CN110196288A - A kind of foundation and application of dynamic Metabolic flux analysis technology - Google Patents
A kind of foundation and application of dynamic Metabolic flux analysis technology Download PDFInfo
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Abstract
The present invention provides the foundation and application of a kind of dynamic Metabolic flux analysis technology, specifically, the present invention provides a kind of dynamic flow metabolic analysis method, the method includes the steps: a) sample making；B) detection of biological sample: the cell extract is analyzed by combined gas chromatography mass spectrometry method, obtains the label ratio of one or more metabolins；C) by one or more metabolite markers ratios, the metabolic flux intracellular of target metabolite is calculated.The method of the present invention has many advantages, such as at low cost, analyzes quick.
Description
Technical field
The present invention relates to molecular biology fields, specifically, the present invention provides a kind of dynamic Metabolic flux analysis technology,
And its application in cell metabolism.
Background technique
Cell metabolism is the basis of required matter and energy of sustaining life, and is specifically completed by metabolism enzymatic intracellular.Metabolism
Flow is the rate for referring to metabolic response intracellular, it can not directly be measured, but passes through combined mathematical module and metabolite
The counted numerical value of isotope labelling information；The method of measurement metabolic flux rate is known as Metabolic flux analysis.Metabolic flux is big
Small is to determine that it can help to parse the machine of cell activities by the interaction synthesis between gene-protein-metabolin
System；Furthermore the parsing of metabolic flux can find potential target spot and provide foundation for the rationality transformation of metabolic engineering.It is metabolized herein
Flow analysis is mutually distinguished with mobile equilibrium analysis.
Traditional Metabolic flux analysis is to utilize cellular uptake13The substrate of C flag and it is intracellular pass through metabolic response pair
One kind that the mark information abundant generated calculates flow intracellular as constraint condition combined mathematical module is reset in carbon skeleton fracture
Method.Due to the feature that metabolic transformation rate intracellular is fast and concentration is low, to the preparation of sample, the separation of metabolin and detector
The sensitivity of device has very high requirement.With13The substrate of C flag passes through long enough as carbon source under conditions of metabolic homeostasis
Time, the biomass such as metabolin and protein intracellular can reach label stable state.Intermediate metabolites intracellular is about in Escherichia coli
The 3% of dry cell weight is accounted for, albumen accounts for about the 50% of dry cell weight.Since the precursor of amino acid is directed to central carbon metabolism simultaneously
And amino acid content in cell protein enriches property and stablizes, and can be calculated by amino acid tag information in detection albumen
The flow of heart carbon metablism, this method are widely used because of the high feature of repeatability easy to operate.
The major advantage of traditional Metabolic flux analysis is that cellular protein content enriches and the property of amino acid is stablized
So that mark information is easily obtained.Disadvantage is that13Long time is cultivated in C flag culture medium can be only achieved egg intracellular
White label stable state, and13C flag substrate is more expensive；It is steady to need to guarantee that cell is in metabolism in prolonged incubation
The condition of state；Can only analytical center carbon metablism and amino acid metabolism approach flow.On the Research foundation of metabolism quasi-stable state, section
Scholars create various conditions and attempt to realize dynamic Metabolic flux analysis.1999, Germany scientist U.Schaefer et al. existed
An entitled Automated Sampling Device for has been delivered on Analytical Biochemistry magazine
The article of Monitoring Intracellular Metabolite Dynamics describes a kind of culture tank combination crawler belt biography
It send the instrument of equipment that automation fast sampling may be implemented, is quenched by the way that thallus to be metabolized in -50 DEG C of 60% methanol, then benefit
Metabolin extraction is carried out to the cell being collected by centrifugation with perchloric acid, sampling frequency can achieve 4.5 seconds every time.This is early stage people
A kind of trial to real-time measurement metabolin intracellular, but methodology equipment cost is high in this way, and when metabolism is quenched have it is thin
The disadvantages of risk of born of the same parents' rupture.Therefore, this field still lacks a kind of low cost, quickly carries out the side of dynamic Metabolic flux analysis
Method.
Summary of the invention
The object of the present invention is to provide a kind of low costs, quickly carry out the method for dynamic Metabolic flux analysis.
The first aspect of the present invention provides a kind of dynamic flow metabolic analysis method, comprising steps of
A) sample making:
(a-1) cell cultivated on filter paper the environment containing isotopic label is transferred to carry out continuing to train
It supports；
(a-2) in sampling time point, the cell on the filter paper is turned over and is buckled into extracting solution, it is mixed to form extracting solution-cell
Close object；
(a-3) cell extract is prepared with the extracting solution-cell mixture；
B) detection of biological sample: the cell extract is analyzed by combined gas chromatography mass spectrometry method, is obtained
The label ratio of one or more metabolins；
C) it by one or more metabolite markers ratios and intracellular concentration, is calculated using Differential Equation Model
To the metabolic flux intracellular of target metabolite.
In another preferred example, before the transfer, the cell is cultivated to nitrogen on filter paper and limits state.
In another preferred example, the isotopic label is Na15NO3。
In another preferred example, this method relies on filter paper culture that being switched fast of cell growing environment, cell may be implemented
Metabolin is quickly quenched and extracts；It can be used for studying the variations such as cell reply external environment such as nutriment, drug-treated
Under the conditions of, regulatory mechanism of the cell on metabolism level.
In another preferred example, this method is parsed with cyanobacteria cytoalgae 6803 in the case where nitrogen limit is switched to nitrogen rich conditions
For arginine route of synthesis metabolic flux intracellular, but it is not limited to the example.
In another preferred example, it is to rely on filter paper culture that the quick of cell growing environment may be implemented the characteristics of this method
Switching, the quick of cell metabolite are quenched and extract.Any side that dynamic Metabolic flux analysis purpose is realized by filter paper culture
Method should all be in claim.
In another preferred example, the method further comprises the steps of: the label for calculating metabolin in organism reaction process
Ratio calculates the flow of target metabolite by metabolic response approach differential equation.
In another preferred example, the target metabolite is citrulling.
In another preferred example, the metabolin includes: citrulling peer, Gln58 segmentSame position
Body, ornithine peer.
In another preferred example, the distribution of the citrulling peer changes with time through following differential equation
Description:
Wherein vOTCIt is the flow by ornithine transcarbamylase, CCITIt is the intracellular concentration of citrulling.
In another preferred example, the label ratio progress further comprised the steps of: before the calculating for metabolin is natural same
The plain abundance correction in position.
In another preferred example, natural isotopic abundance correction includes: the correction of C13 abundance.
In another preferred example, abundance correction includes: and copes in derivating agent when being detected with Gas-phase acidity
Silicon carry out natural isotopic abundance correction.
In another preferred example, C13 abundance correction is carried out by following formula:
In another preferred example, the step (a-1) includes: that will contain Na14NO3In the environment of culture finish
Bacterium solution filters on filter paper, and culture to nitrogen limits state.
In another preferred example, before being transferred to filter medium, the cell is cultivated in BG11 fluid nutrient medium.
In another preferred example, before being transferred to filter medium, the cell is trained in BG11 solid medium agar
It supports.
In another preferred example, the step (a-2) includes: and turns over the filter medium with bacterium colony to be buckled in extraction
In liquid, completes metabolism and terminate, to form extracting solution-cell mixture.
In another preferred example, the extracting solution is the acetonitrile containing formic acid: methanol: water=2:1:1 (v:v:v) mixing is molten
Liquid.
In another preferred example, the cell is selected from the group: cyanobacteria cytoalgae, preferably cyanobacteria cytoalgae
6803。
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
The substrate and product of Fig. 1: N15 label ornithine transcarbamylase；A, the reaction of ornithine transcarbamylase catalysis；
B, the dynamically labeled data of amide nitrogen in glutamine；C, the dynamically labeled data of ornithine；D, the dynamically labeled number of citrulling
According to.
Specific embodiment
The present inventor's in-depth study by long-term provides a kind of completely new dynamic flow metabolic analysis technology.Institute
The technology stated uses filter paper culture combination stable isotope labeling object (such as 13C, 15N, 2H), can in organism including object
Most metabolic pathways including matter metabolism and energy metabolic pathways carry out cell dynamic under conditions of various kinds of cell disturbs
Metabolic flux analysis.This method is at low cost, and intermediate metabolites equilibration time is short, tradition C13 generation existing compared to this field
Thank to flow analysis technology with better applicability.
Dynamic Metabolic flux analysis technology
The real-time detection metabolite markers state intracellular change procedure combination born of the same parents from scratch under conditions of metabolic homeostasis
The concentration data of intracellular metabolite object can calculate metabolic flux intracellular, and this method is known as dynamic Metabolic flux analysis.
Dynamic Metabolic flux analysis has the advantage that unstable state isotope labelling is real relative to traditional Metabolic flux analysis
Testing the required time greatly reduces, this is because the experiment of unstable state isotope labelling directly detects metabolin intracellular rather than albumen
The amino acid in matter source；The biology of one-carbon compound traditional metabolic flux point is utilized for photosynthetic organism, methylotrophic bacteria etc.
Analysis can not apply, and dynamic Metabolic flux analysis can detecte the generation intracellular that metabolite markers degree changes with time and then calculates
Thank to flow；It can analyze other approach other than central carbon metabolism and amino acid pathway.
But dynamic Metabolic flux analysis the production of sample, the detection platform of biological sample, mathematical model processing this 3
A aspect has higher requirement.
A) production of sample: dynamic Metabolic flux analysis requires the flag state of real-time detection intermediate metabolites intracellular, by
It is fast in intermediate metabolites conversion rate intracellular, so needing quickly and effectively to terminate metabolic response and extracting intermediate metabolites: this
Two steps are referred to as the extraction (extraction) that (quenching) and metabolin is quenched of metabolism.
B) detection of biological sample: not only right since intermediate metabolites concentration intracellular is low, type is more, chemical property is various
It extracts and requires height, also have very high requirement to detection.Metabolin intracellular is typically now analyzed with the method for combined gas chromatography mass spectrometry.
C) description metabolite markers degree intracellular, which changes with time, needs to introduce the differential equation, and a large amount of differential equation is to meter
More stringent requirements are proposed for calculation.
Inventor establishes a set of metabolic flux calculation method, including: the 1. method of sample making；2. small molecule generation
Thank to the detection method of object；3. corresponding calculation method.Form a whole set of new method for calculating metabolic flux.
This method is not only suitable for the basic research of research cell reply external environment change mechanism, is also applied for metabolic engineering
The application study of aspect.
Dynamic flow metabolic analysis embodiment of the method for the invention comprising steps of
A) it sample making: is transferred to including being cultivated on filter paper to the cell of nitrogen limit state containing Na15NO3Environment carry out
Continue to cultivate, then in sampling time point, the cell is turned over into button into extracting solution, forms extracting solution-cell mixture, and
Cell extract is prepared with the extracting solution-cell mixture.
In a preferred embodiment, the step includes that will contain Na14NO3In the environment of (such as BG11 training
Support in base) the bacterium solution suction filtration finished is cultivated on filter paper, culture to nitrogen limits state.Then in sampling time point, bacterium will be had
The filter medium fallen is placed in extracting solution, can be readily achieved metabolism and be terminated.Particularly, by filtering cell culture
On paper, fast sampling not only may be implemented, but also can very easily switch the condition of cell growth.
B) detection of biological sample: the cell extract is analyzed by combined gas chromatography mass spectrometry method, is obtained
The concentration and mark information of one or more metabolins；
C) by one or more metabolite concentrations and mark information, mesh is calculated according to Differential Equation Model
Mark the metabolic flux intracellular of metabolin.
Wherein, the metabolin is nitrogenous metabolites, such as amino acid or amino acid fragment.In preferred reality of the invention
It applies in example, by detecting the label ratio of metabolin, corresponding metabolic response approach differential equation is substituted into, to calculate target
The concentration of metabolin.
It is N15 label used in this method, target metabolite can be any nitrogenous biological segment, such as amino acid, phonetic
Pyridine etc. is in an embodiment of the present invention citrulling.In addition to N15 is marked, C13 label, H2 label etc. can also be used.
In an embodiment of the present invention, Biometabolic pathway detected is following interior ornithine transcarbamylase
(OTC) it reacts.In above-mentioned reaction, OTC catalysis carbamyl phosphate synthesizes citrulling with ornithine, and discharges a molecular phosphorus,
Carbamoyl phosphate+L-ornithine=phosphate+L-citrulline.
In the reaction, by combined gas chromatography mass spectrometry method, have detected include amide nitrogen daughter ion fragments
(Gln58), the label ratio of citrulling, ornithine.
More accurate calculated result in order to obtain, before being calculated, can also label ratio for metabolin carry out
Natural isotopic abundance correction.It, can be only right under simplified design conditions since the natural isotopic abundance of H and N are lower
C13 carries out abundance correction.
In the application, the above-mentioned metabolic response of cyanobacteria cytoalgae 6803 is had detected, it should be appreciated that this method can be by
For it is any can be by the metabolic pathway of stable isotope labeling, such as the bird ammonia carried out in other organisms, cell or tissue
Sour transcarbamylase (OTC) reaction.
Existing Metabolic flux analysis needs cell metabolism to be in quasi-stable state.In order to realize dynamic Metabolic flux analysis needs
Intermediate metabolites is directly measured, in order to overcome prepare sample during intermediate metabolites be quickly converted the shortcomings that,
It needs that cell metabolite is quickly quenched and is extracted.The method of existing crawler-tread device combination culture tank have it is at high cost,
The disadvantages of metabolin leakage is quenched in cell.In this regard, the application uses filter paper as culture medium, to improve sample preparation
With the process of processing, allow the concentration of cell metabolism substance quick, be detected in real time, in conjunction with the label of intermediate metabolites
Information can parse the metabolic flux of relational approach.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
1 dynamic flow metabolic analysis technology of embodiment
Reagent and instrument and equipment:
Reagent
● HPLC grades of ultrapure waters
● HPLC grades of acetonitriles
● HPLC grades of methanol
● ammonium hydroxide
● ammonium acetate
● formic acid
● component needed for BG11 culture medium
●Na15NO3
● ultrapure agar powder
● culture dish
● test tube
● shaking flask
● nylon membrane
● 2ml centrifuge tube
● cell scraper
Instrument and equipment
● shaking table
● percival illumination box
● spectrophotometer
● pipettor and 5ml pipette
● tweezers
● centrifuge
● vortex concussion instrument
● combined gas chromatography mass spectrometry equipment
● HPLC sample injection bottle
● -20 DEG C of refrigerators
● steel plate
● vacuum filtration equipment
● Féraud door nh 2 column
Cyanobacteria cytoalgae 6803 is under autotrophic condition, and N metabolic flux especially under disturbed conditions is with traditional 13C generation
Thanking to flow analysis technology can not parse.We are switched to nitrogen from nitrogen limit with parsing cytoalgae 6803 and have ready conditions lower arginine pathway bird
For propylhomoserin transcarbamylase, illustrate the concrete application of dynamic Metabolic flux analysis.
1. Step By Condition:
The production of sample
By 6803 streak inoculation of cytoalgae in glycerol tube on plate, it is placed in 50umol*m2-1*s-1Illumination, 30 degree of trainings
It is cultivated under the conditions of supporting, after confirming no living contaminants, is seeded to the 50ml triangle shake bottle containing 20ml BG11 fluid nutrient medium
In.
When the cyanobacteria OD730 in shaking flask reaches 0.8-1.2, it is transferred to the new fluid nutrient medium of BG11 containing 100ml
In 250ml triangle shake bottle, make its OD730=0.1.
Filter in Suction filtration device, the sterilizing of 5ml pipette is spare.Pour into 20ml BG11 while hot in 10cm culture dish
Solid medium agar, it is spare after its condensation.Configuration extraction liquid: 40% acetonitrile, 40% methanol, 20% moisture are attached to
In 50ml centrifuge tube, 188ul formic acid is added in every pipe.Extracting solution is put into -40 degree pre-coolings.Steel plate is put into -80 degree pre-coolings.
After 12 hours, cyanobacteria grows to 0.2 from OD730=0.1.Pumping and filtering device is assembled, by the bacterium solution of 5ml OD=0.2
The circular filter paper on piece in 9cm is uniformly filtered, and filter paper is seamless applying in the 10cm culture dish containing solid medium, contained
There is the one side of cyanobacteria upward.Culture dish is uniformly put into percival incubator: 50umol*m2-1*s-1Illumination, temperature
30 degree.
Preprepared plate is put into incubator after 45 hours and is preheated.After 48-50 hours, the color of cyanobacteria becomes
Yellow and growth rate is slack-off, and cyanobacteria is in nitrogen limit state at this time.The vacant culture of 10cm is placed on the steel plate of -80 degree pre-coolings
Ware and the extracting solution that 2.5ml pre-cooling is added.
Using tweezers by the related cell of filter paper from 2mM Na14NO3Plate is transferred to 17.6mM Na15NO3And using at this time as
Time zero falls filter paper in 0min, 30s, 1min, 2min, 3min, 5min, 8min, 15min, 30min, 60min, 120min
It is buckled in the culture dish containing extracting solution, completes the termination of metabolism.4 degree of centrifuges are pre-chilled.
Above-mentioned culture dish is put into 30min or so in -20 degree, takes out and is placed on ice.It is incited somebody to action using tweezers, scraper, pipettor
Cell is transferred in 5ml centrifuge tube.Again it draws 1.5ml extracting solution and rinses scraper and filter paper, combined extract.By collection
4ml extracting solution is in 12000rpm, 4 degree of centrifugation 20min.Supernatant is collected into new 5ml centrifuge tube, precipitating is mentioned with new 1ml
Liquid resuspension is taken to be placed in -20 degree 30min.Above-mentioned centrifugal process is repeated, supernatant is merged.
It is spare that extract is stored in -80 degree.
The detection of biological sample
I. column Mobile phase A: ingredient 95%H is prepared2O+5%ACN (20mM NH4OH, 20mM NH4Ac, pH=
9.0)；Mobile phase B: 100%ACN；Mobile phase NH4Ac (100mM, pH 6-7)；Mobile phase: 100% isopropanol.
Ii. Féraud door nh 2 column uses for the first time: HPLC 1. being connect two-way, setting flow velocity is 2ml/min, uses water, second respectively
Nitrile, isopropanol respectively rinse whole system 5min, and solution is into waste.2. pillar is connected, with the stream of 100% isopropanol 0.1ml/min
Fast rinse-system 1h, then with the flow velocity rinse-system 1h of 0.2ml/min, solution is into waste.3. with 100% acetonitrile 0.4ml/min
Flow velocity rinse-system 1h, solution is into waste.4. with the flow velocity rinse-system 40min of+50% water 0.3ml/min of 50% acetonitrile,
Solution is into waste.5. with the flow velocity rinse-system 40min of 50%A+50%B 0.3ml/min, solution is into waste.6. activating
Afterwards, System Testing Pressure is distinguished with 100%A and 100%B.
Iii. Féraud door nh 2 column 80% uses after nitrile saves: HPLC is 1. connect into two-way, setting flow velocity is 2ml/min,
Whole system 5min is respectively rinsed with water, acetonitrile respectively, and solution is into waste.2. connecting pillar, 80%H is first used2O+20%ACN with
The flow velocity of 0.2ml/min rinses 15min.3. using 80%NH again4Ac (100mM, pH 6-7)+20%ACN is with the stream of 0.2ml/min
Speed rinses 30min.4. rinsing 60min with 15%A+85%B with the flow velocity of 0.2ml/min.
Iv. take 300ul extract with 1ml syringe, using 0.22um membrane filtration and be fitted into chromatography column feed materials bottle.Make
It is detected with the mode of LC-MS combined gas chromatography mass spectrometry.
V. combined gas chromatography mass spectrometry detects: 1. liquid phase part uses the UPLC: sampling volume 10ul of waters；Chromatographic column column
15 degree of temperature；Flow velocity 0.3ml/min；Gradient elution program are as follows: 0min, 85%；10min, 45%；15min, 2%；18min, 2%；
18.1min 85%；24min 85%B_ENREF_57.2. mass spectrum (Q is combined using the orbit trap of Thermo in mass spectrum part
Exactive): ion source is the electric spray ion source (source ESI) of heating, is run under holotype or negative mode.Main ginseng
Number: ionization voltage ,+3.8kV/-3.0kV；sheath gas pressure,35arbitrary units；
auxiliary gas,10arbitrary units；auxiliary gas heater temperature,350℃；
capillary temperature,320℃.Mass spectrum acquisition range m/z 70-1000, mass resolution is set as 70,000.
Data analysis
I. the Compound Discoverer 2.0 and 4.0 software of Xcalibur provided with power & light company handles original number
According to.The metabolin that a part identifies uses the accurate molecular weight and retention time confirmation of mark product again.
Ii. the peak area of metabolin is extracted, and calculates the label ratio of metabolin.It needs to correct day when calculating label ratio
Right isotope abundance: H2=0.00015；C13=0.0111；N15=0.0037.The natural isotope indexing of N15 and H2 is smaller
It can not correct, the correction formula of C13 is as follows:
Iii. MATLAB software is utilized, differential equation is write out according to known metabolic response approach, reaction rate is made
For the flag data that the parametric solution differential equation is simulated, the flag data of simulation and the flag data measured are done into least square
It can obtain the size of the reaction rate, that is, the uninterrupted of this step.It can be to avoid according to this local reaction
Metabolic response annotation inaccuracy obtains the result of mistake.
2. expected result:
I. our (Fig. 1 a): the OTC for calculating ornithine transcarbamylase intracellular (OTC) reaction rate are catalyzed carbamyl
Phosphoric acid synthesizes citrulling with ornithine and discharges a molecular phosphorus, carbamoyl phosphate+L-ornithine=
phosphate+L-citrulline.Since carbamyl phosphate chemical property is actively not easy to be detected by LC-MS, and carbamyl phosphate
Nitrogen-atoms derive from glutamine amide nitrogen, so we had detected by the method for MS/MS include amide nitrogen son
Ion fragment (Gln58).It is assumed that metabolic flux quasi-stable state state (flows in and out melon ammonia in entire calculating process
The flow of acid is equal).Peer (Isotopomer) distribution of so citrulling changes with time and can use following differential side
Formula description:
Wherein vOTCIt is the flow by ornithine transcarbamylase, CCITIt is the intracellular concentration of citrulling.
Ii. the sample for using Xcaliburr software analysis time sequence, extracts Gln58, Ornithine, Citrulline
Isotopic peak and integral obtain peak area, using calculated by peak area obtain each peer proportion change with time (figure
1b,1c,1d).It can according to the cell concentration and cell size data of the standard curve of Citrulline in mark product and cytoalgae 6803
With the intracellular concentration data of the Citrulline of calculation.
By vOTCAs parameter, in known Gln58, Ornithine flag data and CCITThat is the intracellular concentration number of citrulling
Under conditions of, the differential equation can be solved according to equation (1) obtain the label degree of citrulling and change with time.By counted melon
His tag degree and actual measured value do minimum two into fitting, it can acquire vOTCValue.All calculating all pass through
What MATLAB was realized.The numerical value of counted vOTC is much larger than the cumulative speed of citrulling intracellular, supports the hypothesis of quasi-stable state.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of dynamic flow metabolic analysis method, which is characterized in that comprising steps of
A) sample making:
(a-1) cell cultivated on filter paper the environment containing isotopic label is transferred to carry out continuing to cultivate；
(a-2) in sampling time point, the cell on the filter paper is turned over and is buckled into extracting solution, form extracting solution-cell mixture；
(a-3) cell extract is prepared with the extracting solution-cell mixture；
B) detection of biological sample: the cell extract is analyzed by combined gas chromatography mass spectrometry method, obtains one
Or the label ratio of multiple metabolins；
C) by one or more metabolite markers ratios and intracellular concentration, mesh is calculated using Differential Equation Model
Mark the metabolic flux intracellular of metabolin.
2. the method as described in claim 1, which is characterized in that the method further comprises the steps of: the biological precursor reactant mistake of calculating
The label ratio of metabolin in journey calculates the flow of target metabolite by metabolic response approach differential equation.
3. method according to claim 2, which is characterized in that the target metabolite is citrulling.
4. method as claimed in claim 3, which is characterized in that the metabolin includes: citrulling peer, Gln58 piece
SectionPeer, ornithine peer.
5. method as claimed in claim 3, which is characterized in that the distribution of the citrulling peer changes with time logical
Cross following differential equation description:
Wherein vOTCIt is the flow by ornithine transcarbamylase, CCITIt is the intracellular concentration of citrulling.
6. method according to claim 2, which is characterized in that further comprise the steps of: the mark for metabolin before the calculating
Note ratio carries out natural isotopic abundance correction.
7. method as claimed in claim 6, which is characterized in that the natural isotopic abundance correction includes: that C13 abundance is rectified
Just.
8. the method for claim 7, which is characterized in that the C13 abundance correction is carried out by following formula:
9. the method as described in claim 1, which is characterized in that the step (a-1) includes: that will contain Na14NO3's
It cultivates the bacterium solution finished under environment to filter on filter paper, culture to nitrogen limits state.
10. method as claimed in claim 9, which is characterized in that the step (a-2) includes: will be with described in bacterium colony
Filter medium, which turns over, to be buckled in extracting solution, is completed metabolism and is terminated, to form extracting solution-cell mixture.
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