JP4742528B2 - Analysis method of intracellular metabolic flux using isotope-labeled substrate - Google Patents

Description

  The present invention relates to a method for analyzing metabolic flux (flux), that is, a metabolic flux analysis method, a program for the method, and a recording medium on which the program is recorded. More specifically, the present invention relates to a metabolic flux analysis method using an isotope-labeled substance, a program for the method, and a recording medium on which the program is recorded.

  Metabolic flux analysis is a method for analyzing the balance of metabolites and isotope-labeled compounds in cells, and for isotopic compound tracing using analytical techniques such as nuclear magnetic resonance (NMR) or mass spectrometry (MS). A method for quantitatively determining intracellular metabolic flux by conducting experiments, and analyzing the stoichiometric ratio (carbon balance) of each metabolite in the metabolic reaction pathway of the target cell. In recent years, it has attracted attention as a technique (Non-Patent Document 1).

Various studies have been conducted in order to develop an accurate analysis method applied to metabolic flux analysis. There have been many research reports regarding the theory of metabolic flux analysis using isotope-labeled substrates (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). Many experiments have been conducted to establish a metabolic flux analysis method, but in order to obtain accurate analysis accuracy, research using a continuous culture method using a synthetic medium, which is an ideal condition, is common (non-patented). Reference 6). Although some examples of metabolic flux analysis in batch culture, a more practical culture method, have been reported, measurement of the isotopic distribution of some substances excreted in the medium has been performed. The calculation based on the isotope distribution measurement of the intracellular substance is not made at all. (Non-patent document 7). On the other hand, as disclosed in Patent Document 1, Patent Document 2 or Patent Document 3, many attempts have been made theoretically to predict metabolic flux. From a practical viewpoint such as application examples, isotopes This is far from the metabolic flux analysis method using a labeled substrate (Non-Patent Document 8, Non-Patent Document 9).
Metabolic Engineering, 2001, Volume 3, p.265-283 Biotechnology and Bioengineering, 1997, Vol. 55, p.101-117 Biotechnology and Bioengineering, 1997, 55, p.118-135 Biotechnology and Bioengineering, 1999, 66, p.69-85 Biotechnology and Bioengineering, 1999, 66, p.86-103 Journal of Biological Chemistry, 2000, 275, p.35932-35941 European Journal of Biochemistry, 2001, Vol. 268, p.2441-2455 International Publication No. WO 00/46405 Pamphlet International Publication No. WO 02/061115 Pamphlet International Publication No. WO 02/055995 Pamphlet Journal of Biotechnology, 2002, 94, p.37-63 Metabolic Engineering, 2001, Volume 3, p.195-205

  Until now, it has been difficult to predict an accurate metabolic flux distribution reflecting the actual state in a culture method or medium used for normal experiments or actual industrial production. In particular, in metabolic flux analysis performed using isotope-labeled compounds, the current situation is that errors that occur due to contamination with unlabeled substrates must be allowed. In actual industrial production, in many cases, nutrients derived from natural materials including nitrogen and carbon sources are added to the medium to increase the initial growth rate, and the effects of these unlabeled carbons are corrected. Therefore, a more accurate metabolic flux analysis method is desired. The present invention relates to a metabolic flux analysis method using an isotope-labeled compound with little analysis error, a method for reducing analysis error in a metabolic flux analysis performed using an isotope-labeled compound, a program for executing the method, and A recording medium storing the program is provided.

  In view of the above problems, the present inventors have conducted intensive studies, and as a result, have found a method for reducing analysis errors in metabolic flux analysis using an isotope-labeled compound. That is, based on the intracellular metabolic flux model constructed for the intracellular metabolic flux to be analyzed, the intracellular metabolic flux is determined from the analytical value of the cells cultured in a medium containing a substrate labeled with an isotope as a carbon source. It has been found that making specific corrections or assumptions in the method is effective in reducing analysis errors.

  More specifically, it has been found that the analysis error can be reduced by correcting the influence of the unlabeled compound in consideration of the exchange reaction occurring between the cellular protein and the intracellular amino acid pool.

  In addition, by constructing formulas that include uptake and degradation pathways of unlabeled compounds for the purpose of improving growth and taking into account the effects of these unlabeled compounds on the isotope distribution of various intracellular substances, It has been found that the accuracy can be improved.

  In addition, as a result of studying the uptake of carbon dioxide, which is the main carbon source other than the isotope-labeled substrate, in the calculation of the carbon balance, the concentration of carbon dioxide generated when the cells consumed the isotope-labeled substrate was very high, and the culture It was found that carbon balance can be calculated as carbon dioxide partial pressure in the liquid as carbon dioxide exhausted by cells.

  In addition, it is effective to correct the uptake of compounds consisting of unlabeled carbon added to the medium into cellular proteins when calculating metabolic flux using analytical values of isotope distribution of cellular protein hydrolyzed amino acids. I found out.

  The present invention has been made based on the above findings, and the gist thereof is as follows.

(1) Based on the intracellular metabolic flux model constructed for the intracellular metabolic flux to be analyzed, the intracellular metabolic flux is determined from the analysis value of cells cultured in a medium containing a substrate labeled with an isotope as a carbon source. A method for analyzing intracellular metabolic flux, comprising:
The said method satisfy | fills at least one of following (a)-(c).
(A) The analysis value of the cell includes the analysis value of the isotope distribution of the intracellular metabolite included in the intracellular metabolic flux model, and the analysis value of the isotope distribution of the intracellular metabolite includes the intracellular metabolite and the cell. It is corrected according to the degree of synthesis and degradation between the cellular constituents produced by taking up internal metabolites.
(B) The intracellular metabolic flux model includes a useful compound and / or its main metabolic intermediate, and the analysis value of the cell is the same compound as the intracellular metabolite in the medium and is labeled with an isotope. Including the rate of uptake of the non-compound into the cell and the analysis of the isotope distribution of the useful compound and / or its main metabolic intermediate, Assuming that the rate of uptake minus the rate of uptake by cellular components is the rate into the metabolic pathway, it is corrected by the effect on the isotopic distribution of useful compounds and / or their major metabolic intermediates Is done.
(C) The intracellular metabolic flux model includes a carbon dioxide fixation reaction and a carbon dioxide generation reaction, and the carbon dioxide used in the fixation reaction is assumed to be carbon dioxide generated in the generation reaction.

(2) Satisfying (a), and the analysis value of the isotope distribution of the intracellular metabolite is the intracellular metabolic flux model, the intracellular metabolite and the cellular constituents produced by the incorporation of the intracellular metabolite The method according to (1), wherein the analysis value of the cell is corrected by including the analysis value of the isotope distribution of the degradation product of the intracellular metabolite and the cell component in the analysis value of the cell.

(3) satisfying (a), and the analysis value of the isotope distribution of the intracellular metabolite is 1) measuring the isotope distribution of the intracellular metabolite and the isotope distribution of the decomposition product of the cell component, 2) The method according to (1), wherein the method is corrected by a step of optimizing the degree of synthesis and decomposition of intracellular metabolites and cell components using the optimization algorithm from the result obtained in step 1).

(4) The method according to (3), wherein the degree of synthesis and decomposition is expressed as a variable defined by an exchange reaction coefficient.

(5) The method according to (3) or (4), wherein the optimization algorithm is an evolution algorithm.

(6) The method according to any one of (3) to (5), wherein the intracellular metabolite is an amino acid and / or an organic acid, and the cell component is a protein.

(7) A compound that satisfies (a) and whose analysis value of the isotope distribution of the degradation product of the cell component is the same compound as the intracellular metabolite in the medium and is not labeled with an isotope The method according to any one of (2) to (6), which is corrected by the influence of uptake into cell components.

(8) The method according to (7), wherein the compound not labeled with an isotope is an amino acid.

(9) The method according to (1), wherein the compound that satisfies (b) and is not labeled with an isotope is an amino acid.

(10) The method according to (9), wherein the amino acid is isoleucine.

(11) The method according to any one of (1) to (10), wherein the cell is a microorganism that retains the ability to produce useful compounds.

(12) The method according to (11), wherein the useful compound is an amino acid and / or an organic acid.

(13) The method according to any one of (1) to (12), wherein the culture is batch culture or fed-batch culture.

(14) The method according to any one of (1) to (13), wherein the intracellular metabolite is an amino acid and / or an organic acid and / or a main metabolic intermediate thereof.

(15) The method according to any one of (1) to (14), wherein the isotope distribution is measured by mass spectrometry.

(16) Means for storing an intracellular metabolic flux model constructed with respect to an intracellular metabolic flux to be analyzed, means for inputting analysis values of cells cultured in a medium containing a substrate labeled with an isotope as a carbon source, cells In order to make the computer function as a means for determining the intracellular metabolic flux model based on the internal metabolic flux model and the analysis value of the cell, and for determining the intracellular metabolic flux and outputting the determined intracellular metabolic flux In the program, an intracellular metabolic flux model is constructed and / or a variable of the intracellular metabolic flux model is calculated so as to satisfy at least one of the following (a) to (c).
(A) The analysis value of the cell includes the analysis value of the isotope distribution of the intracellular metabolite included in the intracellular metabolic flux model, and the analysis value of the isotope distribution of the intracellular metabolite includes the intracellular metabolite and the cell. It is corrected according to the degree of synthesis and degradation between the cellular constituents produced by taking up internal metabolites.
(B) The intracellular metabolic flux model includes a useful compound and / or its main metabolic intermediate, and the analysis value of the cell is the same compound as the intracellular metabolite in the medium and is labeled with an isotope. Including the rate of uptake of the non-compound into the cell and the analysis of the isotope distribution of the useful compound and / or its main metabolic intermediate, Assuming that the rate of uptake minus the rate of uptake by cellular components is the rate into the metabolic pathway, it is corrected by the effect on the isotopic distribution of useful compounds and / or their major metabolic intermediates Is done.
(C) The intracellular metabolic flux model includes a carbon dioxide fixation reaction and a carbon dioxide generation reaction, and the carbon dioxide used in the fixation reaction is assumed to be carbon dioxide generated in the generation reaction.

(17) A computer-readable recording medium on which the program according to (16) is recorded.

  A metabolic flux analysis method using an isotope-labeled compound with a small analysis error is provided.

  Hereinafter, the present invention will be described in detail.

  The intracellular metabolic flux in the present invention is a flux (flux) of intracellular metabolites derived from a stoichiometric model of chemical reaction in cells and a mass action law between metabolites.

  The intracellular metabolite in the present invention is a substance metabolized in the cell. As for intracellular metabolites, a lot of knowledge has been obtained along with their biochemical reactions, and it has been databased (see, for example, Kyoto Encyclopedia of Genes and Genomes (KEGG) (http: //www.genome .ad.jp / kegg /).

  The cell component in the present invention is a substance that constitutes a cell, and is a substance that is produced by taking up an intracellular metabolite. For example, substances such as proteins, carbohydrates, nucleic acids and lipids. Moreover, the degradation product of a cell component means the degradation product of the same level as the intracellular metabolite taken in by it. For example, when the cell constituent is a protein produced by taking up amino acids, the degradation product is an amino acid. In the present specification, when the cell constituent component is a protein produced by incorporating an amino acid, it is also referred to as a cell protein. An amino acid that is a degradation product of cellular protein is also referred to as cellular protein hydrolyzed amino acid.

  The cell to be analyzed in the present invention is any cell, and in particular, cells used for substance production, such as various cultured cells, mold, yeast, various bacteria and the like. Preferred are microorganisms that retain the ability to produce useful compounds such as amino acids, nucleic acids, or organic acids. As microorganisms that retain the ability to produce amino acids, nucleic acids, or organic acids, for example, Escherichia coli, Bacillus bacteria, coryneform bacteria, and the like are preferably used.

The isotope in the present invention is usually a stable isotope, but a radioactive isotope can be used for the same purpose. The substrate labeled with an isotope includes glucose labeled with an isotope, more specifically, glucose labeled with a stable isotope at the first carbon and / or glucose labeled with a stable isotope. Can be mentioned. An isotope includes ¹³ C.

  The intracellular metabolic flux model in the present invention is not particularly limited as long as it is constructed with respect to the metabolic flux to be analyzed, and may be an intracellular metabolic flux model constructed according to a normal construction method. Constructed with respect to metabolic flux means that the metabolic flux reaction (reaction path) to be analyzed is included in the constructed intracellular metabolic flux model.

  Examples of a method for constructing an intracellular metabolic flux model relating to metabolic flux include Metabolic Engineering 3, 265-283, 2001 (Non-patent Document 1), Wiechert, W. and de Graaf, AA Biotechnology and Bioengineering 55, 101-117, 1997 ( Non-Patent Document 2), Metabolic Engineering 3, 195-205, 2001 (Non-Patent Document 9), Metabolic Engineering 3, 173-191, 2001, Biotechnology and Bioengineering 55, 831-840, and the like.

  The reaction pathway for analyzing metabolic flux may be any of the major intracellular metabolic pathways. In particular, glycolysis, TCA cycle, pentose phosphate pathway, and various amino acid synthesis specific pathways are useful for producing useful compounds by microbial fermentation. These are preferably included because they are important for practical use.

  In the construction of an intracellular metabolic flux model, the reaction pathway is simplified, such as treating a series of reactions without branching as one reaction, and treating metabolites before and after the reaction converted by a reaction with a high metabolic rate as one metabolite. May also be performed.

  The analysis value of a cell is a measurable analysis value of a cell cultured in a medium containing a substrate labeled with an isotope as a carbon source. For example, in addition to an analysis value of an isotope distribution in a metabolite, a bacterial cell Examples include production rate and production rate of useful substances. The analysis value of the isotope distribution may be an analysis value reflecting the isotope distribution, and examples thereof include an isotopomer distribution vector (Biotechnology and Bioengineering 55,831-840) and a mass distribution vector (Biotechnology and Bioengineering 62,739-750). . A mass distribution vector is preferable in that it can be measured by mass spectrometry.

  The step of determining the intracellular metabolic flux from the analytical value of cells cultured in a medium containing a substrate labeled with an isotope as a carbon source can be determined according to a commonly used determination method. When the analysis value includes an analysis value of an isotope distribution, the determination is usually performed using an isotopomer balance equation (see, for example, Biotechnology and Bioengineering 66, 69-85, 1999 (Non-patent Document 4)). ).

  If the analysis value of the cell is sufficient to calculate the variables in the metabolic flux model (to obtain a solution when the metabolic flux model is represented by a stoichiometric matrix), Variables can be determined, thereby determining metabolic flux. If the analytical value of the cells is not sufficient to calculate the variables in the metabolic flux model, usually some of the other variables of the isotope distribution in the metabolic flux model are free variables, Calculated from metabolic flux model based on analysis values of cells other than analysis values, and labeling method (position and number of isotopes, and ratio of substrates when using multiple different substrates) on the substrate used Optimization based on the comparison between the calculated value of the isotope distribution and the analysis value of the isotope distribution is performed to determine the variables in the metabolic flux model, thereby determining the metabolic flux. Examples of such optimization methods include Metabolic Engineering 3, 265-283, 2001 (Non-Patent Document 1), Biotechnology and Bioengineering 55, 118-135, 1997 (Non-Patent Document 3), Biotechnology and Bioengineering 66, 69-85, 1999 (nonpatent literature 4) etc. are mentioned.

In the process of determining the intracellular metabolic flux from the analysis value of the isotope distribution of metabolites in cells cultured in a medium containing an isotope-labeled substrate as a carbon source, the method of labeling the substrate is determined by a conventional method. (E.g. Biotechnology and Bioengineering
66, 86-103, 1999 (non-patent document 5), European Journal of Biochemistry 268, 2441-2455, 2001 (non-patent document 7)).

  The method of the present invention is based on an intracellular metabolic flux model constructed with respect to an intracellular metabolic flux to be analyzed. From the analysis value of a cell cultured in a medium containing a substrate labeled with an isotope as a carbon source, the intracellular metabolism is determined. In the method of analyzing a flux, at least one of the following conditions (a) to (c) is satisfied.

(A) The analysis value of the cell includes the analysis value of the isotope distribution of the intracellular metabolite included in the intracellular metabolic flux model, and the analysis value of the isotope distribution of the intracellular metabolite includes the intracellular metabolite and the cell. It is corrected according to the degree of synthesis and degradation between the cellular constituents produced by taking up internal metabolites.

(B) The intracellular metabolic flux model includes a useful compound and / or its main metabolic intermediate, and the analysis value of the cell is the same compound as the intracellular metabolite in the medium and is labeled with an isotope. Including the rate of uptake of the non-compound into the cell and the analysis of the isotope distribution of the useful compound and / or its main metabolic intermediate, Assuming that the rate of uptake minus the rate of uptake by cellular components is the rate into the metabolic pathway, it is corrected by the effect on the isotopic distribution of useful compounds and / or their major metabolic intermediates Is done.

(C) The intracellular metabolic flux model includes a carbon dioxide fixation reaction and a carbon dioxide generation reaction, and the carbon dioxide used in the fixation reaction is assumed to be carbon dioxide generated in the generation reaction.

Hereinafter, each condition will be described.
(A) shows the intracellular metabolism generated by decomposing intracellular constituents (for example, cellular proteins) formed in the cell growth phase based on the isotope distribution of intracellular metabolites (for example, amino acids) when calculating metabolic flux. The correction is made in consideration of the influence received from the product, that is, the exchange reaction between the intracellular metabolite pool and the intracellular metabolite generated by the decomposition of the intracellular constituent components. As a correction method, the analysis value of the isotope distribution of the intracellular metabolite may be corrected based on the exchange reaction, or the exchange reaction may be included in the intracellular metabolic flux model. When an exchange reaction is included in an intracellular metabolic flux model, the intracellular metabolic flux model includes an exchange reaction between an intracellular metabolite and a cellular component generated by the incorporation of the intracellular metabolite. The values include analysis values of isotope distributions of intracellular metabolites and cell component degradation products. When an exchange reaction is included in the intracellular metabolic flux model, the correction value of the analysis value of the isotope distribution of the intracellular metabolite is not directly used, but the intracellular metabolic flux is based on the metabolic flux model. As a result, the analytical value of the isotope distribution of the intracellular metabolite is corrected.

  As a specific example of a method for correcting the isotope distribution of intracellular metabolites, an intracellular metabolic flux model includes an exchange reaction between an intracellular metabolite and a cellular component produced by the incorporation of the intracellular metabolite. The analysis value of the cell includes the analysis value of the isotope distribution of the intracellular metabolite and the decomposition product of the cell component.

  In addition, 1) a step of measuring isotope distribution of intracellular metabolites and isotope distribution of decomposition products of cell components, and 2) intracellular metabolites using an optimization algorithm from the results obtained in step 1). And a step of optimizing the degree of synthesis and decomposition of the cell constituents. In this embodiment, the degree of synthesis and decomposition is preferably expressed as a variable defined by an exchange reaction coefficient. Further, as an optimization method, an evolution algorithm (Journal of theoretical Biology 199, 45-61, 1999) and the like can be mentioned, and an evolution algorithm is preferable. In this embodiment, the intracellular metabolite is preferably an amino acid and / or an organic acid, and the cell constituent is preferably a protein.

  In the embodiment using the analysis value of the isotope distribution of the degradation product of the cell component, the analysis value of the isotope distribution of the degradation product of the cell component is the same compound as the intracellular metabolite in the medium. It is preferable to correct in consideration of the influence of incorporation of a compound that is not labeled with the body into a cell constituent. For example, when the cell component is a protein, cells of amino acids not labeled with isotopes in the medium when calculating metabolic flux using the isotope distribution analysis value of cell protein hydrolyzed amino acids. Correct for protein uptake.

  (B) When the same compounds as intracellular metabolites that are not labeled with isotopes are contained in the medium, the rate at which they are taken into cells is analyzed, and the rate of uptake is used as a cell component. Assuming that the remaining rate minus the applied rate is the flux flowing through the degradation pathway, it corrects the effect on the isotope distribution of useful compounds and / or major metabolic intermediates in the cell. As a correction method, the analysis value of the isotope distribution of the intracellular metabolite may be corrected based on the flux flowing in the decomposition path, or the flow rate may be included in the intracellular metabolic flux model. In this embodiment, the compound that is not labeled with an isotope is preferably an amino acid (preferably isoleucine).

  In the present invention, the useful compound means a compound useful for seasonings, food additives, medicines and the like, and examples include amino acids, organic acids, and nucleic acids.

  In the present invention, the main metabolic intermediate means all metabolic intermediates included in the metabolic flux analysis model. Examples include pyruvic acid, glucose-6-phosphate, fructose-6-phosphate, oxaloacetate. Etc.

  (C) calculates the carbon balance on the assumption that the partial pressure of carbon dioxide in the culture medium is carbon dioxide discharged by the cells consuming the isotope-labeled substrate.

  In the present invention, correction or assumption is performed by satisfying the above conditions. Thereby, the analysis error in the metabolic flux analysis performed using the isotope-labeled compound can be reduced.

  In the present invention, the cells are preferably microorganisms that retain the ability to produce useful compounds. Examples of such cells include Escherichia coli, coryneform bacteria, and Bacillus bacteria. Useful compounds are preferably amino acids and / or organic acids.

  In the present invention, cell culture is preferably batch culture or fed-batch culture. Batch culture is a closed culture method using specific nutrients, and fed-batch culture is a culture method in which a substrate is added continuously or intermittently to an infusion medium in the culture system. The analysis method of the present invention has a great effect of reducing the analysis error when the culture is batch culture or fed-batch culture.

  In the present invention, the intracellular metabolite is preferably an amino acid and / or an organic acid and / or a main metabolic intermediate thereof.

  In the present invention, it is preferable to measure the isotope distribution by mass spectrometry.

  The present invention also provides a program for implementing the analysis method of the present invention. The program of the present invention inputs means for storing an intracellular metabolic flux model constructed with respect to an intracellular metabolic flux to be analyzed, and an analysis value of cells cultured in a medium containing a substrate labeled with an isotope as a carbon source. A computer is used as a means for determining the intracellular metabolic flux model based on the means, the intracellular metabolic flux model and the analysis value of the cell, and for determining the intracellular metabolic flux, and for outputting the determined intracellular metabolic flux. A program for functioning, wherein an intracellular metabolic flux model is constructed and / or a variable of the intracellular metabolic flux model is calculated so as to satisfy at least one of the above (a) to (c) It is characterized by that.

  Another embodiment of the present invention relates to a computer-readable recording medium characterized by recording the above program.

  The intracellular metabolic flux model constructed for the intracellular metabolic flux to be analyzed, and the analysis value of the cells cultured in the medium containing the isotope-labeled substrate as the carbon source are as described for the analysis method of the present invention. It is. The intracellular metabolic flux model is usually stored in the form of data normally used to represent the intracellular metabolic flux model. For example, when a metabolic flux model is represented by a stoichiometric matrix, the model data is stored as a matrix. The means for inputting the analysis value includes means for transferring from the storage medium or via the transmission medium.

  The means for determining the intracellular metabolic flux model based on the intracellular metabolic flux model and the analysis value of the cell and determining the intracellular metabolic flux is suitable for performing the determination process described with respect to the analysis method of the present invention. Any means may be used.

The means for outputting the determined intracellular metabolic flux includes means for transferring to a storage medium or via a transmission medium. The output of the intracellular metabolic flux may be a graph showing a metabolic network in which a metabolic flux model is constructed, and displaying the flux value at a position corresponding to each reaction in the metabolic network in the figure.

  A flowchart of the program of the present invention is shown in FIG. The above (a) to (c) and preferred embodiments thereof are as described for the analysis method of the present invention, and an intracellular metabolic flux model is constructed and / or intracellular metabolism so as to satisfy these. The program of the present invention can be created according to a normal programming method except that the flux model variables are calculated.

  The recording medium on which the program of the present invention is recorded is an arbitrary removable physical medium such as a flexible disk, magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, DVD, ROM, RAM, Includes a “communication medium” that holds a program in a short period of time, such as a communication line or carrier wave when transmitting a program via an arbitrary fixed physical medium such as an HD, or a network represented by a LAN, WAN, or the Internet. Shall be.

  Hereinafter, the present invention will be further described by examples. In the examples, the following strains and culture media were used.

(1) Escherichia coli strain and plasmid strain: WYK050 (Escherichia coli wild strain W3110 S (2-aminoethyl) cysteine resistant and lysine degradation gene ldc and cadA gene deletion strain (Kikuchi, Y. et al. J. Bacteriol. 179, 4486-4492, 1997)).
Plasmid: pCAB1 (E. coli-derived lysC, dapA, dapB genes are loaded on vector RSF1010).
For the culture, a strain obtained by introducing pCAB1 into WYK050 was used.

(2) Medium
LB agar: 1.0% bactotryptone, 0.5% bacto yeast extract, 1% NaCl, 1.5% agar. Streptomycin was added at 20 μg / ml as necessary.
Main culture medium: ammonium sulfate 16g / L, monopotassium dihydrogen phosphate 3g / L, yeast extract 4g / L, iron sulfate heptahydrate 10mg / L, manganese sulfate pentahydrate 10mg / L, isoleucine 400mg / L , Glucose 40g / L, magnesium sulfate heptahydrate 1g / L. The pH was adjusted to 7.0 with potassium hydroxide. Streptomycin was added at 20 μg / ml as necessary. The main culture medium was used for liquid culture of E. coli.
Fed-batch solution: glucose 500g / L, ammonium sulfate 80g / L

(1) Construction of metabolic flux analysis model A stoichiometric formula for calculating metabolic flux was constructed assuming a quasi-steady state of intracellular metabolic intermediates (Savinell and Palsson, Journal of Theoretical Biology 154, 421-454, 1992; Vallino and Stephanopoulos, Biotechnology and Bioengineering 41, 633-646, 1993). The reaction formulas included in this model are as shown in Table 2, and explanation of each abbreviation is shown in Table 1. Several unbranched reactions have been combined into one to simplify the equation. Since the pentose phosphate pathway is complicated, it is expressed in two formulas. Previously reported data were used for biomass composition ratios (Neidhardt et al., Physiology of the Bacterial Cell, 1990). The composition ratio of amino acids in the intracellular protein was determined from the concentration ratio of amino acids obtained by actually hydrolyzing the intracellular protein. The stoichiometric matrix of this model has 8 degrees of freedom, and in order to obtain a solution, 7 fluxes must be determined in addition to the sugar consumption rate. The following were defined as seven free fluxes. Cell production rate, lysine production rate, acetic acid production rate, formic acid production rate, ICL flux, G6PDH flux, malic enzyme (Malic Enzyme) flux. About the cell production rate and various production rates, the result was obtained from the culture experiment. The remaining three fluxes were determined by an optimization algorithm based on measured values of isotope distribution of amino acids (described later). The constructed model also contains 14 reversible reactions. Its reversibility was defined as an exchange coefficient that can take values from 0 to 1 (Dauner et al., Biotechnology and Bioengineering 76, 144-156, 2001; Wiechert and de Graaf, Biotechnology and Bioengineering 55, 101-117, 1997 ). These exchange coefficients are also variables that are determined based on the measured values of the isotope distribution as in the previous three free fluxes. It was assumed that the reversibility of adjacent reactions in the glycolysis, pentose phosphate pathway, and TCA cycle was equal for simplicity. As a result of the sensitivity analysis, it has been clarified that the reactions 9, 29 and 30 in the reaction list of Table 2 hardly affect the isotope distribution. From the above, there were six reversible reactions for which the exchange coefficient had to be determined.

  In order to calculate the IDV (Isotopomer Distribution Vector) of all substances in the model, an isotopomer balance equation was constructed as a function of the free flux and exchange coefficient and the isotopomer distribution of the substrate. The IDV column vector represents the proportion of isotopomers and the sum of the elements is 1 (Schmidt et al., Biotechnology and Bioengineering 55, 831-840, 1997; Wittmann and Heinzle, Biotechnology and Bioengineering 62, 739- 750, 1999). The isotopomer balance equation has been described using the isotopomer mapping matrix (IMM) described in detail by Schmit et al. (Schmidt, et al., Biotechnology and Bioengineering 55, 831-840, 1997). The atom mapping matrix (AMM) is a matrix that describes the movement of carbon atoms from a reactant to a product. Based on this matrix, MATLAB (The MathWorks, Natick, MA) is used as a reactant. An isotopomer mapping matrix (IMM), which is a matrix describing the transfer of isotopomers from to the product, is calculated.

  The isotopomer balance equation can be solved using the Gause-Seidel iteration method with free flux and exchange coefficient as inputs.

In addition to glucose consumption, microbial cells take up carbon dioxide and consume acetic acid during growth. Since carbon dioxide is also generated from the metabolism of isotope-labeled glucose, some percent is ¹³ C-carbon dioxide. This ratio was calculated by a carbon dioxide balance equation that takes into account all reactions that generate carbon dioxide. The exact value varies depending on the intracellular metabolic flux distribution, but is generally around 32%. For this calculation, it was assumed that no carbon dioxide was consumed from the atmosphere. This is because the concentration of carbon dioxide generated by the cells consuming isotope-labeled glucose is very high (in the experiment, the concentration of exhausted carbon dioxide ranged from 4 to 5%), and the carbon dioxide in the fermenter This is because all partial pressures can be considered as carbon dioxide exhausted by cells.

  Although it is not possible to determine the isotopomer distribution of each substance from the analysis by mass spectrometry, the distribution of mass can be determined. This information will be expressed as a mass distribution vector (MDV), and each element contains isotopomers of the same mass (Wittman and Heinzle, Biotechnology and Bioengineering 62, 739-750, 1999). Therefore, there are n + 1 elements of MDV in a substance having n carbon atoms. MDV can be calculated by summing elements of the same mass from IDV. By comparing the MDV calculated in this way with the MDV obtained from the experiment, it can be evaluated how much the result of the model matches the experimental value.

(2) Correction of naturally occurring carbon, hydrogen, nitrogen, oxygen, and isotopes of each atom MDV obtained by experiments is ² H (0.01%), ¹⁵ N (0.37%), ¹⁷ O ( 0.04%) , ¹⁸ O (0.20%) correction was performed. The calculation formula is as follows.

  The natural isotope correction of carbon is not included in the above formula because it is incorporated in the IDV of glucose used as an input value.

(3) Correction of non-labeled amino acids contained in the initial medium In order to increase the initial growth rate in industrial production, naturally derived nutrients containing a nitrogen source and a carbon source are added to the medium. When comparing the MDV calculated by the model with the MDV obtained from the experiment, it is necessary to correct the influence of non-labeled carbon derived from natural components. Different correction methods were used for analysis of intracellular amino acids (free amino acids) and protein hydrolyzed amino acids. It was assumed that all amino acids except for the amino acids remaining at the time of sample acquisition (in this case isoleucine) are taken directly into the intracellular amino acid pool and are not metabolized. That is, they were assumed to be direct protein components. For the remaining amino acids, the rate of uptake is calculated from the experiment, and since it is taken into the cell at a rate higher than the rate taken up by the protein, the excess is incorporated into the model as if it was degraded by metabolism. Identified by calculating from the uptake rate.

Up to about 12 hours at the beginning of the culture, there is uptake of non-labeled amino acids derived from the medium, and they are mixed with an intracellular amino acid pool produced by metabolism of bacteria, which is a substrate, by the bacteria. Cellular proteins are composed of these pools and therefore contain unlabeled amino acids. At the time of initial sample acquisition, the unlabeled amino acids in the medium had already been completely consumed, i.e., the uptake rate was zero, so it was not incorporated into the stoichiometric matrix and isotopomer balance equation. If the exchange of the intracellular pool is not very slow, the intracellular amino acids in the first sample should not contain any unlabeled amino acids from the medium, but in fact they are contained and are the intracellular proteins. It was suggested that there was always an exchange reaction between and the intracellular amino acid pool. In order to consider this point, a coefficient Pex representing this exchange reaction was introduced into a model for analyzing intracellular amino acid analysis data. Pex is a coefficient representing the rate of return from the cellular protein to the intracellular amino acid pool and was assumed to be the same for all amino acids except lysine.

  The MDV of protein hydrolyzed amino acids represents all of the amino acids incorporated into the protein from the beginning of the culture, so the proportion of amino acids derived from the medium is higher than those in intracellular amino acids. Assuming that the concentration of intracellular amino acids is very small compared to the total amount of protein, it is safe to assume that all non-labeled amino acids derived from the medium consumed at the beginning of the culture have been incorporated into the cellular protein.

(4) Metabolic flux optimization MDV is calculated using the isotopomer balance equation with the free flux and exchange reaction flux as input values, so that the sum of squares of the difference from the MDV obtained in the experiment is minimized. A program was developed to optimize the input free flux and exchange reaction flux values by an evolutionary algorithm (Stephani et al, Journal of theoretical Biology 199, 45-61, 1999). Variables to be optimized are ICL, Malic Enzyme, flux of pentose phosphate pathway (G6PDH), six exchange reaction values, and Pex representing exchange reaction between protein and intracellular amino acid pool. The cell yield and lysine yield were set to allow a deviation of 20% from the input value. This is to take into account measurement errors in the experiment. Proteolytic amino acid data and intracellular amino acid data were analyzed separately.

  Some changes were made to the general evolutionary algorithm to reduce computation time. As a result of various studies, it was found that the setting of 50000 elements and 200 generations was optimal for searching for the minimum value in the solution space, and those setting values were used for the analysis.

(5) Sensitivity analysis The confidence interval of the free flux depends not only on the variance of each measured value but also on the Jacobian matrix. The Jacobian matrix expresses how easily each IDV changes when the free flux changes near the optimum value. The variance of each amino acid measurement value was obtained from three analysis values. Based on these, the sensitivity matrix was calculated according to the method of Wiechert et al.

  Prior to conducting the culture experiments, a sensitivity analysis of the analytical model was performed to find the optimal mixing ratio of labeled glucose. The label glucose used was calculated by limiting to 1-13C-Glc and U-13C-Glc. As a result, a mixing ratio of 50% was obtained. This time, because of the cost, we used an 80:20 mixing ratio that provides sufficient information.

(6) Culture experiment
WYK050 / pCAB1 strain cells were applied on LB agar medium, and statically cultured at 37 degrees for 24 hours. Cells from the two stationary culture plates were inoculated into the initial medium. The components of the medium are as described above. A 1 L jar fermenter was used for the culture, and a mixture of 1-13C-Glc and U-13C-Glc at 80:20 was used as the substrate. The mixing ratio was determined by a sensitivity analysis performed in advance. The initial volume of the culture was 300 ml, and the temperature and pH were controlled at 37 ° C. and 6.7, respectively. Ammonia gas was used for pH control. Aeration was controlled at 300 ml / min. The number of agitation was appropriately controlled so that the dissolved oxygen concentration in the culture solution was always maintained at 5% or more. The feeding of the sugar solution started at 17 hours of culture. This is just before complete consumption of the primary sugar. The flow acceleration was appropriately adjusted so that the residual sugar concentration in the medium was 5 g / L or less. The fermentation samples were obtained at 17 hours of culture in the growth phase and at 26 hours of culture in the stationary phase. In each case, intracellular metabolites were extracted by the silicon oil method. Cells for measuring protein hydrolyzed amino acids were also obtained at the same time. The measurement was performed using LC-MS and CE-MS.

FIG. 2 shows the absorbance (OD), specific growth rate μ, specific sugar consumption rate ν, specific lysine production rate ρ, oxygen absorption rate rab, and respiratory quotient RQ of the cultured cells.
The time course of amino acid concentration in the medium is shown in FIGS. 3A and 3B. The amino acids from the yeast extract were almost completely consumed during the 15 hours of culture. After isoleucine was completely consumed around 20 hours of culture, the increase in the oxygen absorption rate rab stopped and μ and ν also decreased. In order to clarify the difference in metabolic flux distribution before and after this, that is, in the growth phase and stationary phase, metabolic flux analysis was performed.
The final fermentation results are shown in Table 3.

(7) Metabolic flux analysis <Metabolic flux analysis using protein hydrolyzed amino acid data>
As a result of analyzing protein hydrolyzed amino acids in cells by LC-MS, the following amino acid isotope ratio data were obtained. They are glycine, alanine, serine, proline, valine, threonine, phenylalanine, tyrosine, leucine, and methionine. For proline, growth phase data was not used because it was less reliable than other analysis values, and only stationary phase data was used.

After correcting for the effects of natural isotopes other than carbon, the IDV of each amino acid was calculated from the experimental results. Since cell proteins contain amino acids biosynthesized from the start of culture to sample acquisition, the analytical data obtained from cell protein hydrolysis is considered to be the average value in this interval. Therefore, the following numerical value, which is the average value of this section, was used for analysis of data acquired in 17 hours. Yield 0.379 g DCW / 10 mmol glucose, lysine yield 2.82 mmol / 10 mmol glucose, acetic acid uptake rate 0.47 mmol / 10 mmol glucose. When the isotopomer balance was calculated, the MDV of the experimental results tended to have a higher proportion of isotopes with lighter masses than the MDV predicted by the calculation. This was considered to be the effect of non-labeled amino acids derived from the yeast extract, which is a natural nutrient source, and was corrected by the method described above. The non-labeled amino acid mentioned here does not mean that it consists of 100% ¹² C, but means that it contains the proportion of ¹³ C that exists in nature.

Free flux optimization using protein hydrolyzed amino acid data was performed several times, all with similar results. In the optimization by the evolutionary algorithm, the number of generations was 200 with 50000 elements. Table 4 shows experimental MDV and calculated MDV. In this calculation, the sum of the error between the experiment and the calculation is 21.19. The optimized free flux values are shown in Table 5. All metabolic flux distributions are shown in FIG. The metabolic flux distributions shown in FIGS. 4 and 5 also include energy metabolism reactions. The energy metabolism reaction was recalculated using the stoichiometric matrix from the results calculated based on the carbon transfer. In summary, 16% of the consumed glucose flows into the pentose phosphate pathway, and this flux is not sufficient to produce lysine and cells, so the flux of NADH to NADPH conversion using transhydrogenase is high. It was. Furthermore, in this analysis, the flux by ICL and Malic Enzyme was zero. The reaction that was highly reversible was a reaction between the glycolysis and the pentose phosphate pathway.

  The same analysis was performed using analysis data of stationary-phase protein hydrolyzed amino acids, but as described above, since this analysis data includes information from the start of the culture, it is greatly different from the growth phase. No results. The results are shown in FIG. 6 and FIG.

<Metabolic flux analysis using intracellular amino acid analysis data>
Intracellular amino acids were analyzed by LC-MS, and the following amino acid MDVs were obtained. Glycine, alanine, serine, proline, valine, threonine, asparagine, glutamine, glutamic acid, lysine, phenylalanine, tyrosine. The input data to the analysis model was used when the sample was acquired. Yield 0.272 g DCW / 10 mmol glucose, lysine yield 3.30 mmol / 10 mmol glucose, acetic acid uptake rate 0 mmol / 10 mmol glucose. Intracellular analysis data is data at the moment when a sample is obtained, and thus differs greatly from the protein hydrolysis data representing the average value shown above.

  When the MDV of each amino acid was predicted using the isotopomer balance equation, it was not as much as when using protein hydrolyzed amino acid data. There was a tendency for the body ratio to be high. At the time of sample acquisition, the unlabeled amino acids from the medium were already completely consumed, but the experimental results suggested that they were affected. Here, in order to consider the influence, the ratio Pex of the exchange reaction with the intracellular amino acid pool due to the degradation of the cellular protein was defined, and the algorithm was changed to optimize the numerical value. Pex was assumed to be the same for all amino acids.

  Based on the above, as a result of optimization by an evolutionary algorithm, the sum of errors between MDV obtained through experiments and MDV obtained through calculations was minimized to 7.57. Again, the number of elements used in the evolutionary algorithm was 50000 and the number of generations was 200. Table 5 shows the MDV obtained by experiment and the MDV obtained by calculation. The optimized free flux values are shown in Table 5. All metabolic flux distributions are shown in FIG. The major difference from the analysis results using protein hydrolyzed amino acid data is that the flux of ICL and G6PDH is large.

  Similarly, as a result of analysis using intracellular amino acid data in the stationary phase, the sum of errors between the MDV obtained by experiment and the MDV obtained by calculation was the smallest at 8.71. The input data to the analysis model was used when the sample was acquired. Cell yield 0.055 g DCW / 10 mmol glucose, lysine yield 4.27 mmol / 10 mmol glucose, acetic acid excretion rate 0.9 mmol / 10 mmol glucose.

  The main difference is that the flux of ICL increased nearly three times in the stationary phase compared to the growth phase, and the flux of PEPC became zero.

The figure which showed the relationship between the uptake | capture of the non-labeled amino acid derived from a culture medium, and the exchange reaction of an intracellular protein and an intracellular amino acid pool. In the initial stage of culture (about 12 hours of culture), unlabeled amino acids derived from the medium are incorporated. In the growth phase (about 17 hours in culture), all unlabeled amino acids derived from the yeast extract are consumed, but isoleucine, a growth promoting factor, remains in the medium. The consumption rate V _Ile was measured and decomposed by metabolism. V _YE indicates the uptake flux of amino acids from the medium to the bacteria. Pex represents an exchange reaction coefficient between an intracellular protein and an intracellular amino acid pool. Pex is a variable determined by the optimization algorithm. Analytical value of the culture. Cell absorbance (OD), specific growth rate μ, specific sugar consumption rate ν, specific lysine production rate ρ, oxygen absorption rate rab, respiratory quotient RQ Amino acid and acetic acid concentrations in the medium. (Asp) Aspartate (Thr) Threonine (Ser) Serine (Leu) Leucine (Gly) Glycine (Ala) Alanine (Cys) Cysteine (Val) Valine (Met) Methionine Continuation of FIG. 3A. (Tyr) Tyrosine (Phe) Phenylalanine (His) Histidine (Arg) Arginine (Glu) Glutamic acid (Ile) Isoleucine (Lys) Lysine (AcOH) Acetic acid Metabolic flux distribution calculated from isotope distribution measurements of protein hydrolyzed amino acids (growth phase: 17 hours after the start of culture). The unit of each numerical value is expressed in mmol of change of each substance with respect to 10 mmol glucose. Metabolic flux distribution calculated from isotope distribution measurements of protein hydrolyzed amino acids (stationary phase: 26 hours after the start of culture). The unit of each numerical value is expressed in mmol of change of each substance with respect to 10 mmol glucose. Metabolic flux distribution calculated from the measured isotope distribution of intracellular amino acids (growth phase: 17 hours after the start of culture). The unit of each numerical value is expressed in mmol of change of each substance with respect to 10 mmol glucose. Metabolic flux distribution calculated from isotope distribution measurements of intracellular amino acids (stationary phase: 26 hours after the start of culture). The unit of each numerical value is expressed in mmol of change of each substance with respect to 10 mmol glucose. The flowchart of the analysis program of intracellular metabolic flux is shown.

Claims (9)

A substrate labeled with an isotope as a carbon source based on an intracellular metabolic flux model built on glycolytic systems, TCA cycle, pentose phosphate pathway, amino acid synthesis intrinsic pathway and intracellular metabolic flux including metabolic flux reaction pathway Determination of intracellular metabolic flux from analysis values including metabolite isotope distribution analysis, cell production rate, amino acid production rate, and organic acid production rate of cells cultured in a medium containing An analysis method of intracellular metabolic flux,
The said method which satisfy | fills all of following (a)-(c).
(A) The intracellular metabolic flux model includes an exchange reaction between an amino acid and a protein produced by the incorporation of the amino acid, and the analysis value of the cell includes the isotope distribution of amino acids and protein degradation included in the intracellular metabolic flux model. include analytical value of isotope distribution in an object, analytical values of isotope distribution of amino acids, 1) measuring the isotopic distribution of the isotope distribution and degradation of proteins in amino acid, obtained in 2) step 1) Using the optimization algorithm, the result is corrected by a step of optimizing the degree of synthesis and decomposition of amino acids and proteins expressed as a variable defined by the exchange reaction coefficient .
(B) an intracellular metabolic flux model includes amino San及 beauty / or its metabolic intermediates, analytical values of cells in the medium, the uptake rate into cells of amino acids that are not isotopically labeled, as well as, include analytical value of isotope distribution in the amino acid and / or its metabolic intermediates, analytical values of isotope distribution of the amino acid and / or its metabolic intermediates, a speed obtained by subtracting a speed that is incorporated into the protein from the uptake rate Assuming a rate to the metabolic pathway, it is corrected by the effect on the isotopic distribution of the amino acid and / or its metabolic intermediate by the rate to the metabolic pathway.
(C) The intracellular metabolic flux model includes a carbon dioxide fixation reaction and a carbon dioxide generation reaction, and the carbon dioxide used in the fixation reaction is assumed to be carbon dioxide generated in the generation reaction. The method of claim 1 , wherein the optimization algorithm is an evolutionary algorithm. The method according to claim 1 or 2 , wherein in (a) , the analysis value of the isotope distribution of the protein degradation product is corrected by the influence of incorporation of amino acids not labeled with isotopes into the protein in the medium. . The method according to claim 1 , wherein the amino acid is isoleucine. The method according to any one of claims 1 to 4 , wherein the cell is a microorganism that retains the ability to produce the amino acid . The cell culture is any one of claims 1 to 5 , wherein the culture is batch culture or fed-batch culture.
The method according to item. Isotope distribution is measured by mass spectrometry The method according to any one of claims 1-6. Glycolytic system, TCA cycle, pentose phosphate pathway, amino acid synthesis intrinsic pathway, a means to memorize intracellular metabolic flux model built on intracellular metabolic flux including metabolic flux reaction pathway , labeled with isotope as carbon source Means for inputting analysis values including metabolite isotope distribution, cell production rate, amino acid production rate, and organic acid production rate of cells cultured in a medium containing a substrate, intracellular metabolic flux model And a program for operating a computer as a means for determining the intracellular metabolic flux model based on the analysis value of the cell and for determining the intracellular metabolic flux, and for outputting the determined intracellular metabolic flux. Te, so as to satisfy all of the following (a) ~ (c), intracellular metabolic flux model is constructed, the cell The program variables metabolic flux model is calculated.
(A) The intracellular metabolic flux model includes an exchange reaction between an amino acid and a protein produced by the incorporation of the amino acid, and the analysis value of the cell includes the isotope distribution of amino acids and protein degradation included in the intracellular metabolic flux model. include analytical value of isotope distribution in an object, analytical values of isotope distribution of amino acids, 1) measuring the isotopic distribution of the isotope distribution and degradation of proteins in amino acid, obtained in 2) step 1) Using the optimization algorithm, the result is corrected by a step of optimizing the degree of synthesis and decomposition of amino acids and proteins expressed as a variable defined by the exchange reaction coefficient .
(B) an intracellular metabolic flux model includes amino San及 beauty / or its metabolic intermediates, analytical values of cells in the medium, the uptake rate into cells of amino acids that are not isotopically labeled, as well as, include analytical value of isotope distribution in the amino acid and / or its metabolic intermediates, analytical values of isotope distribution of the amino acid and / or its metabolic intermediates, a speed obtained by subtracting a speed that is incorporated into the protein from the uptake rate Assuming a rate to the metabolic pathway, it is corrected by the effect on the isotopic distribution of the amino acid and / or its metabolic intermediate by the rate to the metabolic pathway.
(C) The intracellular metabolic flux model includes a carbon dioxide fixation reaction and a carbon dioxide generation reaction, and the carbon dioxide used in the fixation reaction is assumed to be carbon dioxide generated in the generation reaction. 9. A computer-readable recording medium on which the program according to claim 8 is recorded.

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