in skeletal muscle during acute reduction in blood flow and oxygen supply to mitochondria: in-silico studies using a multi-scale, top-down integrated model

Control mechanisms of cellular metabolism and energetics in skeletal muscle that may become evident in response to physiological stresses such as reduction in blood flow and oxygen supply to mitochondria can be quantitatively understood using a multi-scale computational model. The analysis of dynamic responses from such a model can provide insights into mechanisms of metabolic regulation that may not be evident from experimental studies. For the purpose, a physiologically-based, multi-scale computational model of skeletal muscle cellular metabolism and energetics was developed to describe dynamic responses of key chemical species and reaction fluxes to muscle ischemia. The model, which incorporates key transport and metabolic processes and subcellular compartmentalization, is based on dynamic mass balances of 30 chemical species in both capillary blood and tissue cells (cytosol and mitochondria) domains. The reaction fluxes in cytosol and mitochondria are expressed in terms of a general phenomenological Michaelis-Menten equation involving the compartmentalized energy controller ratios ATP/ADP and NADH/NAD(+). The unknown transport and reaction parameters in the model are estimated simultaneously by minimizing the differences between available in vivo experimental data on muscle ischemia and corresponding model outputs in coupled with the resting linear flux balance constraints using a robust, nonlinear, constrained-based, reduced gradient optimization algorithm. With the optimal parameter values, the model is able to simulate dynamic responses to reduced blood flow and oxygen supply to mitochondria associated with muscle ischemia of several key metabolite concentrations and metabolic fluxes in the subcellular cytosolic and mitochondrial compartments, some that can be measured and others that can not be measured with the current experimental techniques. The model can be applied to test complex hypotheses involving dynamic regulation of cellular metabolism and energetics in skeletal muscle during physiological stresses such as ischemia, hypoxia, and exercise.     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

Simultaneous tracers and a unified model of positional and mass isotopomers for quantification of metabolic flux in liver

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of ²H and ¹³C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from ²H and ¹³C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1–C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical by ²H or ¹³C NMR.     

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.     

Metabolic systems maintain stable non‐equilibrium via thermodynamic buffering

Here, we analyze how the set of nucleotides in the cell is equilibrated and how this generates simple rules that help the cell to organize itself via maintenance of a stable non‐equilibrium state. A major mechanism operating to achieve this state is thermodynamic buffering via high activities of equilibrating enzymes such as adenylate kinase. Under stable non‐equilibrium, the ratios of free and Mg‐bound adenylates, Mg²⁺ and membrane potentials are interdependent and can be computed. The adenylate status is balanced with the levels of reduced and oxidized pyridine nucleotides through regulated uncoupling of the pyridine nucleotide pool from ATP production in mitochondria, and through oxidation of substrates non‐coupled to NAD⁺ reduction in peroxisomes. The set of adenylates and pyridine nucleotides constitutes a generalized cell energy status and determines rates of major metabolic fluxes. As the result, fluxes of energy and information become organized spatially and temporally, providing conditions for self‐maintenance of metabolism.     

Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-nitrilotriacetic acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering

1?C labeling experiments performed with kernel cultures showed that developing maize endosperm is more efficient than other non-photosynthetic tissues such as sunflower and maize embryos at converting maternally supplied substrates into biomass. To characterize the metabolic fluxes in endosperm, maize kernels were labeled to isotopic steady state using 13C-labeled glucose. The resultant labeling in free metabolites and biomass was analyzed by NMR and GC-MS. After taking into account the labeling of substrates supplied by the metabolically active cob, the fluxes through central metabolism were quantified by computer-aided modeling. The flux map indicates that 51-69% of the ATP produced is used for biomass synthesis and up to 47% is expended in substrate cycling. These findings point to potential engineering targets for improving yield and increasing oil contents by, respectively, reducing substrate cycling and increasing the commitment of plastidic carbon into fatty acid synthesis at the level of pyruvate kinase.     

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.     

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using ¹³C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO₂ via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.     

Vacuolar compartmentation complicates the steady-state analysis of glucose metabolism and forces reappraisal of sucrose cycling in plants

Steady-state stable isotope labelling provides a method for generating flux maps of the compartmented network of central metabolism in heterotrophic plant tissues. Theoretical analysis of the contribution of the vacuole to the regeneration of glucose by endogenous processes shows that numerical fitting of isotopomeric data will only generate an accurate map of the fluxes involving intracellular glucose if information is available on the labelling of both the cytosolic and vacuolar glucose pools. In the absence of this information many of the calculated fluxes are at best unreliable or at worst indeterminate. This result suggests that the anomalously high rates of sucrose cycling and glucose resynthesis that have been reported in earlier steady-state analyses of tissues labelled with (13)C-glucose precursors may be an artefact of assuming that the labelling pattern of extracted glucose reflected the labelling of the cytosolic pool. The analysis emphasises that although subcellular information can sometimes be deduced from a steady-state analysis without recourse to subcellular fractionation, the success of this procedure depends critically on the structure of the metabolic network. It is concluded that methods need to be implemented that will allow measurement of the subcellular labelling pattern of glucose and other metabolites, as part of the routine analysis of the redistribution of label in steady-state stable isotope labelling experiments, if the true potential of network flux analysis for generating metabolic phenotypes is to be realized.     

Kinetic flux profiling for quantitation of cellular metabolic fluxes

This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatography-mass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t(1/2)相似文献