KR20090101652A - Systems-oriented approach for predicting drug targets in vibrio family - Google Patents

Abstract

PURPOSE: A prediction of drug target of Vibrio sp. using system method is provided to narrow the number of drug target candidate to diseases and combine effective drug target. CONSTITUTION: A method for screening Vibrio sp. drug target enzyme or its gene comprises: a step of constructing metabolic network model of Vibrio sp.; a step of selecting drug target enzyme candidates (I) and (II); a step of selecting overlapped drug target enzyme candidate as a first drug target enzyme group; a step of selecting a second drug target enzyme group; a step of additionally selecting essential metabolites comprising metabolites of which enzyme reaction formula is not related to any human protein; and a step of variously combining among a group comprising the first and second drug target genes.

Description

System-oriented Approach for Predicting Drug Targets in Vibrio family}

The present invention utilizes computer system techniques to provide Vibrio A method for predicting drug targets of the genus microorganisms, and more specifically, to build a metabolic network model of V. vulnificus , and then chokepoint analysis, single enzyme deletion and / or essential Each method of simulating metabolite essentiality is applied to metabolic models to predict drug targets.

To date, efforts are underway to find drug targets for developing effective antipathogenic drugs.

However, the methodologies so far do not fully utilize the biological information currently available. Determining which gene product is the ideal drug target to kill pathogenic microorganisms is difficult, and it is technically difficult to determine the lethality of a gene by deleting every single gene in the pathogenic microorganism. . In addition, drug targets are mostly determined by interactions between complex cell components, not one gene, and even when each gene is not lethal, the combination is very complicated, such as lethality in a plurality of gene deletions.

Therefore, it is very important to understand cellular mechanisms and interactions between microbial cell components in order to develop effective drugs that target pathogenic microorganisms. Therefore, the development of metabolites and metabolic networks through the development of genomic information and functional genomics has added the importance in understanding the interaction of genes and proteins to compose cellular components and constructing metabolic networks to develop effective drugs. .

Indeed, when metabolic network information through genomic information is used to identify new metabolic pathways that are not found in mammalian cells in pathogenic microorganisms, we have developed therapies that target these metabolic properties and thus are not pathogenic to human cells. Only cells can attack specifically. On the other hand, if it is found that certain metabolic pathways are essential for survival of pathogenic microorganisms, it is possible to develop drugs that inhibit the metabolic pathways. Once drugs for pathogenic microorganisms are developed, it is expected that drugs that inhibit other similar pathogenic microorganisms can be easily obtained using similar compounds.

Analytical and predictive techniques through metabolic networks have shown promise with rapidly growing genomic information. In particular, the metabolic network models of each microorganism are combined with mathematical models and optimization techniques, making it possible to predict the response of the metabolic network after removal or addition of genes (Lee et al., Trends Biotechnol ., 23: 349 , 2005). . In addition, metabolic flow analysis using metabolic networks shows the ideal metabolic flow of cells even when dynamic information is not required, and it is known to accurately simulate and predict cell behavior (Papin, J. et al., Nat. Rev. Mol. Cell Biol . , 6:99, 2005).

Metabolic flow analysis uses the mass balance and cell composition information of biochemical equations to obtain the ideal metabolic flow space that cells can reach, and aims to maximize or minimize specific objective functions through optimization methods (maximization of cell growth rate or specific perturbation). Minimization of metabolic regulation by In addition, metabolic flow analysis can generally be used to confirm the lethality of specific genes of a desired metabolite through strain improvement, and can be used to determine the metabolic network characteristics within the strain. In addition, various studies have been reported applying metabolic flow analysis methods to predict the flow changes in metabolic networks caused by the removal or addition of genes.

Vibrio , a type of pathogenic microorganism that causes a variety of infectious diseases in humans, is an anaerobic gram-negative bacillus that exists in the oceans and estuaries and is isolated from fresh water, rivers, ponds, and lakes. More than 30 species belong to the genus Vibrio , 12 of which infect humans. Intestinal infections are caused by V. cholerae and V. parahaemolyticus . Intestinal infections such as blood, wounds, eyes, ears, and bile can also occur. In Korea, Vibrio sepsis often occurs in summer, which is caused by V. vulnificus , which is mainly caused by cirrhosis and cancer patients and has a poor prognosis. .

Vibrio vulnificus is mainly found in estuaries and is a pathogen microorganism that infects a variety of animals and seafood, including humans (Gulig et al., J. Mcirobiol . , 43: 118, 2005). Ingestion of seafood infected with V. vulnificus or contact with the microorganisms of the human body can cause pneumonia, gastroenteritis, and wound infection. In particular, V. vulnificus is known to be very fast in human body when infected. Contact with the pathogen can kill within 24 hours. It is known that mortality due to pulmonary disease is up to 75%, and mortality due to wound infection is reported to be up to 50%.

However, it is known that antibiotics that can effectively cope with the pathogen are not yet developed (Chonnam National University of Medicine), and therefore, there is an urgent need for systematic drug target prediction and corresponding anti-pathogenic drug development.

In particular, in the case of V. vulnificus , the genome sequence translation of the two strains is completed (Chen et al., Genome Res . , 13: 2577, 2003), the present inventors look at the metabolism of V. vulnificus from the whole point of view, rather than the strain manipulation using partial metabolic information using metabolic flow analysis techniques, and manipulates specific genes in the overall metabolic flow. By identifying the effects, we have found the possibility of developing a method to accurately predict the drug target of pathogenic microorganisms.

Therefore, the present inventors established a metabolic network model of V. vulnificus , and then simulated three kinds of simulation methods: single enzyme deletion based on chokepoint and metabolic flow analysis, and metabolite essentiality analysis. Are respectively applied to metabolic models to predict drug targets, and further, enzyme reaction groups overlapping each other in the chokepoint and essential enzyme reaction schemes; And said Vibrio by the enzyme to reaction scheme (reaction outgoing) group consuming the essential metabolite (metabolite essential) reduce the number of the next likely candidate in the following, different combinations of these evaluates the result of the network structure analysis Theoretically, the present inventors have found that an ideal combination for efficiently controlling genus microorganisms can be made, and the present invention has been completed.

Based on the purpose of Vibrio (Vibrio) in microbial metabolic network model structure of the present invention, the choke point (chokepoint), the required enzyme Scheme analysis based on the metabolic flux analysis (single enzyme deletion) and / or the required metabolite analysis (metabolite essentiality) The present invention provides a method for screening an enzyme or a gene encoding the enzyme that is a drug target of the microorganism using three simulations.

Another object of the present invention is to provide drug target enzymes for Vibrio genus microorganisms and gene groups encoding them obtained by the above method.

In order to achieve the above object, in the present invention

comprising the steps of: (a) establish the metabolic network model of the microorganism of the genus Vibrio (Vibrio);

(b) the established Vibrio (Vibrio) spp Selecting chokepoints, which are the only enzymes that consume or produce a particular metabolite in the metabolic network, as drug target enzyme candidates (I);

(c) the microorganism of the genus Vibrio Selecting the drug target enzyme candidates (II) as blocked enzyme reactions when cell growth does not occur when the linear programming is applied while blocking the enzyme reactions one by one with respect to the enzyme reactions constituting the metabolic network;

(d) comparing the drug target enzyme candidates (I) and (II) which are the results obtained in steps (b) and (c) and selecting the overlapping drug target enzyme candidates as the first drug target enzyme group;

(e) from the group of primary drug target enzymes selected from (d) above

(i) selecting those that do not have homology with human proteins and do not have isozymes as the second drug target enzyme group, and the genes encoding the second target enzymes as the first drug target gene group do or,

(ii) a microorganism that is not homologous to human protein and is of the genus Vibrio Selecting those involved in one or more enzymatic reaction schemes forming a metabolic network as a second drug target enzyme group and selecting genes encoding the second target enzymes as a first drug target gene group;

(f) a Vibrio (Vibrio) spp built in the step (a) Determining that certain metabolites as essential metabolites (I) when the growth rate of the cell is zero when blocking all enzymatic reactions of specific metabolites on the metabolic network;

(g) further screening for essential metabolites where all of the essential metabolites (I) determined in (f) above consume each essential metabolite only if they do not have homology with human proteins, Selecting genes encoding enzymes for use in the enzyme reaction consuming the further selected essential metabolites (II) as a second drug target gene group;

(h) creating various combinations of genes selected from the group consisting of the first drug target gene group selected in step (e) and the second drug target gene group selected in step (g); And

(i) For the reaction schemes in which the enzymes encoding the genes forming the combinations made in step (h) are involved, the average distance on the metabolic network is calculated and the genes forming the combinations of cases where the average distance is far from the drug targets are used. provides the selection or, whereby the enzyme encoded by comprising the step of selecting the drug target enzyme, Vibrio (Vibrio) drug target of the enzyme in microorganisms, or the screening method of the gene to gene.

At this time, the secondary drug target enzyme selected in the step (e) is not a human protein homologous relationship without having the isozyme (isozyme), the Vibrio (Vibrio) spp It is most preferable to select among the first drug target enzyme candidates satisfying the above three conditions when participating in one or more enzyme reaction schemes constituting the metabolic network.

In the above method, step (b) or step (c) may alternatively be selected and applied.

The three simulations of chokepoint, single enzyme deletion based on metabolic flow analysis, and metabolite essentiality according to the present invention are to look at different aspects of cells with different approaches. By applying these drug targeting methods simultaneously to the Vibrio genus microbial metabolism model, systematic and rational drug targets can be predicted. In other words, the results of the three simulation methods can be selected to further narrow the number of candidate drug target candidate groups for diseases caused by Vibrio genus pathogens, and to make effective drug target combinations from these diseases. It is useful for treatment and prevention.

FIG. 1 is a schematic diagram illustrating a process of creating a combination of drug targets from drug target prediction through chokepoint, essential enzyme reaction analysis based on metabolic flow analysis, and essential metabolite analysis simulation.

Figure 2 is a diagram illustrating the definition of chokepoints, the oval refers to the enzyme scheme, the square represents the metabolite.

3 is a schematic diagram illustrating a method of predicting a drug target according to an essential enzyme reaction scheme obtained from an enzyme reaction blocking simulation using metabolic flow analysis.

Figure 4 is a schematic diagram illustrating an essential metabolite analysis simulation using metabolic flow analysis, the oval refers to the enzyme reaction scheme, the square represents the metabolite.

5 shows a method of making three types of drug target combinations.

FIG. 6 is a diagram illustrating a process of creating a reaction node on a line representing a reaction between metabolites to calculate an average distance between enzyme reactions present in each drug target combination, wherein an ellipse refers to an enzyme reaction, and a rectangle represents metabolism. Indicates a product

The present invention relates to a screening method of a drug target enzyme or gene of the microorganism of the genus In one aspect, Vibrio (Vibrio). A schematic process is shown in FIG. 1 and the detailed description of each step is as follows.

(1) First, build a Vibrio (Vibrio) metabolic network models in microorganisms.

In one embodiment of the invention it was used to establish the metabolic network model of Vibrio vulnificus from the Vibrio (Vibrio) spp. In the following description, the metabolic network model of V. vulnificus is constructed.

At this time, it is desirable to construct a genome-level metabolic network using various known databases and experimental results.

(2) Next, the constructed V. vulnificus Chocopoints, the only enzymes that consume or produce certain metabolites in the metabolic network, are selected as drug target enzyme candidates (I).

In the present invention, "chokepoint" refers to an enzyme reaction scheme that uniquely consumes or produces a specific metabolite in a metabolic network model, and was devised by the inventors for drug targets (Yeh et al., Genome Res. 14 ( 5): 917, 2004). The chokepoint is based on the assumption that inhibiting these enzymatic reactions would make it impossible to consume or produce the specific metabolites involved in the enzyme and thus eventually stop the cell growth of the target microorganism.

Mass preservation formulas are established for all the metabolites constituting the constructed V. vulnificus metabolic network model, from which the enzymes that consume and produce each metabolite are selected as chokepoints (FIG. 2).

(3) On the other hand, when the linear scheme is applied while blocking the enzyme reactions one by one for the enzyme reactions constituting the V. vulnificus metabolic network, the blocked enzyme reaction (required enzyme reaction when the cell growth does not occur) ) Are selected as drug target enzyme candidates (II).

At this time, in order to mathematically express the constructed metabolic network, all metabolites constituting the constructed metabolic network model, the metabolic pathway of the metabolite and the stoichiometric matrix S (stoichiometric matrix) in the metabolic pathway ( S _The metabolic flow vector ( ν _j , the metabolic flow of the j- th metabolic reaction) can be calculated using the stoichiometric coefficient of the i- th metabolite in the _ij , j- th reaction.

Here, the change in the metabolite concentration X over time can be expressed as the sum of the flows of all metabolic reactions. Assuming that the amount of change of X with time is constant, that is, under the assumption of a quasi-steady state, the amount of change of the metabolite concentration with time may be defined by Equation 1 below.

Where Sν : Change in X over time, X : concentration of metabolite, t: time)

In the above-described stoichiometric matrix, the reaction scheme to be optimized, that is, maximized or minimized, is set as the objective function and the metabolic flow in the cell is predicted using linear programming (Kim et al., Mol Biosyst . 4 (2)). : 113, 2008). In one embodiment of the present invention, the cell growth rate was optimized by setting an enzyme scheme representing the constituents of the cells in the matrix S as the objective function.

In addition, in applying the linear programming method for metabolic flow analysis, it should be performed under the assumption that all the nutrients necessary for the growth of the cell can be ingested. This is because when pathogenic microorganisms grow in the host, various nutrients can be taken from the host. Enzyme reaction may appear to be essential only under certain conditions. Thus, if metabolic flow analysis is applied on the assumption that all the nutrients can be ingested, it is possible to predict an enzyme reaction which is essential at all times.

In the present invention, in the constructed metabolic network model, the enzyme reaction blocking simulation is performed to increase the cell growth rate in a state in which the metabolic flow of the enzyme reaction to be blocked in the metabolic flow vector ν is fixed at 0 (= v _j ). Set as objective function and execute linear programming.

When the linear programming is performed with all the enzyme reactions constituting the model blocked one by one, if the cell growth becomes zero despite applying the reaction that maximizes the cell growth rate, the blocked enzyme reaction is necessary. Define by reaction scheme and select it as a drug target candidate (II). A schematic process of this step is shown in FIG.

(4) Compare drug target candidates (I) and (II), which are the results obtained in steps (2) and (3), to select overlapping drug target enzyme candidates as primary drug target enzyme groups.

In other words, this step is to integrate the drug target enzyme candidates obtained from the enzyme reaction blocking simulation using chokepoint analysis and metabolic flow analysis.

Only the overlapping targets are selected by combining the drug target results according to the essential enzyme reaction scheme based on the chokepoint analysis in step (2) and the metabolic flow analysis in step (3) (primary drug target enzyme group). This is to significantly reduce the number of targets with only the biologically most probable candidate drug target enzymes.

The integrated primary drug target enzyme group is a result of considering both the network topology of the metabolic network and the metabolic function reflecting the metabolic flow of each enzyme reaction constituting the metabolic network. have.

In one embodiment of the present invention, only the step (2) may be performed or the step (3) may be performed to obtain drug target enzyme candidates. That is, steps (2) and (3) may be alternatively performed. However, it is preferable to obtain the overlapping drug target enzyme candidates most preferably through the above (4) step of performing both the above (2) and (3) and synthesizing the results.

If only step (2) or step (3) is performed, each of the results will be selected as the primary drug target enzyme group, and will be skipped to step (5) described below.

(5) there is no homology with human proteins among the primary drug target enzyme groups selected by the above method; It does not have a homogeneous enzyme (isozyme) and / or the Vibrio (Vibrio) spp Those involved in one or more enzyme reactions that make up the metabolic network are selected and selected as secondary drug target enzyme groups.

In this step, duplicate primary drug target enzymes are further screened based on the following three criteria in the method of enzymatic reaction blocking simulation based on chokepoint analysis and metabolic flow analysis. This is to screen only with enzymes that are more effective as drug targets.

(i) First of all, essentially, the genes encoding the primary drug target enzymes or the amino acid sequence are selected as those having no homology to all proteins present in humans.

Since the present invention is directed to humans as hosts, human genomic information is used as a database.

Drugs developed by targeting specific genes or enzymes act on the basis of the sequence of the genes or enzymes, so if the predicted genes or enzymes exist in humans, the developed drugs may act on human proteins and cause side effects. Because. In examining the homology, any data can be used as long as those skilled in the art can identify the homology regardless of the amino acid sequence or the gene sequence. At this time, a conventional BLASTP (when using an amino acid sequence) or BLAST (when using a gene sequence) program can be used. In one embodiment of the present invention used the BLASTP program.

(ii) remove isozymes present among the primary drug target enzymes.

This is because isozymes having biochemical functions such as enzymes essential for inhibiting cell growth are very inefficient for drug targets because these enzymes must be removed simultaneously.

(iii) among the primary drug target enzymes or genes encoding them, previously constructed V. vulnificus Only those with multifunctionality that participate in more than one scheme on the metabolic network are selected.

This allows for more efficient drug targeting since the target enzyme or genes are deleted by the drug, causing one or more related reactions to stop in the vibrio pathogen metabolism.

At this stage, in order to prevent side effects of the drug, the conditions of (i) must be essentially satisfied, but the conditions of (ii) and (iii) may alternatively be satisfied. However, most preferably, a group of drug target enzymes satisfying all of the conditions (i), (ii) and (iii) is selected.

Drug target enzymes satisfying the above conditions are selected as the second drug target enzyme group, and genes encoding the second drug target enzymes are selected as the first drug target gene group.

(6) As described above, by virtue of chokepoint analysis and essential enzyme reaction results, potential drug target candidates are selected, while the vibrio genus microorganism constructed in step (a) is obtained . When blocking all enzymatic reactions of specific metabolites consumed on the metabolic network, determining the specific metabolites as essential metabolites (I) when the growth rate of the cell is zero is performed.

In general, the method of determining the cell growth rate according to a specific gene deletion in the conventional metabolic flow analysis uses a method of inactivating each corresponding reaction scheme. However, in this case, in order to identify a cell growth deterioration phenomenon caused by two or more gene deletions, there has been a disadvantage in that all cases of two or more combinations must be calculated.

On the contrary, in the present invention, by defining the 'essentiality' of each metabolite and examining the properties of each metabolite, it is easy to identify the phenomenon of cell growth caused by the deletion of two or more genes. That is, the present invention provides a method of defining and using 'essentiality' of metabolites constituting the metabolic network of the target microorganism as follows.

The 'essentiality' of metabolites is the effect of cells on the growth of cells when they are not consumed by metabolism. The rate of cell growth for each metabolite under certain conditions is determined by metabolic flow analysis. The essentiality of metabolites can be determined by investigating (Kim et al., Proc . Natl . Acad . Sci . USA , 104: 13638, 2007).

That is, in the metabolic flow analysis process of metabolites constituting the metabolic network of the target microorganism, all metabolic reactions consuming each metabolite are simultaneously blocked (deleted), and the metabolic flow value of the corresponding reaction equation is fixed to 0, If the growth rate of the cell at this time is selected as an essential metabolite (Fig. 4).

Where j _m is the consumption equation of each metabolite and V _jm represents the metabolic flow value of the consumption equation.

Essential metabolite analysis applies Equation 2 as an additional constraint while simultaneously blocking (deleting) all metabolic reactions consuming each metabolite in the stoichiometric matrix. By fixing the metabolic flow value of the consumption equation to 0, the case where the cell growth rate is 0 is selected as an essential metabolite.

During the analytical process to determine the essentiality, the reason for not inactivating the metabolism produced without consuming a given metabolite is that even if the metabolite is non-essential, the metabolism that produces the metabolite is Since other essential metabolites are also likely to be produced, if cell growth is inhibited due to inactivation of the metabolic reaction, it may be misunderstood that non-essential metabolites are essential.

(7) Next, essential metabolites (II) in the case where all of the essential metabolites (I) determined in (6) above consume all the essential metabolites, only those that do not have homology with human proteins (II) Perform the step of screening.

In this step, further screening of enzyme reactions that consume essential metabolites predicted through the metabolic flow analysis, based on homology with humans, further reduces the number of possible next essential metabolites.

If any of the enzymes in the consumption schemes of the essential metabolites are statistically similar to human proteins, those essential metabolites and their consumption schemes are no longer considered as drug targets.

In examining the homology, a human genome information can be used as a database, and a BLASTP program can be used when using an amino acid sequence or a BLAST program can be used when using a gene sequence. However, any data can be used as long as those skilled in the art can identify homology regardless of amino acid sequence or gene sequence. In one embodiment of the present invention used the BLASTP program.

The genes and amino acid sequences encoding all of the enzymes consuming each of these additionally selected essential metabolites (II) are significantly different from those of the host protein and, as a result, are structurally different from human proteins.

This process inhibits multiple enzyme consumption reactions simultaneously with a single drug, such as when the metabolite analog is used as a drug, but minimizes the possibility of side effects from the drug because the protein does not exist in the host human. It's work.

In this step, a group of genes encoding all enzymes consuming each of the essential metabolites (II) further selected is selected as the second drug target gene group.

(8) Various drug target combinations are constructed using genes selected from the group consisting of the first drug target gene group selected in step (5) and the second drug target gene group selected in step (7).

In one embodiment of the present invention, the combination of the drug target genes is a combination consisting of only the first drug target gene group predicted in step (5), as shown in Figure 5 (Fig. 5A), the step (5) and The combination consisting of the first and second drug target gene groups predicted in step (7) (FIG. 5BCDE), the combination consisting only of the second drug target gene group predicted in step (7) (FIG. 5F), etc. can be made. have.

When a drug target combination is made using the gene group predicted in step (7), in the case of a nonessential reaction in which some of the essential metabolites have their consumption reactions blocked at the same time, cell growth is inhibited. Genes encoding enzymes involved in the consumption scheme are always configured to belong to the same combination.

As used herein, the term "non-essential reaction formula" refers to the above enzyme reaction when there are two or more enzyme reactions related to the consumption of a specific essential metabolite, in which case all related enzyme reactions should be blocked simultaneously. However, cell growth is inhibited. Therefore, the genes encoding these enzymes must also be configured to belong to the same combination.

As such, the genes encoding the enzymes involved in the non-essential reaction equation among the gene groups predicted in the step (7) must be configured in the same combination, but otherwise may be separated and configured in different combinations.

However, most preferably, the genes involved in the consumption equations of the essential metabolite predicted in step (7) are always configured in the same set to belong to the same combination. This is because when metabolite analogues are used as drugs, the consuming enzymes can attack simultaneously in the same group.

As such, when the combination is made of the drug target gene group of the present invention, there is an advantage of simultaneously targeting multiple sites of vibrio pathogen metabolism.

Typically, the biggest problem with anti-pathogen drugs is that the resistance of the pathogen to the drug occurs quickly, which is mainly caused by a single endogenous mutation of the enzyme-targeting enzyme gene. Combination has the advantage of being able to simultaneously target several sites of vibrio pathogen metabolism, thereby minimizing the resistance of vibrio pathogens, and reliably controlling the growth of vibrio pathogens in humans.

(9) Finally, with respect to the various gene combinations created in step (8), the average distance on the metabolic network is calculated, and the genes forming the combination when the average distance is far are selected as drug target genes.

This step uses the metabolic network structure of the pathogen to calculate the distance and find the ideal combination of enzyme reactions corresponding to the genes that make up the drug target combination.

In general, distant enzyme reactions in metabolic networks are believed to participate in different cellular metabolic functions (Ma et al. Bioinformatics , 20: 1870, 2004). In inhibiting cell growth, targeting multiple sites in cell metabolism with different functions simultaneously is more effective than intensively targeting only one part in cell metabolism (Kitano. Nat. Rev. Drug. Discov. 6: 202, 2007). Therefore, among the drug target combinations made in the above 8, the longest average distance between the combination schemes is regarded as an ideal combination. Therefore, the combination with the longest average distance is the most effective drug combination.

The distance between enzyme reactions can be calculated by analyzing the metabolic network structure of the pathogen (Ma et al. Bioinformatics , 20: 1870, 2004) (FIG. 6). In general metabolic networks, nodes point to metabolites, and edges show enzymatic reaction schemes (FIG. 6A).

In the present invention, the term "shortest path" in the network structure analysis refers to the number of lines passing in the shortest path among all possible paths existing between two nodes.

The criterion for calculating the distance between two reaction schemes using a network structure can be variously applied. However, in one embodiment of the present invention, in order to calculate the distance between enzyme reaction schemes, a node of the reaction scheme is additionally added to a line representing the reaction between metabolites. The distances of these reaction nodes were obtained (FIG. 6B). For example, in FIG. 6A the distance between the metabolite node A and node F is two. In conclusion, the average distance of enzyme reactions that form each drug target combination is calculated as the average of the distances between each reaction.

Of the various drug target combinations obtained in the present invention, those with far average distances of enzymatic reaction schemes are considered as more possible combinations.

The enzymes and genes selected by this method are determined as drug target enzymes and drug target genes for Vibrio genus microorganisms, respectively.

As described above, the present invention relates to a method for predicting a drug target of Vibrio sp. Microorganisms, as an embodiment, a metabolic network model of V. vulnificus is constructed, and then (A) a metabolic network constructed by chokepoint analysis. Predicts only enzyme reactions that consume or produce specific metabolites in drug targets, and (B) enzymes that form a metabolic network model based on the distribution of metabolic flows in cells using enzyme reaction deletion methods using metabolic flow analysis. By blocking the reactions one by one, the enzyme reactions and genes that are essential for cell growth are identified, and (C) essential metabolite analysis methods are used to simultaneously block all consumption reactions of each metabolite to maintain cell growth. Metabolites and genes involved in them are screened.

Among these methods (A), (B) and (C), if necessary, (A) and (C); Or (B) and (C); Or (A), (B) and by the combination of (C) reduce the number of the candidates that likely next following, various combinations thereof can effectively control the Vibrio spp evaluates the result of the network structure analysis Predict the ideal combination.

The present invention also provides, in another aspect, a drug target enzyme candidate selected by the chokepoint analysis of step (2) and a gene group encoding the same; Drug target enzyme candidates selected by applying the linear programming method according to the metabolic flow analysis of step (3) and a gene group encoding the same; And, the drug target enzyme candidate and the gene group encoding the overlapping essential enzyme reaction results using the chokepoint analysis and linear programming of step (4); And drug target enzyme candidates and gene groups encoding the same, which are selected as more probable ones based on the presence or absence of isozymes, homology with human proteins, and multifunctionality in step (5).

The present invention also provides, in another aspect, essential metabolites determined using metabolic flow analysis in step (6), enzymes used to consume them, and a gene group encoding the same; And essential metabolites consumed by enzymes not homologous to human proteins, further selected in step (7), enzymes used to consume them and gene groups encoding them.

The present invention also, in another aspect, a variety of drug target combinations made in step (8); And a combination in the case where the average distance is farther determined in step (9), and further comprises a drug target gene and enzyme encoded by the combination.

For various combinations of drug target candidates according to the present invention, selecting an ideal combination based on network analysis can yield a drug target combination that can effectively inhibit pathogens.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, the following examples are only illustrated for the drug target screening method using V. vulnificus as a model system, but it is apparent to those skilled in the art from the present disclosure that the present invention also applies to vibrio genus pathogenic microorganisms other than V. vulnificus. Do.

Example  One: V. vulnificus The metabolic network

To predict the drug target of V. vulnificus using a computer, a genome-level metabolic network was constructed using various databases and experimental results.

KEGG (Kanehisa et al. Nucleic Acids Res , 34: D354, 2006), TransportDB (Ren et al., PLoS Comput . Biol . , 1: e27, 2005), MetaCyc (Caspi et al. Nucleic Acids Res. , 36: D623, 2008), to build an early version of the metabolic network and to clarify the relationship between the directionality, gene and protein of enzyme reactions based on genomic information.

The metabolic network of V. vulnificus consists of 945 biochemical equations and 765 metabolites. The information of the metabolic network contains the following 670 gene information. The predicted drug targets were selected from these genes.

VV10014 (dGTP triphosphohydrolase),  VV10053 (Histidine ammonia-lyase),  VV10060 (Putative beta-ketoacyl-ACP reductase),  VV10061 (Putative beta-ketoacyl-ACP synthase),  VV10136 (Long-chain-fatty-acid-CoA ligase),  VV10143 (Formyltetrahydrofolate hydrolase),  VV10145 (Arginyl-tRNA synthetase),  VV10154 (Succinyl-CoA synthetase,  alpha subunit),  VV10155 (Succinyl-CoA synthetase,  beta subunit),  VV10156 (2-oxoglutarate dehydrogenase complex,  E2 component,  dihydrolipoamide succinyltransferase),  VV10157 (2-oxoglutarate dehydrogenase complex,  E1 component),  VV10158 (Succinate dehydrogenase,  iron-sulfur protein),  VV10159 (Succinate dehydrogenase,  flavoprotein subunit),  VV10160 (Succinate dehydrogenase,  hydrophobic membrane anchor protein),  VV10161 (Succinate dehydrogenase,  cytochrome b556 subunit),  VV10162 (Citrate synthase),  VV10169 (Phosphoglucomutase),  VV10176 (Glutaminyl-tRNA synthetase),  VV10177 (# N / A),  VV10179 (PTS system,  N-acetylglucosamine-specific IIBC component),  VV10180 (N-acetylglucosamine-6-phosphate deacetylase),  VV10183 (Asparagine synthase B,  glutamine-hydrolyzing),  VV10187 (Ferrochelatase),  VV10188 (Adenylate kinase),  VV10209 (Cysteine synthase A),  VV10212 (PTS system,  glucose-specific IIA component),  VV10236 (Glutamyl-tRNA synthetase),  VV10246 (Pseudouridine synthase family 1 protein),  VV10248 (5'-nucleotidase precursor),  VV10249 (2-dehydro-3-deoxyphosphooctonate aldolase),  VV10254 (Glutamyl-tRNA reductase),  VV10256 (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK)),  VV10257 (Ribose-phosphate pyrophosphokinase),  VV10265 (2-polyprenyl-6-methoxyphenol hydroxylase and related FAD-dependent oxidoreductase),  VV10272 (Leucyl-tRNA synthetase),  VV10286 (Serine hydroxymethyltransferase),  VV10288 (Trehalose-6-phosphate hydrolase),  VV10289 (PTS system,  trehalose-specific IIBC component),  VV10291 (Histidinol phosphatase and related phosphatase),  VV10314 (Geranylgeranyl pyrophosphate synthase),  VV10315 (1-deoxyxylulose-5-phosphate synthase),  VV10316 (Phosphatidylglycerophosphatase A),  VV10317 (Thiamine monophosphate kinase),  VV10319 (Riboflavin synthase beta-chain),  VV10321 (3, 4-dihydroxy-2-butanone 4-phosphate synthaseGTP cyclohydrolase II),  VV10322 (Riboflavin synthase alpha chain),  VV10323 (Riboflavin-specific deaminase),  VV10325 (Gamma-glutamyl phosphate reductase),  VV10326 (Glutamate 5-kinase),  VV10329 (Xanthine-guanine phosphoribosyltransferase),  VV10333 (Aminoacyl-histidine dipeptidase),  VV10340 (Phosphoribosylformylglycinamidine (FGAM) synthase),  VV10344 (Zn-dependent alcohol dehydrogenase,  class III),  VV10366 (Putative inorganic polyphosphateATP-NAD kinase),  VV10414 (Transcriptional regulator,  LysR family),  VV10418 (GMP synthase (glutamine-hydrolyzing)),  VV10419 (GMP synthase (glutamine-hydrolyzing)),  VV10426 (Histidyl-tRNA synthetase),  VV10427 (Enzyme involved in the deoxyxylulose pathway of isoprenoid biosynthesis GcpE),  VV10430 (Nucleoside diphosphate kinase),  VV10449 (Isocitrate lyase),  VV10450 (Malate synthase A),  VV10465 (Exopolyphosphatase),  VV10484 (Pseudouridylate synthase,  23S RNA-specific),  VV10487 (Chorismate mutasepephenate dehydratase),  VV10494 (Chorismate mutaseprephenate dehydrogenase),  VV10495 (Phospho-2-dehydro-3-deoxyheptonate aldolase,  tyr-sensitive),  VV10504 (Penicillin tolerance protein LytB),  VV10507 (Isoleucyl-tRNA synthetase),  VV10508 (FAD synthase),  VV10516 (Thymidylate synthase),  VV10526 (Lysyl-tRNA synthetase (class II)),  VV10543 (Threonine synthase),  VV10544 (Homoserine kinase),  VV10545 (Aspartokinasehomoserine dehydrogenase,  threonine-sensitive),  VV10553 (Glutamate synthase,  large subunit),  VV10554 (Glutamate synthase,  small subunit),  VV10555 (Glutamate synthase,  large subunit),  VV10556 (Glutamate synthase,  small subunit),  VV10558 (Nucleoside phosphorylase),  VV10559 (Cobalamin biosynthesis protein CobDCbiB),  VV10565 (Carbamoylphosphate synthase large subunit (split gene in MJ)),  VV10566 (Carbamoylphosphate synthase small subunit),  VV10567 (Dihydrodipicolinate reductase),  VV10571 (UDP-3-O-acyl-N-acetylglucosamine deacetylase),  VV10577 (UDP-N-acetylmuramate-alanine ligase),  VV10578 (UDP-N-acetylglucosamine-N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase),  VV10580 (UDP-N-acetylmuramoylalanine-D-glutamate ligase),  VV10581 (Phospho-N-acetylmuramoyl-pentapeptide-transferase),  VV10582 (UDP-N-acetylmuramoylalanyl-D-glutamyl-2,  6-diaminopimelate-D-alanyl-D-alanyl ligase),  VV10583 (UDP-N-acetylmuramylalanyl-D-glutamate--2,  6-diaminopimelate ligase),  VV10591 (Phosphoheptose isomerase),  VV10595 (Ubiquinol--cytochrome c reductase,  cytochrome c1),  VV10596 (Ubiquinol--cytochrome c reductase,  cytochrome B),  VV10597 (Ubiquinol--cytochrome c reductase,  iron-sulfur subunit),  VV10610 (MutTnudix family protein),  VV10613 (ADP-heptose synthase,  bifunctional sugar kinaseadenylyltransferase),  VV10623 (Putative undecaprenol kinase (Bacitracin resistance protein)),  VV10625 (Dihydroneopterin aldolase FolB),  VV10638 (PTS system,  mannitol-specific IIABC component),  VV10639 (Mannitol-1-phosphate 5-dehydrogenase),  VV10641 (Glucosamine--fructose-6-phosphate aminotransferase),  VV10644 (Pyruvate kinase),  VV10647 (Acetolactate synthase,  small (regulatory) subunit),  VV10648 (Acetolactate synthase III,  large subunit),  VV10649 (Long-chain acyl-CoA synthetase (AMP-forming)),  VV10654 (2-isopropylmalate synthase),  VV10655 (3-isopropylmalate dehydrogenase),  VV10656 (3-isopropylmalate dehydratase,  large subunit),  VV10657 (3-isopropylmalate dehydratase,  small subunit),  VV10662 (Pyridoxal phosphate biosynthesis protein PdxA),  VV10665 (Bis (5`-nucleosyl) -tetraphosphatase),  VV10666 (Dihydrofolate reductase),  VV10673 (Malate dehydrogenase),  VV10678 (1-acyl-sn-glycerol-3-phosphate acyltransferase),  VV10679 (UDP-N-acetylglucosamine enolpyruvyl transferase),  VV10688 (Low specificity phosphatase (HAD superfamily)),  VV10705 (3-polyprenyl-4-hydroxybenzoate decarboxylase),  VV10707 (Fructose-1, 6-bisphosphatase),  VV10708 (Inorganic pyrophosphatase),  VV10723 (Adenylylsulfate kinase),  VV10725 (Sulfate adenylate transferase subunit 1),  VV10726 (Sulfate adenylate transferase,  subunit 2),  VV10727 (Uroporphyrinogen-III methylase),  VV10728 (2`, 3`-cyclic-nucleotide 2`-phosphodiesterase),  VV10774 (Nucleotide sugar dehydrogenase),  VV10779 (UDP-N-acetyl-D-mannosaminuronate dehydrogenase),  VV10780 (UDP-N-acetylglucosamine 2-epimerase),  VV10796 (ADP-L-glycero-D-mannoheptose-6-epimerase),  VV10797 (Lipid A biosynthesis (kdo) 2- (lauroyl) -lipid IVA acyltransferase),  VV10799 (3-deoxy-D-manno-octulosonic-acid transferase (KDO transferase)),  VV10803 (CMP-N-acetylneuraminic acid synthetase),  VV10804 (Putative nucleoside-diphosphate-sugar pyrophosphorylase containing CBS domain),  VV10808 (Sialic acid synthase),  VV10814 (Putative KDO kinase WavC),  VV10815 (Diacylglycerol kinase),  VV10819 (Phosphopantetheine adenylyltransferase),  VV10828 (DNApantothenate metabolism flavoprotein),  VV10830 (Lipid A biosynthesis lauroyl acyltransferase),  VV10831 (Orotate phosphoribosyltransferase),  VV10850 (Guanylate kinase),  VV10852 (Guanosine-3 ', 5'-bis (diphosphate) 3'-pyrophosphohydrolase SpoT),  VV10854 (ATP-dependent DNA helicase RecG),  VV10881 (Phosphoenolpyruvate carboxykinase (ATP)),  VV10889 (Glutamine synthetase (glutamate-ammonia ligase)),  VV10894 (Oxygen-independent coproporphyrinogen III oxidase),  VV10902 (Delta-aminolevulinic acid dehydratase),  VV10907 (Putative ubiquinone biosynthesis protein AarF),  VV10909 (Ubiquinonemenaquinone biosynthesis methlytransferase UbiE),  VV10933 (NAD (P) H-flavin reductase),  VV10935 (3-polyprenyl-4-hydroxybenzoate decarboxylase and related decarboxylase),  VV10940 (Guanosine-5'-triphosphate, 3'-diphosphate pyrophosphatase),  VV10963 (Thiamine monophosphate synthase ThiE),  VV10964 (Thiamine biosynthesis protein ThiC),  VV10978 (Protoporphyrinogen oxidase),  VV10981 (Fatty oxidation complex,  alpha subunit),  VV10982 (Fatty oxidation complex,  beta subunit),  VV10989 (Glycyl-tRNA synthetase,  alpha subunit),  VV10990 (Glycyl-tRNA synthetase,  beta subunit),  VV10992 (Valine-pyruvate aminotransferase),  VV11015 (ATP synthase F0,  A subunit),  VV11016 (ATP synthase F0,  C subunit),  VV11017 (ATP synthase F0,  B subunit),  VV11018 (ATP synthase F1,  delta subunit),  VV11019 (ATP synthase F1,  alpha subunit),  VV11020 (ATP synthase F1,  gamma subunit),  VV11021 (ATP synthase F1,  beta subunit),  VV11022 (ATP synthase F1,  epsilon subunit),  VV11023 (UDP-N-acetylglucosamine pyrophosphorylase),  VV11028 (Threonine dehydratase),  VV11029 (Dihydroxy-acid dehydratase (DAD)),  VV11030 (Branched-chain amino acid aminotransferase),  VV11031 (Acetolactate synthase II,  small (regulatory) subunit),  VV11032 (Acetolactate synthase II,  large subunit),  VV11047 (Methionyl-tRNA formyltransferase),  VV11053 (Phosphoribosylaminoimidazole carboxylase,  ATPase subunit),  VV11054 (Phosphoribosylaminoimidazole carboxylase,  catalytic subunit),  VV11056 (Coproporphyrinogen III oxidase,  aerobic),  VV11057 (Shikimate 5-dehydrogenase),  VV11077 (Ketol-acid reductoisomerase IlvC),  VV11083 (# N / A),  VV11099 (Phosphogluconate dehydratase),  VV11100 (Thermoresistant gluconokinase),  VV11102 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV11105 (Glutathione reductase),  VV11120 (Uncharacterized enzyme of heme biosynthesis HemX),  VV11121 (Uroporphyrinogen-III synthase HemD),  VV11122 (Porphobilinogen deaminase),  VV11123 (Adenylate cyclase Cya),  VV11126 (Diaminopimelate decarboxylase),  VV11127 (Diaminopimelate epimerase DapF),  VV11141 (Glyceraldehyde-3-phosphate dehydrogenase),  VV11153 (N6-adenine-specific_methylase),  VV11163 (Chorismate-pyruvate lyase),  VV11164 (4-hydroxybenzoate octaprenyltransferase),  VV11165 (Glycerol-3-phosphate acyltransferase),  VV11168 (Pyridine nucleotide-disulfide oxidoreductase,  class I),  VV11175 (Glutamate racemase),  VV11195 (Phosphatidylserine synthase),  VV11197 (UDP-N-acetylenolpyruvoylglucosamine reductase),  VV11198 (BirA bifunctional protein),  VV11199 (# N / A),  VV11200 (Pantothenate kinase),  VV11218 (Uroporphyrinogen decarboxylase),  VV11226 (Phosphoribosylamine-glycine ligase PurD),  VV11227 (Phosphoribosylaminoimidazolecarboxamide formyltransperaseIMP cyclohydrolase PurH),  VV11234 (Acetyl-CoA carboxylase,  biotin carboxylase),  VV11235 (Acetyl-CoA carboxylase,  biotin carboxyl carrier protein),  VV11236 (3-dehydroquinate dehydratase II),  VV11237 (Acetyl-coenzyme A synthetase),  VV11249 (Aspartate ammonia-lyase),  VV11257 (6-phosphofructokinase,  isozyme I),  VV11266 (Fumarate reductase,  13 kDa hdrophobic protein),  VV11267 (Fumarate reductase,  15 kDa hdrophobic protein),  VV11268 (Fumarate reductase,  Fe-S protein),  VV11269 (Fumarate reductase,  flavoprotein subunit),  VV11270 (Putative lysyl-tRNA synthetase),  VV11276 (Serine acetyltransferase),  VV11277 (Glycerol-3-phosphate dehydrogenase),  VV11281 (2, 3-bisphosphoglycerate-independent phosphoglycerate mutase),  VV11284 (Phosphatidylserine decarboxylase),  VV11291 (N-acetylmuramoyl-L-alanine amidase),  VV11299 (Adenylosuccinate synthetase),  VV11306 (Phosphoglycolate phosphatase),  VV11307 (Tryptophanyl-tRNA synthetase),  VV11311 (Para-aminobenzoate synthase glutamine amidotransferase,  component II),  VV11312 (# N / A),  VV11313 (Acetylornithine aminotransferase),  VV11314 (Arginineornithine N-succinyltransferase beta subunit),  VV11315 (NAD-dependent aldehyde dehydrogenase),  VV11328 (Asparaginase 2),  VV11342 (UDP-glucose 4-epimerase),  VV11343 (Triosephosphate isomerase),  VV11345 (5-carboxymethyl-2-hydroxymuconate isomerase),  VV11349 (Glycerol metabiolism protein GlpX),  VV11353 (1, 4-dihydroxy-2-naphthoate octaprenyltransferase),  VV11361 (Putative malate oxidoreductase),  VV11364 (Cystathionine gamma-synthase),  VV11365 (Aspartokinase IIhomoserine dehydrogenase,  methionine-sensitive),  VV11366 (5, 10-methylenetetrahydrofolate reductase),  VV11369 (Phosphoenolpyruvate carboxylase),  VV11370 (Acetylornithine deacetylase),  VV11371 (N-acetyl-gamma-glutamyl-phosphate reductase),  VV11372 (Acetylglutamate kinase),  VV11373 (Argininosuccinate synthase),  VV11374 (Bifunctional protein ArgH {Includes:  Argininosuccinate lyase (Arginosuccinase) (ASAL);  Probable acetyltransferase}),  VV11382 (Shikimate kinase),  VV11383 (3-dehydroquinate synthetase),  VV11386 (Ribulose-phosphate-3-epimerase),  VV11393 (Alanine racemase),  VV11396 (Glucose-6-phosphate isomerase),  VV11402 (Sulfite reductase (NADPH) flavoprotein alpha subunit),  VV11403 (Sulfite reductase (NADPH) hemoprotein beta subunit),  VV11404 (3'-phosphoadenosine 5'-phosphosulfate sulfotransferase (PAPS reductase)),  VV11423 (Cobalamin-dependent methionine synthase),  VV11424 (Aspartokinase III,  lysine-sensitive),  VV11425 (Aminotransferase,  class V),  VV11428 (UDP--glucose-1-phosphate uridylyltransferase),  VV11453 (Ribonucleases G and E),  VV11461 (Ribosomal large subunit pseudouridine synthase A),  VV11464 (Aspartate carbamoyltransferase,  regulatory subunit),  VV11465 (Aspartate carbamoyltransferase,  catalytic chain),  VV11466 (Ornithine carbamoyltransferase),  VV11467 (Arginine deiminase),  VV11474 (Valyl-tRNA synthetase),  VV11485 (6-phospho-beta-glucosidase),  VV11517 (Glutaminase family protein),  VV11519 (Putative oxygen-independent coproporphyrinogen III oxidase),  VV11524 (Pyrroline-5-carboxylate reductase),  VV11530 (Glutathione synthetase),  VV11536 (S-adenosylmethionine synthetase),  VV11537 (Transketolase 1),  VV11539 (Erythrose-4-phosphate dehydrogenase),  VV11540 (3-phosphoglycerate kinase),  VV11541 (Fructose-bisphosphate aldolase,  class II),  VV11546 (D-3-phosphoglycerate dehydrogenase),  VV11547 (Ribose 5-phosphate isomerase),  VV11552 (2-Polyprenyl-6-methoxyphenol hydroxylase UbiH),  VV11558 (L-aspartate oxidase),  VV11568 (Pyridoxal phosphate biosynthesis protein PdxJ),  VV11569 (Holo- (acyl-carrier-protein) synthase),  VV11575 (GTP pyrophosphokinase (ppGpp synthetase) SpoT),  VV11576 (# N / A),  VV11578 (CTP synthase (UTP-ammonia lyase)),  VV11579 (Enolase),  VV11582 (4-diphosphocytidyl-2-methyl-D-erythritol synthase),  VV11583 (2-C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS)),  VV11584 (tRNA pseudouridine synthase D),  VV11585 (Acid phosphatase SurE (survival protein SurE)),  VV11593 (Alanyl-tRNA synthetase),  VV11594 (Aspartokinase,  alpha and beta subunits),  VV11600 (Oxaloacetate decarboxylase,  gamma subunit),  VV11601 (Oxaloacetate decarboxylase,  alpha subunit),  VV11602 (Oxaloacetate decarboxylase,  beta subunit),  VV11606 (Glutamate--cysteine ligase),  VV11608 (Autoinducer-2 synthase LuxS),  VV11621 (# N / A),  VV11622 (Dephospho-CoA kinase),  VV11627 (Nicotinate-nucleotide pyrophosphorylase,  NadC),  VV11630 (Pyruvate dehydrogenase complex E1 component),  VV11631 (Pyruvate dehydrogenase complex E2 component,  dihydrolipoamide acyltransferase),  VV11632 (Pyruvate dehydrogenase complex E3 component,  dihydrolipoamide dehydrogenase),  VV11635 (# N / A),  VV11636 (Hypoxanthine phosphoribosyltransferase),  VV11637 (Putative carbonic anhydrase),  VV11642 (Pantoate--beta-alanine ligase),  VV11643 (3-methyl-2-oxobutanoate hydroxymethyltransferase),  VV11644 (2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase),  VV11653 (Aconitase hydratase B),  VV11654 (Glycerate kinase),  VV11664 (Phosphomannomutase),  VV11678 (Glutamate-1-semialdehyde 2, 1-aminomutase (GSA)),  VV11683 (Tyrosyl-tRNA synthetase),  VV11691 (Dihydropteroate synthase),  VV11692 (Phosphomannomutase),  VV11698 (tRNA pseudouridine synthase B),  VV11716 (Predicted acetyltransferase),  VV11725 (Deoxyribose-phosphate aldolase),  VV11726 (Thymidine phosphorylase),  VV11727 (Phosphopentomutase),  VV11728 (Purine-nucleoside phosphorylase),  VV11730 (Phosphoserine phosphatase),  VV11766 (Evolved beta-D-galactosidase,  beta subunit),  VV11767 (Evolved beta-D-galactosidase,  alpha-subunit),  VV11770 (UDP-glucose 4-epimerase),  VV11771 (Galactose-1-phosphate uridylyltransferase),  VV11772 (Galactokinase),  VV11773 (Galactose-1-epimerase),  VV11785 (Aerobic glycerol-3-phosphate dehydrogenase),  VV11787 (Glycerol kinase),  VV11790 (Tetrahydrodipicolinate N-succinyltransferase),  VV11799 (Amino-acid acetyltransferase (N-acetylglutamate synthase)),  VV11810 (2-dehydropantoate 2-reductase),  VV11838 (Prolyl-tRNA synthetase),  VV11846 (FabH,  3-oxoacyl- {acyl-carrier-protein}),  VV11855 (tRNA pseudouridine synthase C (Pseudouridylate synthase)),  VV11865 (CDP-diglyceride synthetase),  VV11866 (1-deoxy-D-xylulose 5-phosphate reductoisomerase),  VV11870 (UDP-3-O- {3-hydroxymyristoyl} glucosamine N-acyltransferase),  VV11872 (Acyl- (acyl-carrier-protein)-UDP-N-acetylglucosamine O-acyltransferase),  VV11873 (Lipid A disaccharide synthetase),  VV11876 (Acetyl-CoA carboxylase alpha subunit),  VV11883 (Putative hydroxyacylglutathione hydrolase GloB),  VV11896 (Oxidoreductase,  acyl-CoA dehydrogenase family),  VV11897 (Phosphoheptose isomerase),  VV11899 (Folate-dependent phosphoribosylglycinamide formyltransferase PurN),  VV11900 (Phosphoribosylformylglycinamidine cyclo-ligase),  VV11901 (Uracil phosphoribosyltransferase),  VV11912 (Dihydrodipicolinate synthase DapA),  VV11916 (Succinyl-diaminopimelate desuccinylase),  VV11975 (Fatty oxidation complex,  beta subunit),  VV11976 (Fatty oxidation complex,  alpha subunit),  VV11978 (Phosphohistidine phosphatase SixA),  VV11981 (Chorismate synthase (5-enolpyruvylshikimate-3-phosphate phospholyase)),  VV11986 (3-oxoacyl- (acyl-carrier-protein) synthase I),  VV11988 (Erythronate-4-phosphate dehydrogenase),  VV11989 (Aspartate-semialdehyde dehydrogenase Asd),  VV11992 (tRNA pseudouridine synthase A),  VV11993 (Acetyl-CoA carboxylase,  carboxyl transferase beta subunit),  VV11994 (Folylpolyglutamate synthasedihydrofolate synthase),  VV11997 (Glutamine phosphoribosylpyrophosphate amidotransferase),  VV12002 (Adenine phosphoribosyltransferase),  VV12016 (Putative lactoylglutathione lyase),  VV12022 (5, 10-methylene-tetrahydrofolate dehydrogenasemethenyl tetrahydrofolate cyclohydrolase),  VV12064 (Uridine phosphorylase),  VV12074 (NADH dehydrogenase,  FAD-containing subunit),  VV12075 (# N / A),  VV12086 (Tetraacyldisaccharide 4`-kinase),  VV12088 (3-deoxy-manno-octulosonate cytidylyltransferase),  VV12098 (Formate acetyltransferase),  VV12116 (Pseudouridine synthase family 1 protein),  VV12118 (Isocitrate dehydrogenase),  VV12126 (# N / A),  VV12127 (3-phosphoshikimate 1-carboxyvinyltransferase),  VV12131 (Glucose-1-phosphate adenylyltransferase),  VV12132 (Glycogen synthase),  VV12156 (Aspartyl-tRNA synthetase),  VV12162 (Cytochrome d ubiquinol oxidase,  subunit I),  VV12163 (Cytochrome d ubiquinol oxidase,  subunit II),  VV12173 (Quinolinate synthetase A),  VV12200 (D-Lactate dehydrogenase),  VV12219 (5-Methyltetrahydropteroyltriglutamate--homocysteine methyltransferase),  VV12220 (Phosphate acetyltransferase),  VV12221 (Acetate kinase),  VV12227 (Glycosidase),  VV12234 (GTP cyclohydrolase II),  VV12248 (Aspartate aminotransferase),  VV12254 (Asparaginyl-tRNA synthetase (Asparagine-tRNA ligase) (AsnRS)),  VV12257 (6-pyruvoyl tetrahydrobiopterin synthase),  VV12260 (L-serine dehydratase 1),  VV12265 (Para-Aminobenzoate synthase,  component I),  VV12266 (Fumarate hydratase,  class I),  VV12341 (Acyl carrier protein phosphodiesterase),  VV12349 (Lactonizing lipase),  VV12355 (Agmatinase),  VV12356 (Biosynthetic arginine decarboxylase),  VV12357 (# N / A),  VV12370 (Phenylalanyl-tRNA synthetase,  alpha chain),  VV12371 (Phenylalanyl-tRNA synthetase,  beta chain),  VV12372 (Nicotinate phosphoribosyltransferase),  VV12374 (Nicotinamidasepyrazinamidase),  VV12378 (Phosphoribosylglycinamide formyltransferase 2),  VV12379 (Cytidine deaminase),  VV12389 (Histidine ammonia-lyase),  VV12390 (Urocanate hydratase),  VV12391 (Formiminoglutamase),  VV12392 (Imidazolonepropionase),  VV12397 (Threonyl-tRNA synthetase),  VV12448 (Putative acetyltransferase),  VV12560 (Riboflavin synthase alpha chain),  VV12590 (Putative formate dehydrogenase large subunit),  VV12591 (Formate dehydrogenase,  iron-sulfur subunit),  VV12592 (Formate dehydrogenase,  cytochrome b556 subunit),  VV12599 (NAD-dependent protein deacetylase,  SIR2 family),  VV12614 (Cation transport ATPase),  VV12617 (Cytochrome c oxidase,  subunit CcoP),  VV12618 (Cytochrome c oxidase,  subunit CcoQ),  VV12619 (Cytochrome c oxidase,  subunit CcoO),  VV12620 (Cytochrome c oxidase,  subunit CcoN),  VV12637 (Dihydroorotate dehydrogenase),  VV12641 (Aminopeptidase N),  VV12654 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV12682 (6-phosphogluconate dehydrogenase),  VV12683 (6-phosphogluconolactonase),  VV12684 (Glucose-6-phosphate 1-dehydrogenase),  VV12699 (1-acyl-sn-glycerol-3-phosphate acyltransferase),  VV12702 (Uroporphyrinogen-III methylase),  VV12711 (Acylphos phatase),  VV12730 (Aconitate hydratase 1),  VV12731 (Methylcitrate synthase),  VV12732 (Carboxyphosphonoenolpyruvate phosphonomutase),  VV12754 (Glycerophosphoryl diester phosphodiesterase),  VV12755 (Catalase-peroxidase KatG),  VV12765 (Glutathione S-transferase),  VV12768 (Hemolysin VllY),  VV12771 (Putative 2 ', 3'-cyclic-nucleotide 2'-phosphodiesterase),  VV12772 (Homoserine O-succinyltransferase),  VV12783 (Succinylglutamate desuccinylase),  VV12785 (Fructose-2, 6-bisphosphatase),  VV12786 (Adenosyl cobinamide kinaseadenosyl cobinamide phosphate guanylyltransferase),  VV12787 (Cobalamin-5-phosphate synthase),  VV12788 (NaMN: DMB phosphoribosyltransferase),  VV12797 (Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase),  VV12799 (Outer membrane phospholipase A),  VV12801 (Malate oxidoreductase),  VV12810 (Thioredoxin reductase),  VV12813 (Phosphoserine aminotransferase),  VV12824 (Putative glutamate decarboxylase),  VV12826 (Alcohol dehydrogenase),  VV12843 (Ribosomal small subunit pseudouridine synthase A),  VV12871 (Cardiolipin synthase),  VV12872 (Cystathionine beta-lyase),  VV12888 (Inorganic pyrophosphataseexopolyphosphatase),  VV12890 (Putative alpha-1,  6-galactosidase),  VV12907 (Thymidine kinase),  VV12908 (Cysteinyl-tRNA synthetase),  VV12910 (Conserved hypothetical protein),  VV12913 (Phosphoribosyl-AMP cyclohydrolasephosphoribosyl-ATP pyrophosphohydrolase),  VV12914 (Imidazole glycerol phosphate synthase subunit HisF),  VV12915 (Phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide (ProFAR) isomerase),  VV12916 (Imidazole glycerol phosphate synthase subunit HisH),  VV12917 (Histidine biosynthesis bifunctional protein HisB {Includes:  Histidinol-phosphatase;  Imidazoleglycerol-phosphate dehydratase (IGPD)}),  VV12918 (Histidine biosynthesis bifunctional protein HisB {Includes:  Histidinol-phosphatase;  Imidazoleglycerol-phosphate dehydratase (IGPD)}),  VV12919 (Histidinol dehydrogenase),  VV12920 (ATP phosphoribosyltransferase),  VV12924 (Inosine-guanosine kinase),  VV12928 (Adenylosuccinate lyase),  VV12940 (Dethiobiotin synthetase),  VV12942 (8-amino-7-oxononanoate synthase),  VV12943 (Biotin synthase),  VV12944 (Adenosylmethionine-8-amino-7-oxononanoate aminotransferase),  VV12945 (# N / A),  VV12946 (Seryl-tRNA synthetase),  VV12952 (Alanine dehydrogenase),  VV12977 (Orotidine-5'-phosphate decarboxylase),  VV12983 (Cytidylate kinase),  VV12992 (Pyruvate kinase II),  VV12999 (PTS system,  glucose-specific IIBC component),  VV13002 (Thymidylate kinase),  VV13005 (4-amino-4-deoxychorismate lyase),  VV13006 (3-oxoacyl- (acyl-carrier-protein) synthase II),  VV13007 (Hypothetical protein),  VV13009 (3-oxoacyl- (acyl-carrier-protein) reductase),  VV13010 (Malonyl CoA-acyl carrier protein transacylase),  VV13011 (3-oxoacyl- (acyl-carrier-protein) synthase III),  VV13016 (23S rRNA ribosomal pseudouridine synthase),  VV13018 (Ribonuclease E),  VV13022 (Cob (I) alamin adenosyltransferase),  VV13025 (Uridine kinase),  VV13028 (Methionyl-tRNA synthetase),  VV13035 (Formate-dependent nitrite reductase,  periplasmic cytochrome c552 subunit NrfA),  VV13040 (3-demethylubiquinone-9 3-methyltransferase),  VV13041 (Ribonucleoside-diphosphate reductase,  alpha subunit),  VV13042 (Ribonucleoside-diphosphate reductase,  beta subunit),  VV13050 (Diaminobutyrate-pyruvate transaminaseL-2, 4-diaminobutyrate decarboxylase),  VV13052 (Phosphatidylglycerophosphate synthase),  VV13060 (Pseudouridine synthase family 1 protein),  VV13064 (Anthranilate synthase component I),  VV13065 (Anthranilate synthase component II),  VV13066 (Anthranilate phosphoribosyltransferase),  VV13067 (Indole-3-glycerol phosphate synthase IgpSphosphoribosylanthranilate isomerase TrpF),  VV13068 (Tryptophan synthase beta chain),  VV13069 (Tryptophan synthase alpha chain),  VV13100 (Lactoylglutathione lyase),  VV13111 (Alcohol dehydrogenaseacetaldehyde dehydrogenase),  VV13115 (Aspartate-semialdehyde dehydrogenase),  VV13135 (L-asparaginase I),  VV13140 (Glyceraldehyde 3-phosphate dehydrogenase),  VV13153 (Cysteine synthase),  VV13168 (O-succinylbenzoic acid-CoA ligase),  VV13169 (O-succinylbenzoate-CoA synthase),  VV13170 (Dihydroxynaphthoic acid synthase),  VV13172 (2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1-carboxylate synthase),  VV13173 (Menaquinone-specific isochorismate synthase),  VV13174 (Putative aspartate aminotransferase),  VV20005 (Phosphoenolpyruvate synthase),  VV20010 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit A),  VV20011 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit B),  VV20012 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit C),  VV20019 (Alcohol dehydrogenase,  class IV),  VV20053 (L-allo-threonine aldolase),  VV20065 (Ribokinase),  VV20117 (Hydroxymethylglutaryl-CoA reductase),  VV20123 (Methylglyoxal synthase),  VV20148 (Acetate kinase 2),  VV20186 (Glycine cleavage system P protein (pyridoxal-binding)),  VV20188 (Serine hydroxymethyltransferase),  VV20190 (Glycine cleavage system T protein (aminomethyltransferase)),  VV20198 (PTS system,  fructose-specific IIBC component),  VV20199 (1-Phosphofructokinase),  VV20200 (PTS system,  fructose-specific IIAFPR component),  VV20206 (Pyruvate kinase II),  VV20214 (Glucose-1-phosphate adenylyltransferase 2),  VV20216 (Formate-tetrahydrofolate ligase),  VV20217 (# N / A),  VV20218 (Inosine-guanosine kinase),  VV20237 (Putative 5'-nucleotidase),  VV20256 (Glycosidase),  VV20280 (D-alanine-D-alanine ligase),  VV20315 (# N / A),  VV20316 (NAD (P) transhydrogenase beta subunit),  VV20317 (NAD (P) transhydrogenase alpha subunit),  VV20330 (Cobyric acid synthase),  VV20334 (Amino acid biosynthesis aminotransferase),  VV20337 (Anareobic ribonucleoside-triphosphate reductase),  VV20349 (3-Oxoacyl- (acyl-carrier-protein) synthase III),  VV20367 (Uroporphyrinogen-III methylase),  VV20369 (Nitrite reductase {NAD (P) H},  small subunit),  VV20370 (Nitrite reductase {NAD (P) H},  large subunit),  VV20389 (Ferredoxin subunits of nitrite reductase and ring-hydroxylating dioxygenase),  VV20390 (NAD (P) H-nitrite reductase),  VV20397 (Uroporphyrinogen-III methylase),  VV20398 (Anaerobic dehydrogenase,  typically selenocysteine-containing),  VV20400 (Alpha-amylase),  VV20407 (Glycerophosphoryl diester phosphodiesterase),  VV20455 (Phenylalanine-4-hydroxylase),  VV20456 (Acetyl-CoA synthase),  VV20468 (Adenosine deaminase),  VV20469 (Putative pyruvate dehydrogenase E1 component,  alpha subunit),  VV20470 (Putative pyruvate dehydrogenase E1 component,  beta subunit),  VV20471 (Putative dihydrolipoamide acetyltransferase),  VV20478 (Alanine racemase 2),  VV20488 (Putative short-hanin alcohol dehydrogenase),  VV20489 (3-hydroxyisobutyrate dehydrogenase),  VV20490 (Enoyl-CoA hydrataseisomerase family),  VV20491 (Enoyl-CoA hydrataseisomerase family),  VV20493 (NAD-dependent aldehyde dehydrogenase),  VV20494 (Acetyl-CoA acetyltransferase),  VV20496 (Acyl-CoA dehydrogenase),  VV20497 (Acetyl-CoA carboxylase,  carboxyltransferase component),  VV20498 (Enoyl-CoA hydratasecarnithine racemase),  VV20499 (Putative hydroxymethylglutaryl-CoA lyase),  VV20500 (# N / A),  VV20514 (# N / A),  VV20515 (Mannose-6-phosphate isomerase),  VV20531 (Deoxycytidylate deaminase),  VV20532 (Peptidase T),  VV20543 (GTP cyclohydrolase I),  VV20552 (Transaldolase B),  VV20553 (Transketolase 1),  VV20558 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV20560 (Polyprenyltransferase (cytochrome oxidase assembly factor)),  VV20561 (Uncharacterized protein required for cytochrome oxidase assembly),  VV20565 (Cytochrome C oxidase,  subunit III),  VV20566 (Cytochrome C oxidase assembly factor CtaG),  VV20567 (Cytochrome C oxidase,  subunit I),  VV20568 (Cytochrome C oxidase,  subunit II),  VV20569 (Phosphomannomutase),  VV20712 (GMP reductase),  VV20721 (Periplasmic nitrate reductase),  VV20730 (Putative N-acetylneuraminate lyase),  VV20734 (Putative N-acetylmannosamine-6-phosphate epimerase),  VV20735 (Putative N-acetylmannosamine kinase),  VV20736 (N-acetylglucosamine-6-phosphate deacetylase),  VV20741 (Acetyl-CoA acetyltransferase),  VV20742 (Acetoacetyl-CoA reductase),  VV20752 (Autoinducer 2 sensor kinasephosphatase LuxQ),  VV20768 (Adenylosuccinate synthase),  VV20789 (Cytosine deaminase),  VV20833 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV20835 (Vulnibactin-specific isochorismate synthase),  VV20854 (Tryptophanase),  VV20869 (NAD-dependent aldehyde dehydrogenase),  VV20878 (Tyrosyl-tRNA synthetase),  VV20903 (Alpha-amylase),  VV20904 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV20905 (2-keto-3-deoxygluconate kinase),  VV20914 (2-deoxy-D-gluconate 3-dehydrogenase),  VV20966 (Oxygen-insensitive NAD (P) H nitroreductase),  VV20996 (Methionine synthase II (cobalamin-independent)),  VV21024 (Predicted tagatose 6-phosphate kinase),  VV21030 (Diaminopimelate decarboxylase),  VV21050 (6-phospho-beta-glucosidase,  Family 4 glycosyl hydrolase),  VV21062 (Bifunctional PLP-dependent enzyme with beta-cystathionase and maltose regulon repressor activities),  VV21064 (D-mannonate dehydratase),  VV21069 (Mannonate oxidoreductase),  VV21070 (Uronate isomerase),  VV21071 (Sugar kinase,  ribokinase family),  VV21072 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV21084 (3-hexulose-6-phosphate synthase SgbH),  VV21085 (Putative hexulose-6-phosphate isomerase SgbU),  VV21093 (Dehydrogenase with different specificities (related to short-chain alcohol dehydrogenases)),  VV21094 (Galactose-1-phosphate uridylyltransferase),  VV21095 (UDP-glucose 4-epimerase),  VV21118 (Putative proline dehydrogenase),  VV21122 (Pyridoxamine-phosphate oxidase),  VV21136 (Putative acetyltransferase (isoleucine patch superfamily)),  VV21142 (Putative PTS system sucrose-specific IIBC component),  VV21180 (GTP cyclohydrolase II),  VV21200 (Glucosamine-6-phosphate isomerase),  VV21204 (Glutathione synthase),  VV21235 (Ornithine decarboxylase,  inducible),  VV21237 (Putative pyridoxine kinase),  VV21250 (Maltodextrin phosphorylase),  VV21251 (4-alpha-glucanotransferase),  VV21266 (NAD-dependent aldehyde dehydrogenase),  VV21287 (Glycosyl hydrolase family 1),  VV21318 (Pseudouridylate synthase,  23S RNA-specific),  VV21327 (Beta-galactosidase LacZ),  VV21330 (Alpha-galactosidase),  VV21348 (Mannose-6-phosphate isomerase),  VV21349 (PTS system,  fructose-specific IIABC component),  VV21352 (PTS system fructose-specific component IIB),  VV21353 (PTS system,  fructose-specific IIABC component ),  VV21356 (PTS system,  fructose-specific IIBC component),  VV21357 (PTS system,  fructose-specific IIA component),  VV21373 (Uridine phosphorylase),  VV21395 (Probable taurine catabolism dioxygenase),  VV21412 (Diacylglycerol kinase),  VV21426 (3, 4-dihydroxy-2-butanone 4-phosphate synthase (DHBP synthase)),  VV21432 (Putative membrane-associated phospholipid phosphatase),  VV21433 (Phosphomethylpyrimidine kinase),  VV21457 (Lactate dehydrogenase),  VV21473 (Catalase KatE),  VV21484 (2-amino-3-ketobutyrate coenzyme A ligase),  VV21485 (L-threonine 3-dehydrogenase),  VV21520 (Carbonic anhydrase),  VV21540 (Purine-nucleoside phosphorylase),  VV21596 (Dihydroorotase),  VV21599 (NH (3) -dependent NAD (+) synthetase),  VV21615 (Coproporphyrinogen III oxidase),  VV21622 (Alpha-amylase),  VV21635 (Spermidine synthase),  VV21637 (3-hydroxyisobutyrate dehydrogenase),  VV21651 (Hexapeptide-repeat containing-acetyltransferase),  VV21663 (4-aminobutyrate aminotransferase),  VV21664 (2-aminoethylphosphonate: pyruvate aminotransferase),  VV21677 (L-serine deaminase),  VV21687 (NAD-dependent aldehyde dehydrogenase),  VV21688 (Choline dehydrogenase),  

Example 2: Selection of Chokepoints

In the metabolic network constructed in Example 1, chokepoint analysis was performed using MetaFluxNet (Lee et al., Bioinformatics , 19: 2144, 2003) and Excel on 765 metabolites of V. vulnificus .

The analysis revealed a total of 530 chokepoints, the only enzyme reactions that consume or produce specific metabolites. This corresponds to 487 genes of Table 2 below among the 670 genes.

VV10053, VV10060, VV10061, VV10136, VV10145, VV10156, VV10157, VV10176, VV10177, VV10179, VV10187, VV10209, VV10236, VV10246, VV10248, VV10249, VV10254, VV10256, VV10254, VV10265, V2102 VV10316, VV10319, VV10321, VV10322, VV10323, VV10325, VV10326, VV10333, VV10340, VV10344, VV10414, VV10426, VV10427, VV10430, VV10450, VV10465, VV10484, VV10487, VV10494, V508 V10 VV10544, VV10545, VV10558, VV10559, VV10565, VV10566, VV10571, VV10577, VV10578, VV10580, VV10581, VV10582, VV10583, VV10591, VV10610, VV10613, VV10623, VV10625, VV10638, V647 V10 VV10656, VV10657, VV10662, VV10665, VV10678, VV10679, VV10688, VV10705, VV10723, VV10725, VV10726, VV10727, VV10728, VV10774, VV10779, VV10796, VV10797, VV10799, VV1073, V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 V108 VV10831, VV10852, VV10854, VV10889, VV10902, VV10907, VV10909, VV10933, VV10935, VV10940, VV10963, V V10964, VV10978, VV10981, VV10982, VV10989, VV10990, VV11023, VV11029, VV11030, VV11031, VV11032, VV11047, VV11053, VV11054, VV11057, VV11077, VV11083, VV11100, VV11102, VV11120, VV11121, VV11122, VV11123, VV11127, VV11141, VV11153, VV11163, VV11164, VV11165, VV11195, VV11197, VV11198, VV11199, VV11200, VV11218, VV11226, VV11227, VV11234, VV11235, VV11236, VV11237, VV11257, VV11270, VV11276, V11, V11276, V112 VV11312, VV11313, VV11314, VV11315, VV11342, VV11345, VV11353, VV11364, VV11365, VV11370, VV11371, VV11372, VV11373, VV11374, VV11382, VV11383, VV11386, VV11396, V11411 VV11461, VV11464, VV11465, VV11474, VV11485, VV11517, VV11530, VV11536, VV11537, VV11539, VV11546, VV11552, VV11568, VV11569, VV11575, VV11576, VV11579, VV11582, VV11583, V115 V11 VV11622, VV11627, VV11630, VV11631, VV11632, VV11637, VV11642, VV11643, VV11644, VV11653, VV11654, VV 11664, VV11678, VV11683, VV11691, VV11692, VV11698, VV11716, VV11725, VV11726, VV11727, VV11728, VV11730, VV11766, VV11767, VV11770, VV11772, VV11787, VV11790, VV11799, V118 V118 V118 V118 V118 V11 VV11870, VV11872, VV11873, VV11876, VV11883, VV11896, VV11897, VV11900, VV11912, VV11916, VV11975, VV11976, VV11978, VV11981, VV11986, VV11988, VV11989, VV11992, VV11993, V11994V V11993 VV12074, VV12075, VV12086, VV12088, VV12116, VV12126, VV12127, VV12131, VV12132, VV12156, VV12173, VV12219, VV12220, VV12227, VV12234, VV12248, VV12254, VV12257, VV12260, V123 V123 V123 V123 VV12370, VV12371, VV12378, VV12379, VV12389, VV12390, VV12391, VV12392, VV12397, VV12448, VV12560, VV12599, VV12614, VV12641, VV12654, VV12682, VV12683, VV12699, VV126702, VV12702, V12712 VV12768, VV12771, VV12772, VV12783, VV12785, VV12786, VV12787, VV12788, VV12797, VV12799, VV12810, VV1 2813, VV12824, VV12826, VV12843, VV12871, VV12872, VV12890, VV12908, VV12910, VV12913, VV12914, VV12915, VV12916, VV12917, VV12918, VV12919, VV12920, VV12924, VV12928, V12, V12, V12, V12, V12, V12, V12, V12, V12, V12, V12, V12 VV12977, VV12983, VV13005, VV13006, VV13007, VV13009, VV13010, VV13011, VV13016, VV13022, VV13028, VV13040, VV13050, VV13052, VV13060, VV13066, VV13067, VV13068, VV13069, V13 V13 VV13169, VV13170, VV13172, VV13173, VV13174, VV20019, VV20053, VV20065, VV20117, VV20123, VV20186, VV20190, VV20198, VV20200, VV20214, VV20218, VV20237, VV20256, VV20237, VV20256, VV20280, V203, V203, V203, V203, V203, V203, V203, V203, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, V20, VV, VV20280, V20, V20, V20, An example VV20407, VV20455, VV20456, VV20468, VV20469, VV20470, VV20471, VV20488, VV20490, VV20491, VV20493, VV20496, VV20497, VV20498, VV20499, VV20500, VV20532, VV20543, VV20552, V5220, V2020, V20, V20 VV20768, VV20789, VV20833, VV20835, VV20854, VV20869, VV20878, VV20904, VV20914, VV20996, VV21024, VV21 050, VV21062, VV21064, VV21069, VV21070, VV21072, VV21084, VV21085, VV21093, VV21095, VV21118, VV21136, VV21142, VV21180, VV21204, VV21237, VV21250, VV21251, VV21287, V213 V21 V21 V21 V21 V21 V21 VV21356, VV21357, VV21373, VV21395, VV21426, VV21432, VV21433, VV21457, VV21473, VV21520, VV21540, VV21596, VV21599, VV21635, VV21651, VV21663, VV21664, VV21677

Example  3: Metabolic flow analysis  Selection of Essential Enzyme Reaction by Simulating Enzyme Blocking Simulation

Essential enzyme reaction analysis based on metabolic flow analysis was performed using the metabolic network of V. vulnificus constructed in Example 1 above.

For all of the 945 enzyme reactions that make up the metabolic network of V. vulnificus, the objective function was set to maximize the rate of cell growth, and the model was analyzed every metabolic flow analysis to find the essential enzyme reactions that stopped cell growth. The linear programming method was carried out by breaking down all the reaction equations that constitute 945 reactions one by one.

When the human body is infected with V. vulnificus , V. vulnificus is able to ingest various nutrients present in the human body. It was set up to be able to ingest the ingredients simultaneously.

In addition, a small amount of oxygen (O ₂ ), phosphate (phosphate), sulfate (sulfate), nitrate (nitrate), nitrite (nitrite), sodium (sodium) intake was also possible.

Carbon component Nitrogen component 2-Oxoglutarate Adenosine * 2-Phospho-D-glycerate Cytidine * 3-Phospho-D-glycerate Cytosine (S) -Lactate D-alanine * (S) -Malate Deoxyadenosine * Acetate Deoxycytidine * alpha, alpha-Trehalose Deoxyguanosine * alpha-D-Glucose Deoxyuridine * Citrate D-Glutamate * D-Fructose Glycine * D-Gluconate Guanosine * D-Mannitol L-Alanine * Fumarate L-Arginine * Glycerol L-Asparagine * Glycolate L-Aspartate * Isocitrate L-Cysteine Isomaltose L-Glutamate * Maltose L-Glutamine * Melibiose L-Histidine sn-Glycerol 3-phosphate L-Homoserine * Succinate L-Isoleucine Sucrose L-Leucine L-Lysine L-Methionine L-Ornithine * L-Phenylalanine L-Proline * L-Serine * L-Threonine * L-Tryptophan * L-Tyrosine L-Valine N-Acetyl-D-glucosamine * NH ₃ Putrescine * Spermidine * Thymidine Uracil Urea Uridine * Xanthine

As a result, 138 of the total 945 reactions were predicted to be essential for cell growth. This corresponds to the following 136 genes of the 670 genes reflected in the metabolic network.

VV10060, VV10061, VV10136, VV10256, VV10291, VV10314, VV10315, VV10316, VV10319, VV10321, VV10322, VV10323, VV10366, VV10427, VV10430, VV10495, VV10504, VV10508, VV10558, V105 V, V105, V105, V105 VV10591, VV10613, VV10623, VV10625, VV10649, VV10662, VV10678, VV10679, VV10796, VV10819, VV10828, VV10850, VV10854, VV10889, VV10909, VV10981, VV10982, VV11057, VV11083, V11 V11 VV11235, VV11236, VV11284, VV11311, VV11312, VV11353, VV11382, VV11383, VV11423, VV11536, VV11539, VV11547, VV11558, VV11568, VV11582, VV11583, VV11608, VV11621, VV11 220, V11 V, V11610, V11, V11, V11, V11, V11, V2, V11, V11, V11, V11, V11, V11, V11, V11, V11, 12, 12, 12, 12 VV11865, VV11866, VV11876, VV11896, VV11897, VV11912, VV11916, VV11975, VV11976, VV11981, VV11986, VV11988, VV11993, VV11994, VV12098, VV12126, VV12127, VV12131, V7312, V1212, V1212, V12 VV12699, VV12799, VV12810, VV12813, VV12824, VV12983, VV13002, VV13005, VV13006, VV13007, VV13009, V V13010, VV13011, VV13052, VV13168, VV13169, VV13170, VV13172, VV13173, VV20065, VV20214, VV20280, VV20349, VV20488, VV20490, VV20491, VV20498, VV20543, VV20558, VV20552, V215 V21, V21

Example  4: Chalk point  Drugs from Analysis and Simulation of Enzyme Deletion table Integration of enemy candidates

Only the overlapping targets were selected by combining the drug target results using the enzyme reaction deletion simulation based on the chokepoint analysis and metabolic flow analysis. This drastically reduced the number of drug targets with the most biologically probable ones.

The integrated drug target takes into account both the network topology of the metabolic network and the metabolic function that reflects the metabolic flow of each enzyme reaction constituting the metabolic network.

The predicted chokepoints overlap 126 out of a total of 530 enzymatic reactions and 138 enzymatic reactions predicted in the essential enzyme reaction analysis, which corresponds to 126 genes in Table 5 below among the 670 genes reflected in the metabolic network. It was.

VV10060, VV10061, VV10136, VV10256, VV10291, VV10314, VV10315, VV10316, VV10319, VV10321, VV10322, VV10323, VV10427, VV10430, VV10495, VV10504, VV10508, VV10558, VV10583, V105 VV10613, VV10623, VV10625, VV10649, VV10662, VV10678, VV10679, VV10796, VV10819, VV10828, VV10854, VV10889, VV10981, VV10982, VV11057, VV11083, VV11127, VV11165, VV1195, V11V, V111 VV11311, VV11312, VV11382, VV11383, VV11423, VV11536, VV11539, VV11568, VV11582, VV11583, VV11608, VV11621, VV11622, VV11627, VV11642, VV11643, VV11790, VV11810, VV11846, V118 V11 VV11916, VV11975, VV11976, VV11981, VV11986, VV11988, VV11993, VV11994, VV12126, VV12127, VV12131, VV12132, VV12173, VV12234, VV12265, VV12560, VV12614, VV12654, VV12699, V13, V13, V12, V130, V12, V12, V12, V, V12, V, V12, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V VV13009, VV13010, VV13011, VV13052, VV13168, VV13169, VV13170, VV13172, VV13173, VV20065, VV20214, V V20280, VV20349, VV20488, VV20490, VV20491, VV20498, VV20543, VV20558, VV20752, VV20833, VV20835, VV21180, VV21426, VV21432, VV21599

Example  5: Chalk point  From analysis and essential enzyme reactions equally  Additional Screening of Released Drug Targets

The overlapping predicted drug target candidates from the method of enzyme reaction deletion simulation based on chokepoint analysis and metabolic flow analysis were further screened based on the following three criteria. This was selected with only enzymes that were more effective as drug targets.

(i) First of all the enzymes selected as drug targets and their corresponding genes were removed from the presence of isozymes. (ii) Next, the gene or amino acid sequence of the drug target was selected as one that has no homology to all proteins present in humans. (iii) Finally, the genes or enzymes predicted to be drug targets were selected to have multifunctionality involved in one or more reaction schemes.

As a result, of the 126 genes predicted in Example 4, only one of the following VV10323 was predicted to be the most effective drug. VV10323 has no isoenzyme, no homology with human proteins, and promotes two reactions.

gene Ambassador E.C. number Enzyme name VV10323 Riboflavin metabolism 3.5.4.26 diaminohydroxyphosphoribosylaminopyrimidine deaminase VV10323 Riboflavin metabolism 1.1.1.193 5-amino-6- (5-phosphoribosylamino) uracil reductase

Example  6: Metabolic flow analysis  Mandatory Metabolite  Essential by Analytical Simulation versus Still-product determination

Metabolic flow analysis was conducted to determine the essentiality of metabolites for each metabolite by examining the effect of cells on cell growth when the metabolite was not consumed through metabolic reactions.

That is, in the metabolic flow analysis process of metabolites constituting the metabolic network of the target microorganism, all metabolic reactions consuming each metabolite are deleted, that is, the metabolic flow value of the corresponding reaction equation is fixed to 0 (= ν _j ). In this case, the cell growth rate of 0 was selected as an essential metabolite.

As a result, a total of 107 metabolites in Table 7 were determined as essential metabolites by metabolic flow analysis.

1,4-dihydroxy-2-naphthoate, 1-Deoxy-D-xylulose 5-phosphate, 2,3-Dihydrodipicolinate, 2-Amino-4-hydroxy-6- (erythro-1,2,3-trihydroxypropyl) -dihydropteridine triphosphate, 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, 2-Demethylmenaquinone, 2-Oxobutanoate, 2-Oxoglutarate, 3-Dehydroshikimate, 3-Methyl-2-oxobutanoic acid, 4-Aminobenzoate, 5 , 10-Methylenetetrahydrofolate, 5-Phospho-alpha-D-ribose 1-diphosphate, Acetyl- [acyl-carrier protein], Acetyl-CoA, Acyl-carrier protein, all-trans-Octaprenyl diphosphate, AMP, ATP, beta-Alanine , beta-D-Fructose 6-phosphate, CDP-diacylglycerol, Chorismate, CO2, coenzyme A (CoA), CTP, D-alanine, dATP, dCTP, D-Erythrose 4-phosphate, D-Glucose 1-phosphate, D- Glutamate, D-Glyceraldehyde 3-phosphate, dGTP, Dihydrofolate, Dodecanoyl- [acyl-carrier protein], D-Ribose 5-phosphate, D-Ribulose 5-phosphate, dTMP, dTTP, Flavin adenine dinucleotide (FAD), Flavin mononucleotide ( FMN), Fumarate, GDP, Glycerone phosphate, Glycine, Glycogen, GMP, GTP, Hexadecanoyl -[acyl-carrier protein], Hexadecenoyl- [acyl-carrier protein], Isopentenyl diphosphate, L-Alanine, L-Arginine, L-Asparagine, L-Aspartate, L-Aspartate 4-semialdehyde, L-Cysteine, L-Glutamate , L-Glutamine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L -Valine, Malonyl- [acyl-carrier protein], menaquinol, menaquinone, meso-2,6-Diaminopimelate, NADH, NADPH, NH3, Nicotinamide adenine dinucleotide (NAD +), Nicotinamide adenine dinucleotide phosphate (NADP +), Nicotinate D-ribonucleotide, Octadecanoyl- [acyl-carrier protein], Octadecenoyl- [acyl-carrier protein], O-Phospho-4-hydroxy-L-threonine, Pentadecanoyl- [acyl-carrier protein], Phosphatidylethanolamine, Phosphatidylglycerol, Phosphatidylseryl, Phosphoenolpyruvate, Prop acyl-carrier protein], Propanoyl-CoA, Pyridoxine, Pyridoxine 5'-phosphate, Pyruvate, S-Adenosyl-L-methionine, Sedoheptulose 7-phosphate, sn-Glycerol 3-phosphate, Succinyl-CoA, Te tradecanoyl- [acyl-carrier protein], Tetrahydrofolate, Thioredoxin, UDP, UDPglucose, UDP-N-acetyl-D-glucosamine, UMP, UTP

Example  7: required Metabolite  Additional screening

For the essential metabolites determined through metabolic flow analysis in Example 6, their consumption reactions were further screened based on homology with humans to further reduce the number of possible essential metabolites.

If any of the enzymes in the consumption schemes of the essential metabolites are statistically similar to human proteins, the essential metabolites and their consumption schemes are no longer considered drug targets.

As a result, the gene and amino acid sequences associated with all consumption schemes of each of the next essential metabolites selected are significantly different from those of human proteins and are structurally different from human proteins.

Essential Metabolites Ambassador Gene of consumption equation E.C. of Consumption Schemes number Enzyme of consumption 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine Folate biosynthesis VV11644 2.7.6.3 2-amino-4-hydroxy-6-hydroxymethyldihydropteridinepyrophosphokinase Folate biosynthesis VV11691 2.5.1.15 dihydropteroate synthase D-Glutamate D-Glutamine and D-glutamate metabolism VV11175 5.1.1.3 glutamate racemase Peptidoglycan biosynthesis VV10580 6.3.2.9 UDP-N-acetylmuramoylalanine--D-glutamate ligase 2,3-Dihydrodipicolinate Lysine biosynthesis VV10567 1.3.1.26 dihydrodipicolinate reductase Lysine biosynthesis VV10567 1.3.1.26 dihydrodipicolinate reductase 1-Deoxy-D-xylulose 5-phosphate Biosynthesis of steroids VV11866 1.1.1.267 1-deoxy-D-xylulose-5-phosphate reductoisomerase Vitamin B6 metabolism VV11568 PdxJ pyridoxine 5-phosphate synthase 4-Aminobenzoate Folate biosynthesis VV11691 2.5.1.15 dihydropteroate synthase Folate biosynthesis VV11691 2.5.1.15 dihydropteroate synthase

Example  8: Various drug target combinations by genes encoding selected drug target enzymes

Possible combinations were made with genes selected from the group consisting of drug target genes finally selected in Example 5 and genes involved in the consumption equation of the essential metabolite finally selected in Example 7 (FIG. 5) .

Typically, the drug target combination is a combination consisting of only the drug targets predicted in Example 5 (FIG. 5A), a combination of the drug targets predicted in Examples 5 and 7 (FIG. 5BCDE), and the drug predicted in Example 7 A combination of only targets was made (FIG. 5F). These are referred to as Type I, Type II, and Type III, respectively, for convenience.

When making a drug target combination, the genes involved in the consumption equations of the essential metabolite predicted in Example 7 were always configured to belong to the same combination, and the final drug target gene predicted in Example 5 in the present invention was one We did not make Type I because it is a dog. Type II and Type III are shown in Table 9 below. Essential metabolites consumed by enzymes corresponding to each combination of genes are also indicated. In Type II, genes from chokepoint and essential enzyme reaction assays were designated as abnormal essential enzyme genes.

Combination ID Drug Target Combination Type II II-1 VV10323 (above essential enzyme gene), VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine) II-2 VV10323 (above essential enzyme gene), VV11175, VV10580 (above D-Glutamate) II-3 VV10323 (above essential enzyme gene), VV10567 (more than 2,3-Dihydrodipicolinate) II-4 VV10323 (above essential enzyme gene), VV11866, VV11568 (above 1-Deoxy-D-xylulose 5-phosphate) II-5 VV10323 (over essential enzyme gene), VV11691 (over 4-Aminobenzoate)

Combination ID Drug target combination Type III III-1 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11175, VV10580 (more than D-Glutamate) III-2 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV10567 (more than 2,3-Dihydrodipicolinate) III-3 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate) III-4 VV11644 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, 4-Aminobenzoate) III-5 VV11175, VV10580 (or higher D-Glutamate), VV10567 (or higher 2,3-Dihydrodipicolinate) III-6 VV11175, VV10580 (or higher D-Glutamate), VV11866, VV11568 (or higher 1-Deoxy-D-xylulose 5-phosphate) III-7 VV11175, VV10580 (or higher D-Glutamate), VV11691 (or higher 4-Aminobenzoate) III-8 VV10567 (more than 2,3-Dihydrodipicolinate), VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate) III-9 VV10567 (more than 2,3-Dihydrodipicolinate), VV11691 (more than 4-Aminobenzoate) III-10 VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate), VV11691 (more than 4-Aminobenzoate)

Example 9: V. vulnificus Calculate the distance and find the ideal combination of enzyme reactions corresponding to the genes that make up the drug target combination

In order to be able to target several sites in the metabolic network at the same time, we wanted to find a combination that is far from the metabolic network. That is, among the drug target combinations made in Example 8, those with a long average distance between the combination schemes were regarded as ideal combinations.

As shown in FIG. 6, in order to calculate the distance between enzyme reaction schemes, nodes of the corresponding reaction schemes were additionally formed in a line representing the reaction between metabolites, and the distances of the reaction scheme nodes were obtained.

After the respective distances were calculated, the average distances of the enzyme reactions forming the respective drug target combinations were calculated as the average values of the distances between the reaction equations. Higher average distances of enzyme reactions were employed in a more likely combination. It is summarized as follows based on the average distance between the enzyme inequalities corresponding to the genes of the drug target combination.

Combination ID Drug Target Combination Type II and III Average distance II-1 VV10323 (above essential enzyme gene), VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine) 7.33 II-2 VV10323 (above essential enzyme gene), VV11175, VV10580 (above D-Glutamate) 7.33 II-3 VV10323 (above essential enzyme gene), VV10567 (more than 2,3-Dihydrodipicolinate) 6.67 II-4 VV10323 (above essential enzyme gene), VV11866, VV11568 (above 1-Deoxy-D-xylulose 5-phosphate) 6.67 II-5 VV10323 (over essential enzyme gene), VV11691 (over 4-Aminobenzoate) 6.67 III-5 VV11175, VV10580 (or higher D-Glutamate), VV10567 (or higher 2,3-Dihydrodipicolinate) 6.00 III-6 VV11175, VV10580 (or higher D-Glutamate), VV11866, VV11568 (or higher 1-Deoxy-D-xylulose 5-phosphate) 6.00 III-2 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV10567 (more than 2,3-Dihydrodipicolinate) 5.33 III-3 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate) 5.33 III-1 VV11644, VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11175, VV10580 (more than D-Glutamate) 4.67 III-8 VV10567 (more than 2,3-Dihydrodipicolinate), VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate) 4.67 III-9 VV10567 (more than 2,3-Dihydrodipicolinate), VV11691 (more than 4-Aminobenzoate) 4.67 III-10 VV11866, VV11568 (more than 1-Deoxy-D-xylulose 5-phosphate), VV11691 (more than 4-Aminobenzoate) 4.67 III-7 VV11175, VV10580 (or higher D-Glutamate), VV11691 (or higher 4-Aminobenzoate) 2.00 III-4 VV11644 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine), VV11691 (more than 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, 4-Aminobenzoate) 2.00

As a result, combinations of VV10323 (essential enzyme gene), VV11644, and VV11691 (consumption scheme involved 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine) of combination II-1 and combination II-2 VV10323 (above essential enzyme genes), VV11175 and VV10580 (consumption reaction genes of D-Glutamate) were screened for optimal drug target genes.

In addition, diaminohydroxyphosphoribosylaminopyrimidine deaminase, 5-amino-6- (5-phosphoribosylamino) uracil reductase, 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase, dihydropteroate synthase enzyme combination (II-1) encoded by these genes and diaminohydroxyphosphoribosylaminopyrimidine deaminase , 5-amino-6- (5-phosphoribosylamino) uracil reductase, glutamate racemase, and UDP-N-acetylmuramoylalanine--D-glutamate ligase enzyme combination (II-2) were screened as optimal drug target enzymes.

Claims (20)

Comprising the steps of, Vibrio (Vibrio) a screening method of a drug target enzyme or gene of the microorganism of the genus: comprising the steps of: (a) establish the metabolic network model of the microorganism of the genus Vibrio (Vibrio); (b) the established Vibrio (Vibrio) spp Selecting chokepoints, which are the only enzymes that consume or produce a particular metabolite in the metabolic network, as drug target enzyme candidates (I); (c) the Vibrio (Vibrio) spp Selecting the drug target enzyme candidates (II) as blocked enzyme reactions when cell growth does not occur when the linear programming is applied while blocking the enzyme reactions one by one with respect to the enzyme reactions constituting the metabolic network; (d) comparing the drug target enzyme candidates (I) and (II) which are the results obtained in steps (b) and (c) and selecting the overlapping drug target enzyme candidates as the first drug target enzyme group; (e) from the group of primary drug target enzymes selected from (d) above Selecting a second drug target enzyme group that is not homologous to a human protein and does not have isozymes, and selects genes encoding the second target enzymes as a first drug target gene group; (f) Vibrio genus microorganism constructed in step (a) Determining that certain metabolites as essential metabolites (I) when the growth rate of the cell is zero when blocking all enzymatic reactions of specific metabolites on the metabolic network; (g) further screening for essential metabolites where all of the essential metabolites (I) determined in (f) above consume each essential metabolite only if they do not have homology with human proteins, Selecting genes encoding enzymes for use in the enzyme reaction consuming the further selected essential metabolites (II) as a second drug target gene group; (h) creating various combinations of genes selected from the group consisting of the first drug target gene group selected in step (e) and the second drug target gene group selected in step (g); And (i) For the reaction schemes in which the enzymes encoding the genes forming the combinations made in step (h) are involved, the average distance on the metabolic network is calculated and the genes forming the combinations of cases where the average distance is far from the drug targets are used. Selecting the enzymes encoded by the genes or encoded by the drug target enzymes. The method of claim 1, wherein the selection of step (e) the secondary drug target enzyme is without no human protein homologous relationship have the isozyme (isozyme), the Vibrio (Vibrio) spp A first drug target enzyme candidate when selected for one or more enzyme reactions comprising a metabolic network. Comprising the steps of, Vibrio (Vibrio) a screening method of a drug target enzyme or gene of the microorganism of the genus: comprising the steps of: (a) establish the metabolic network model of the microorganism of the genus Vibrio (Vibrio); (b) Vibrio genus microorganisms Selecting chokepoints, which are the only enzymes that consume or produce a particular metabolite in the metabolic network, as drug target enzyme candidates (I); (c) the microorganism of the genus Vibrio Selecting the drug target enzyme candidates (II) as blocked enzyme reactions when cell growth does not occur when the linear programming is applied while blocking the enzyme reactions one by one with respect to the enzyme reactions constituting the metabolic network; (d) comparing the drug target enzyme candidates (I) and (II) which are the results obtained in steps (b) and (c) and selecting the overlapping drug target enzyme candidates as the first drug target enzyme group; (e) from the group of primary drug target enzymes selected from (d) above There is no homology between the human protein and the Vibrio (Vibrio) spp Selecting those involved in one or more enzymatic reaction schemes forming a metabolic network as a second drug target enzyme group and selecting genes encoding the second target enzymes as a first drug target gene group; (f) Vibrio genus microorganism constructed in step (a) Determining that certain metabolites as essential metabolites (I) when the growth rate of the cell is zero when blocking all enzymatic reactions of specific metabolites on the metabolic network; (g) further screening for essential metabolites where all of the essential metabolites (I) determined in (f) above consume each essential metabolite only if they do not have homology with human proteins, Selecting genes encoding enzymes for use in the enzyme reaction consuming the further selected essential metabolites (II) as a second drug target gene group; (h) creating various combinations of genes selected from the group consisting of the first drug target gene group selected in step (e) and the second drug target gene group selected in step (g); And (i) For the reaction schemes in which the enzymes encoding the genes forming the combinations made in step (h) are involved, the average distance on the metabolic network is calculated and the genes forming the combinations of cases where the average distance is far from the drug targets are used. Selecting the enzymes encoded by the genes or encoded by the drug target enzymes. The method of any one of claims 1 to 3, wherein the microorganism of the genus Vibrio is Vibrio vulnificus . The method according to any one of claims 1 to 3,  Metabolic network construction in step (a) is VV10014 (dGTP triphosphohydrolase),  VV10053 (Histidine ammonia-lyase),  VV10060 (Putative beta-ketoacyl-ACP reductase),  VV10061 (Putative beta-ketoacyl-ACP synthase),  VV10136 (Long-chain-fatty-acid-CoA ligase),  VV10143 (Formyltetrahydrofolate hydrolase),  VV10145 (Arginyl-tRNA synthetase),  VV10154 (Succinyl-CoA synthetase,  alpha subunit),  VV10155 (Succinyl-CoA synthetase,  beta subunit),  VV10156 (2-oxoglutarate dehydrogenase complex,  E2 component,  dihydrolipoamide succinyltransferase),  VV10157 (2-oxoglutarate dehydrogenase complex,  E1 component),  VV10158 (Succinate dehydrogenase,  iron-sulfur protein),  VV10159 (Succinate dehydrogenase,  flavoprotein subunit),  VV10160 (Succinate dehydrogenase,  hydrophobic membrane anchor protein),  VV10161 (Succinate dehydrogenase,  cytochrome b556 subunit),  VV10162 (Citrate synthase),  VV10169 (Phosphoglucomutase),  VV10176 (Glutaminyl-tRNA synthetase),  VV10177 (# N / A),  VV10179 (PTS system,  N-acetylglucosamine-specific IIBC component),  VV10180 (N-acetylglucosamine-6-phosphate deacetylase),  VV10183 (Asparagine synthase B,  glutamine-hydrolyzing),  VV10187 (Ferrochelatase),  VV10188 (Adenylate kinase),  VV10209 (Cysteine synthase A),  VV10212 (PTS system,  glucose-specific IIA component),  VV10236 (Glutamyl-tRNA synthetase),  VV10246 (Pseudouridine synthase family 1 protein),  VV10248 (5'-nucleotidase precursor),  VV10249 (2-dehydro-3-deoxyphosphooctonate aldolase),  VV10254 (Glutamyl-tRNA reductase),  VV10256 (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK)),  VV10257 (Ribose-phosphate pyrophosphokinase),  VV10265 (2-polyprenyl-6-methoxyphenol hydroxylase and related FAD-dependent oxidoreductase),  VV10272 (Leucyl-tRNA synthetase),  VV10286 (Serine hydroxymethyltransferase),  VV10288 (Trehalose-6-phosphate hydrolase),  VV10289 (PTS system,  trehalose-specific IIBC component),  VV10291 (Histidinol phosphatase and related phosphatase),  VV10314 (Geranylgeranyl pyrophosphate synthase),  VV10315 (1-deoxyxylulose-5-phosphate synthase),  VV10316 (Phosphatidylglycerophosphatase A),  VV10317 (Thiamine monophosphate kinase),  VV10319 (Riboflavin synthase beta-chain),  VV10321 (3, 4-dihydroxy-2-butanone 4-phosphate synthaseGTP cyclohydrolase II),  VV10322 (Riboflavin synthase alpha chain),  VV10323 (Riboflavin-specific deaminase),  VV10325 (Gamma-glutamyl phosphate reductase),  VV10326 (Glutamate 5-kinase),  VV10329 (Xanthine-guanine phosphoribosyltransferase),  VV10333 (Aminoacyl-histidine dipeptidase),  VV10340 (Phosphoribosylformylglycinamidine (FGAM) synthase),  VV10344 (Zn-dependent alcohol dehydrogenase,  class III),  VV10366 (Putative inorganic polyphosphateATP-NAD kinase),  VV10414 (Transcriptional regulator,  LysR family),  VV10418 (GMP synthase (glutamine-hydrolyzing)),  VV10419 (GMP synthase (glutamine-hydrolyzing)),  VV10426 (Histidyl-tRNA synthetase),  VV10427 (Enzyme involved in the deoxyxylulose pathway of isoprenoid biosynthesis GcpE),  VV10430 (Nucleoside diphosphate kinase),  VV10449 (Isocitrate lyase),  VV10450 (Malate synthase A),  VV10465 (Exopolyphosphatase),  VV10484 (Pseudouridylate synthase,  23S RNA-specific),  VV10487 (Chorismate mutasepephenate dehydratase),  VV10494 (Chorismate mutaseprephenate dehydrogenase),  VV10495 (Phospho-2-dehydro-3-deoxyheptonate aldolase,  tyr-sensitive),  VV10504 (Penicillin tolerance protein LytB),  VV10507 (Isoleucyl-tRNA synthetase),  VV10508 (FAD synthase),  VV10516 (Thymidylate synthase),  VV10526 (Lysyl-tRNA synthetase (class II)),  VV10543 (Threonine synthase),  VV10544 (Homoserine kinase),  VV10545 (Aspartokinasehomoserine dehydrogenase,  threonine-sensitive),  VV10553 (Glutamate synthase,  large subunit),  VV10554 (Glutamate synthase,  small subunit),  VV10555 (Glutamate synthase,  large subunit),  VV10556 (Glutamate synthase,  small subunit),  VV10558 (Nucleoside phosphorylase),  VV10559 (Cobalamin biosynthesis protein CobDCbiB),  VV10565 (Carbamoylphosphate synthase large subunit (split gene in MJ)),  VV10566 (Carbamoylphosphate synthase small subunit),  VV10567 (Dihydrodipicolinate reductase),  VV10571 (UDP-3-O-acyl-N-acetylglucosamine deacetylase),  VV10577 (UDP-N-acetylmuramate-alanine ligase),  VV10578 (UDP-N-acetylglucosamine-N-acetylmuramyl- (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase),  VV10580 (UDP-N-acetylmuramoylalanine-D-glutamate ligase),  VV10581 (Phospho-N-acetylmuramoyl-pentapeptide-transferase),  VV10582 (UDP-N-acetylmuramoylalanyl-D-glutamyl-2,  6-diaminopimelate-D-alanyl-D-alanyl ligase),  VV10583 (UDP-N-acetylmuramylalanyl-D-glutamate--2,  6-diaminopimelate ligase),  VV10591 (Phosphoheptose isomerase),  VV10595 (Ubiquinol--cytochrome c reductase,  cytochrome c1),  VV10596 (Ubiquinol--cytochrome c reductase,  cytochrome B),  VV10597 (Ubiquinol--cytochrome c reductase,  iron-sulfur subunit),  VV10610 (MutTnudix family protein),  VV10613 (ADP-heptose synthase,  bifunctional sugar kinaseadenylyltransferase),  VV10623 (Putative undecaprenol kinase (Bacitracin resistance protein)),  VV10625 (Dihydroneopterin aldolase FolB),  VV10638 (PTS system,  mannitol-specific IIABC component),  VV10639 (Mannitol-1-phosphate 5-dehydrogenase),  VV10641 (Glucosamine--fructose-6-phosphate aminotransferase),  VV10644 (Pyruvate kinase),  VV10647 (Acetolactate synthase,  small (regulatory) subunit),  VV10648 (Acetolactate synthase III,  large subunit),  VV10649 (Long-chain acyl-CoA synthetase (AMP-forming)),  VV10654 (2-isopropylmalate synthase),  VV10655 (3-isopropylmalate dehydrogenase),  VV10656 (3-isopropylmalate dehydratase,  large subunit),  VV10657 (3-isopropylmalate dehydratase,  small subunit),  VV10662 (Pyridoxal phosphate biosynthesis protein PdxA),  VV10665 (Bis (5`-nucleosyl) -tetraphosphatase),  VV10666 (Dihydrofolate reductase),  VV10673 (Malate dehydrogenase),  VV10678 (1-acyl-sn-glycerol-3-phosphate acyltransferase),  VV10679 (UDP-N-acetylglucosamine enolpyruvyl transferase),  VV10688 (Low specificity phosphatase (HAD superfamily)),  VV10705 (3-polyprenyl-4-hydroxybenzoate decarboxylase),  VV10707 (Fructose-1, 6-bisphosphatase),  VV10708 (Inorganic pyrophosphatase),  VV10723 (Adenylylsulfate kinase),  VV10725 (Sulfate adenylate transferase subunit 1),  VV10726 (Sulfate adenylate transferase,  subunit 2),  VV10727 (Uroporphyrinogen-III methylase),  VV10728 (2`, 3`-cyclic-nucleotide 2`-phosphodiesterase),  VV10774 (Nucleotide sugar dehydrogenase),  VV10779 (UDP-N-acetyl-D-mannosaminuronate dehydrogenase),  VV10780 (UDP-N-acetylglucosamine 2-epimerase),  VV10796 (ADP-L-glycero-D-mannoheptose-6-epimerase),  VV10797 (Lipid A biosynthesis (kdo) 2- (lauroyl) -lipid IVA acyltransferase),  VV10799 (3-deoxy-D-manno-octulosonic-acid transferase (KDO transferase)),  VV10803 (CMP-N-acetylneuraminic acid synthetase),  VV10804 (Putative nucleoside-diphosphate-sugar pyrophosphorylase containing CBS domain),  VV10808 (Sialic acid synthase),  VV10814 (Putative KDO kinase WavC),  VV10815 (Diacylglycerol kinase),  VV10819 (Phosphopantetheine adenylyltransferase),  VV10828 (DNApantothenate metabolism flavoprotein),  VV10830 (Lipid A biosynthesis lauroyl acyltransferase),  VV10831 (Orotate phosphoribosyltransferase),  VV10850 (Guanylate kinase),  VV10852 (Guanosine-3 ', 5'-bis (diphosphate) 3'-pyrophosphohydrolase SpoT),  VV10854 (ATP-dependent DNA helicase RecG),  VV10881 (Phosphoenolpyruvate carboxykinase (ATP)),  VV10889 (Glutamine synthetase (glutamate-ammonia ligase)),  VV10894 (Oxygen-independent coproporphyrinogen III oxidase),  VV10902 (Delta-aminolevulinic acid dehydratase),  VV10907 (Putative ubiquinone biosynthesis protein AarF),  VV10909 (Ubiquinonemenaquinone biosynthesis methlytransferase UbiE),  VV10933 (NAD (P) H-flavin reductase),  VV10935 (3-polyprenyl-4-hydroxybenzoate decarboxylase and related decarboxylase),  VV10940 (Guanosine-5'-triphosphate, 3'-diphosphate pyrophosphatase),  VV10963 (Thiamine monophosphate synthase ThiE),  VV10964 (Thiamine biosynthesis protein ThiC),  VV10978 (Protoporphyrinogen oxidase),  VV10981 (Fatty oxidation complex,  alpha subunit),  VV10982 (Fatty oxidation complex,  beta subunit),  VV10989 (Glycyl-tRNA synthetase,  alpha subunit),  VV10990 (Glycyl-tRNA synthetase,  beta subunit),  VV10992 (Valine-pyruvate aminotransferase),  VV11015 (ATP synthase F0,  A subunit),  VV11016 (ATP synthase F0,  C subunit),  VV11017 (ATP synthase F0,  B subunit),  VV11018 (ATP synthase F1,  delta subunit),  VV11019 (ATP synthase F1,  alpha subunit),  VV11020 (ATP synthase F1,  gamma subunit),  VV11021 (ATP synthase F1,  beta subunit),  VV11022 (ATP synthase F1,  epsilon subunit),  VV11023 (UDP-N-acetylglucosamine pyrophosphorylase),  VV11028 (Threonine dehydratase),  VV11029 (Dihydroxy-acid dehydratase (DAD)),  VV11030 (Branched-chain amino acid aminotransferase),  VV11031 (Acetolactate synthase II,  small (regulatory) subunit),  VV11032 (Acetolactate synthase II,  large subunit),  VV11047 (Methionyl-tRNA formyltransferase),  VV11053 (Phosphoribosylaminoimidazole carboxylase,  ATPase subunit),  VV11054 (Phosphoribosylaminoimidazole carboxylase,  catalytic subunit),  VV11056 (Coproporphyrinogen III oxidase,  aerobic),  VV11057 (Shikimate 5-dehydrogenase),  VV11077 (Ketol-acid reductoisomerase IlvC),  VV11083 (# N / A),  VV11099 (Phosphogluconate dehydratase),  VV11100 (Thermoresistant gluconokinase),  VV11102 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV11105 (Glutathione reductase),  VV11120 (Uncharacterized enzyme of heme biosynthesis HemX),  VV11121 (Uroporphyrinogen-III synthase HemD),  VV11122 (Porphobilinogen deaminase),  VV11123 (Adenylate cyclase Cya),  VV11126 (Diaminopimelate decarboxylase),  VV11127 (Diaminopimelate epimerase DapF),  VV11141 (Glyceraldehyde-3-phosphate dehydrogenase),  VV11153 (N6-adenine-specific_methylase),  VV11163 (Chorismate-pyruvate lyase),  VV11164 (4-hydroxybenzoate octaprenyltransferase),  VV11165 (Glycerol-3-phosphate acyltransferase),  VV11168 (Pyridine nucleotide-disulfide oxidoreductase,  class I),  VV11175 (Glutamate racemase),  VV11195 (Phosphatidylserine synthase),  VV11197 (UDP-N-acetylenolpyruvoylglucosamine reductase),  VV11198 (BirA bifunctional protein),  VV11199 (# N / A),  VV11200 (Pantothenate kinase), VV11218 (Uroporphyrinogen decarboxylase),  VV11226 (Phosphoribosylamine-glycine ligase PurD),  VV11227 (Phosphoribosylaminoimidazolecarboxamide formyltransperaseIMP cyclohydrolase PurH),  VV11234 (Acetyl-CoA carboxylase,  biotin carboxylase),  VV11235 (Acetyl-CoA carboxylase,  biotin carboxyl carrier protein),  VV11236 (3-dehydroquinate dehydratase II),  VV11237 (Acetyl-coenzyme A synthetase),  VV11249 (Aspartate ammonia-lyase),  VV11257 (6-phosphofructokinase,  isozyme I),  VV11266 (Fumarate reductase,  13 kDa hdrophobic protein),  VV11267 (Fumarate reductase,  15 kDa hdrophobic protein),  VV11268 (Fumarate reductase,  Fe-S protein),  VV11269 (Fumarate reductase,  flavoprotein subunit),  VV11270 (Putative lysyl-tRNA synthetase),  VV11276 (Serine acetyltransferase),  VV11277 (Glycerol-3-phosphate dehydrogenase),  VV11281 (2, 3-bisphosphoglycerate-independent phosphoglycerate mutase),  VV11284 (Phosphatidylserine decarboxylase),  VV11291 (N-acetylmuramoyl-L-alanine amidase),  VV11299 (Adenylosuccinate synthetase),  VV11306 (Phosphoglycolate phosphatase),  VV11307 (Tryptophanyl-tRNA synthetase),  VV11311 (Para-aminobenzoate synthase glutamine amidotransferase,  component II),  VV11312 (# N / A),  VV11313 (Acetylornithine aminotransferase),  VV11314 (Arginineornithine N-succinyltransferase beta subunit),  VV11315 (NAD-dependent aldehyde dehydrogenase),  VV11328 (Asparaginase 2),  VV11342 (UDP-glucose 4-epimerase),  VV11343 (Triosephosphate isomerase),  VV11345 (5-carboxymethyl-2-hydroxymuconate isomerase),  VV11349 (Glycerol metabiolism protein GlpX),  VV11353 (1, 4-dihydroxy-2-naphthoate octaprenyltransferase),  VV11361 (Putative malate oxidoreductase),  VV11364 (Cystathionine gamma-synthase),  VV11365 (Aspartokinase IIhomoserine dehydrogenase,  methionine-sensitive),  VV11366 (5, 10-methylenetetrahydrofolate reductase),  VV11369 (Phosphoenolpyruvate carboxylase),  VV11370 (Acetylornithine deacetylase),  VV11371 (N-acetyl-gamma-glutamyl-phosphate reductase),  VV11372 (Acetylglutamate kinase),  VV11373 (Argininosuccinate synthase),  VV11374 (Bifunctional protein ArgH {Includes:  Argininosuccinate lyase (Arginosuccinase) (ASAL);  Probable acetyltransferase}),  VV11382 (Shikimate kinase),  VV11383 (3-dehydroquinate synthetase),  VV11386 (Ribulose-phosphate-3-epimerase),  VV11393 (Alanine racemase),  VV11396 (Glucose-6-phosphate isomerase),  VV11402 (Sulfite reductase (NADPH) flavoprotein alpha subunit),  VV11403 (Sulfite reductase (NADPH) hemoprotein beta subunit),  VV11404 (3'-phosphoadenosine 5'-phosphosulfate sulfotransferase (PAPS reductase)),  VV11423 (Cobalamin-dependent methionine synthase),  VV11424 (Aspartokinase III,  lysine-sensitive),  VV11425 (Aminotransferase,  class V),  VV11428 (UDP--glucose-1-phosphate uridylyltransferase),  VV11453 (Ribonucleases G and E),  VV11461 (Ribosomal large subunit pseudouridine synthase A),  VV11464 (Aspartate carbamoyltransferase,  regulatory subunit),  VV11465 (Aspartate carbamoyltransferase,  catalytic chain),  VV11466 (Ornithine carbamoyltransferase),  VV11467 (Arginine deiminase),  VV11474 (Valyl-tRNA synthetase),  VV11485 (6-phospho-beta-glucosidase),  VV11517 (Glutaminase family protein),  VV11519 (Putative oxygen-independent coproporphyrinogen III oxidase),  VV11524 (Pyrroline-5-carboxylate reductase),  VV11530 (Glutathione synthetase),  VV11536 (S-adenosylmethionine synthetase),  VV11537 (Transketolase 1),  VV11539 (Erythrose-4-phosphate dehydrogenase),  VV11540 (3-phosphoglycerate kinase),  VV11541 (Fructose-bisphosphate aldolase,  class II),  VV11546 (D-3-phosphoglycerate dehydrogenase),  VV11547 (Ribose 5-phosphate isomerase),  VV11552 (2-Polyprenyl-6-methoxyphenol hydroxylase UbiH),  VV11558 (L-aspartate oxidase),  VV11568 (Pyridoxal phosphate biosynthesis protein PdxJ),  VV11569 (Holo- (acyl-carrier-protein) synthase),  VV11575 (GTP pyrophosphokinase (ppGpp synthetase) SpoT),  VV11576 (# N / A),  VV11578 (CTP synthase (UTP-ammonia lyase)),  VV11579 (Enolase),  VV11582 (4-diphosphocytidyl-2-methyl-D-erythritol synthase),  VV11583 (2-C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS)),  VV11584 (tRNA pseudouridine synthase D),  VV11585 (Acid phosphatase SurE (survival protein SurE)),  VV11593 (Alanyl-tRNA synthetase),  VV11594 (Aspartokinase,  alpha and beta subunits),  VV11600 (Oxaloacetate decarboxylase,  gamma subunit),  VV11601 (Oxaloacetate decarboxylase,  alpha subunit),  VV11602 (Oxaloacetate decarboxylase,  beta subunit),  VV11606 (Glutamate--cysteine ligase),  VV11608 (Autoinducer-2 synthase LuxS),  VV11621 (# N / A),  VV11622 (Dephospho-CoA kinase),  VV11627 (Nicotinate-nucleotide pyrophosphorylase,  NadC),  VV11630 (Pyruvate dehydrogenase complex E1 component),  VV11631 (Pyruvate dehydrogenase complex E2 component,  dihydrolipoamide acyltransferase),  VV11632 (Pyruvate dehydrogenase complex E3 component,  dihydrolipoamide dehydrogenase),  VV11635 (# N / A),  VV11636 (Hypoxanthine phosphoribosyltransferase),  VV11637 (Putative carbonic anhydrase),  VV11642 (Pantoate--beta-alanine ligase),  VV11643 (3-methyl-2-oxobutanoate hydroxymethyltransferase),  VV11644 (2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase),  VV11653 (Aconitase hydratase B),  VV11654 (Glycerate kinase),  VV11664 (Phosphomannomutase),  VV11678 (Glutamate-1-semialdehyde 2, 1-aminomutase (GSA)),  VV11683 (Tyrosyl-tRNA synthetase),  VV11691 (Dihydropteroate synthase),  VV11692 (Phosphomannomutase),  VV11698 (tRNA pseudouridine synthase B),  VV11716 (Predicted acetyltransferase),  VV11725 (Deoxyribose-phosphate aldolase),  VV11726 (Thymidine phosphorylase),  VV11727 (Phosphopentomutase),  VV11728 (Purine-nucleoside phosphorylase),  VV11730 (Phosphoserine phosphatase),  VV11766 (Evolved beta-D-galactosidase,  beta subunit),  VV11767 (Evolved beta-D-galactosidase,  alpha-subunit),  VV11770 (UDP-glucose 4-epimerase),  VV11771 (Galactose-1-phosphate uridylyltransferase),  VV11772 (Galactokinase),  VV11773 (Galactose-1-epimerase),  VV11785 (Aerobic glycerol-3-phosphate dehydrogenase),  VV11787 (Glycerol kinase),  VV11790 (Tetrahydrodipicolinate N-succinyltransferase),  VV11799 (Amino-acid acetyltransferase (N-acetylglutamate synthase)),  VV11810 (2-dehydropantoate 2-reductase),  VV11838 (Prolyl-tRNA synthetase),  VV11846 (FabH,  3-oxoacyl- {acyl-carrier-protein}),  VV11855 (tRNA pseudouridine synthase C (Pseudouridylate synthase)),  VV11865 (CDP-diglyceride synthetase),  VV11866 (1-deoxy-D-xylulose 5-phosphate reductoisomerase),  VV11870 (UDP-3-O- {3-hydroxymyristoyl} glucosamine N-acyltransferase),  VV11872 (Acyl- (acyl-carrier-protein)-UDP-N-acetylglucosamine O-acyltransferase),  VV11873 (Lipid A disaccharide synthetase),  VV11876 (Acetyl-CoA carboxylase alpha subunit),  VV11883 (Putative hydroxyacylglutathione hydrolase GloB),  VV11896 (Oxidoreductase,  acyl-CoA dehydrogenase family),  VV11897 (Phosphoheptose isomerase),  VV11899 (Folate-dependent phosphoribosylglycinamide formyltransferase PurN),  VV11900 (Phosphoribosylformylglycinamidine cyclo-ligase),  VV11901 (Uracil phosphoribosyltransferase),  VV11912 (Dihydrodipicolinate synthase DapA),  VV11916 (Succinyl-diaminopimelate desuccinylase),  VV11975 (Fatty oxidation complex,  beta subunit),  VV11976 (Fatty oxidation complex,  alpha subunit),  VV11978 (Phosphohistidine phosphatase SixA),  VV11981 (Chorismate synthase (5-enolpyruvylshikimate-3-phosphate phospholyase)),  VV11986 (3-oxoacyl- (acyl-carrier-protein) synthase I),  VV11988 (Erythronate-4-phosphate dehydrogenase),  VV11989 (Aspartate-semialdehyde dehydrogenase Asd),  VV11992 (tRNA pseudouridine synthase A),  VV11993 (Acetyl-CoA carboxylase,  carboxyl transferase beta subunit),  VV11994 (Folylpolyglutamate synthasedihydrofolate synthase),  VV11997 (Glutamine phosphoribosylpyrophosphate amidotransferase),  VV12002 (Adenine phosphoribosyltransferase),  VV12016 (Putative lactoylglutathione lyase),  VV12022 (5, 10-methylene-tetrahydrofolate dehydrogenasemethenyl tetrahydrofolate cyclohydrolase),  VV12064 (Uridine phosphorylase),  VV12074 (NADH dehydrogenase,  FAD-containing subunit),  VV12075 (# N / A),  VV12086 (Tetraacyldisaccharide 4`-kinase),  VV12088 (3-deoxy-manno-octulosonate cytidylyltransferase),  VV12098 (Formate acetyltransferase),  VV12116 (Pseudouridine synthase family 1 protein),  VV12118 (Isocitrate dehydrogenase),  VV12126 (# N / A),  VV12127 (3-phosphoshikimate 1-carboxyvinyltransferase),  VV12131 (Glucose-1-phosphate adenylyltransferase),  VV12132 (Glycogen synthase),  VV12156 (Aspartyl-tRNA synthetase),  VV12162 (Cytochrome d ubiquinol oxidase,  subunit I),  VV12163 (Cytochrome d ubiquinol oxidase,  subunit II),  VV12173 (Quinolinate synthetase A),  VV12200 (D-Lactate dehydrogenase),  VV12219 (5-Methyltetrahydropteroyltriglutamate--homocysteine methyltransferase),  VV12220 (Phosphate acetyltransferase),  VV12221 (Acetate kinase),  VV12227 (Glycosidase),  VV12234 (GTP cyclohydrolase II),  VV12248 (Aspartate aminotransferase),  VV12254 (Asparaginyl-tRNA synthetase (Asparagine-tRNA ligase) (AsnRS)),  VV12257 (6-pyruvoyl tetrahydrobiopterin synthase),  VV12260 (L-serine dehydratase 1),  VV12265 (Para-Aminobenzoate synthase,  component I),  VV12266 (Fumarate hydratase,  class I),  VV12341 (Acyl carrier protein phosphodiesterase),  VV12349 (Lactonizing lipase),  VV12355 (Agmatinase),  VV12356 (Biosynthetic arginine decarboxylase),  VV12357 (# N / A),  VV12370 (Phenylalanyl-tRNA synthetase,  alpha chain),  VV12371 (Phenylalanyl-tRNA synthetase,  beta chain),  VV12372 (Nicotinate phosphoribosyltransferase),  VV12374 (Nicotinamidasepyrazinamidase),  VV12378 (Phosphoribosylglycinamide formyltransferase 2),  VV12379 (Cytidine deaminase),  VV12389 (Histidine ammonia-lyase),  VV12390 (Urocanate hydratase),  VV12391 (Formiminoglutamase),  VV12392 (Imidazolonepropionase),  VV12397 (Threonyl-tRNA synthetase),  VV12448 (Putative acetyltransferase),  VV12560 (Riboflavin synthase alpha chain),  VV12590 (Putative formate dehydrogenase large subunit),  VV12591 (Formate dehydrogenase,  iron-sulfur subunit),  VV12592 (Formate dehydrogenase,  cytochrome b556 subunit),  VV12599 (NAD-dependent protein deacetylase,  SIR2 family),  VV12614 (Cation transport ATPase),  VV12617 (Cytochrome c oxidase,  subunit CcoP),  VV12618 (Cytochrome c oxidase,  subunit CcoQ),  VV12619 (Cytochrome c oxidase,  subunit CcoO),  VV12 620 (Cytochrome c oxidase,  subunit CcoN),  VV12637 (Dihydroorotate dehydrogenase),  VV12641 (Aminopeptidase N),  VV12654 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV12682 (6-phosphogluconate dehydrogenase),  VV12683 (6-phosphogluconolactonase),  VV12684 (Glucose-6-phosphate 1-dehydrogenase),  VV12699 (1-acyl-sn-glycerol-3-phosphate acyltransferase),  VV12702 (Uroporphyrinogen-III methylase),  VV12711 (Acylphosphatase),  VV12730 (Aconitate hydratase 1),  VV12731 (Methylcitrate synthase),  VV12732 (Carboxyphosphonoenolpyruvate phosphonomutase),  VV12754 (Glycerophosphoryl diester phosphodiesterase),  VV12755 (Catalase-peroxidase KatG),  VV12765 (Glutathione S-transferase),  VV12768 (Hemolysin VllY),  VV12771 (Putative 2 ', 3'-cyclic-nucleotide 2'-phosphodiesterase),  VV12772 (Homoserine O-succinyltransferase),  VV12783 (Succinylglutamate desuccinylase),  VV12785 (Fructose-2, 6-bisphosphatase),  VV12786 (Adenosyl cobinamide kinaseadenosyl cobinamide phosphate guanylyltransferase),  VV12787 (Cobalamin-5-phosphate synthase),  VV12788 (NaMN: DMB phosphoribosyltransferase),  VV12797 (Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase),  VV12799 (Outer membrane phospholipase A),  VV12801 (Malate oxidoreductase),  VV12810 (Thioredoxin reductase),  VV12813 (Phosphoserine aminotransferase),  VV12824 (Putative glutamate decarboxylase),  VV12826 (Alcohol dehydrogenase),  VV12843 (Ribosomal small subunit pseudouridine synthase A),  VV12871 (Cardiolipin synthase),  VV12872 (Cystathionine beta-lyase),  VV12888 (Inorganic pyrophosphataseexopolyphosphatase),  VV12890 (Putative alpha-1,  6-galactosidase),  VV12907 (Thymidine kinase),  VV12908 (Cysteinyl-tRNA synthetase),  VV12910 (Conserved hypothetical protein),  VV12913 (Phosphoribosyl-AMP cyclohydrolasephosphoribosyl-ATP pyrophosphohydrolase),  VV12914 (Imidazole glycerol phosphate synthase subunit HisF),  VV12915 (Phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide (ProFAR) isomerase),  VV12916 (Imidazole glycerol phosphate synthase subunit HisH),  VV12917 (Histidine biosynthesis bifunctional protein HisB {Includes:  Histidinol-phosphatase;  Imidazoleglycerol-phosphate dehydratase (IGPD)}),  VV12918 (Histidine biosynthesis bifunctional protein HisB {Includes:  Histidinol-phosphatase;  Imidazoleglycerol-phosphate dehydratase (IGPD)}),  VV12919 (Histidinol dehydrogenase),  VV12920 (ATP phosphoribosyltransferase),  VV12924 (Inosine-guanosine kinase),  VV12928 (Adenylosuccinate lyase),  VV12940 (Dethiobiotin synthetase),  VV12942 (8-amino-7-oxononanoate synthase),  VV12943 (Biotin synthase),  VV12944 (Adenosylmethionine-8-amino-7-oxononanoate aminotransferase),  VV12945 (# N / A),  VV12946 (Seryl-tRNA synthetase),  VV12952 (Alanine dehydrogenase),  VV12977 (Orotidine-5'-phosphate decarboxylase),  VV12983 (Cytidylate kinase),  VV12992 (Pyruvate kinase II),  VV12999 (PTS system,  glucose-specific IIBC component),  VV13002 (Thymidylate kinase),  VV13005 (4-amino-4-deoxychorismate lyase),  VV13006 (3-oxoacyl- (acyl-carrier-protein) synthase II),  VV13007 (Hypothetical protein),  VV13009 (3-oxoacyl- (acyl-carrier-protein) reductase),  VV13010 (Malonyl CoA-acyl carrier protein transacylase),  VV13011 (3-oxoacyl- (acyl-carrier-protein) synthase III),  VV13016 (23S rRNA ribosomal pseudouridine synthase),  VV13018 (Ribonuclease E),  VV13022 (Cob (I) alamin adenosyltransferase),  VV13025 (Uridine kinase),  VV13028 (Methionyl-tRNA synthetase),  VV13035 (Formate-dependent nitrite reductase,  periplasmic cytochrome c552 subunit NrfA),  VV13040 (3-demethylubiquinone-9 3-methyltransferase),  VV13041 (Ribonucleoside-diphosphate reductase,  alpha subunit),  VV13042 (Ribonucleoside-diphosphate reductase,  beta subunit),  VV13050 (Diaminobutyrate-pyruvate transaminaseL-2, 4-diaminobutyrate decarboxylase),  VV13052 (Phosphatidylglycerophosphate synthase),  VV13060 (Pseudouridine synthase family 1 protein),  VV13064 (Anthranilate synthase component I),  VV13065 (Anthranilate synthase component II),  VV13066 (Anthranilate phosphoribosyltransferase),  VV13067 (Indole-3-glycerol phosphate synthase IgpSphosphoribosylanthranilate isomerase TrpF),  VV13068 (Tryptophan synthase beta chain),  VV13069 (Tryptophan synthase alpha chain),  VV13100 (Lactoylglutathione lyase),  VV13111 (Alcohol dehydrogenaseacetaldehyde dehydrogenase),  VV13115 (Aspartate-semialdehyde dehydrogenase),  VV13135 (L-asparaginase I),  VV13140 (Glyceraldehyde 3-phosphate dehydrogenase),  VV13153 (Cysteine synthase),  VV13168 (O-succinylbenzoic acid-CoA ligase),  VV13169 (O-succinylbenzoate-CoA synthase),  VV13170 (Dihydroxynaphthoic acid synthase),  VV13172 (2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1-carboxylate synthase),  VV13173 (Menaquinone-specific isochorismate synthase),  VV13174 (Putative aspartate aminotransferase),  VV20005 (Phosphoenolpyruvate synthase),  VV20010 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit A),  VV20011 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit B),  VV20012 (Anaerobic glycerol-3-phosphate dehydrogenase,  subunit C),  VV20019 (Alcohol dehydrogenase,  class IV),  VV20053 (L-allo-threonine aldolase),  VV20065 (Ribokinase),  VV20117 (Hydroxymethylglutaryl-CoA reductase),  VV20123 (Methylglyoxal synthase),  VV20148 (Acetate kinase 2),  VV20186 (Glycine cleavage system P protein (pyridoxal-binding)),  VV20188 (Serine hydroxymethyltransferase),  VV20190 (Glycine cleavage system T protein (aminomethyltransferase)),  VV20198 (PTS system,  fructose-specific IIBC component),  VV20199 (1-Phosphofructokinase),  VV20200 (PTS system,  fructose-specific IIAFPR component),  VV20206 (Pyruvate kinase II),  VV20214 (Glucose-1-phosphate adenylyltransferase 2),  VV20216 (Formate-tetrahydrofolate ligase),  VV20217 (# N / A),  VV20218 (Inosine-guanosine kinase),  VV20237 (Putative 5'-nucleotidase),  VV20256 (Glycosidase),  VV20280 (D-alanine-D-alanine ligase),  VV20315 (# N / A),  VV20316 (NAD (P) transhydrogenase beta subunit),  VV20317 (NAD (P) transhydrogenase alpha subunit),  VV20330 (Cobyric acid synthase),  VV20334 (Amino acid biosynthesis aminotransferase),  VV20337 (Anareobic ribonucleoside-triphosphate reductase),  VV20349 (3-Oxoacyl- (acyl-carrier-protein) synthase III),  VV20367 (Uroporphyrinogen-III methylase),  VV20369 (Nitrite reductase {NAD (P) H},  small subunit),  VV20370 (Nitrite reductase {NAD (P) H},  large subunit),  VV20389 (Ferredoxin subunits of nitrite reductase and ring-hydroxylating dioxygenase),  VV20390 (NAD (P) H-nitrite reductase),  VV20397 (Uroporphyrinogen-III methylase),  VV20398 (Anaerobic dehydrogenase,  typically selenocysteine-containing),  VV20400 (Alpha-amylase),  VV20407 (Glycerophosphoryl diester phosphodiesterase),  VV20455 (Phenylalanine-4-hydroxylase),  VV20456 (Acetyl-CoA synthase),  VV20468 (Adenosine deaminase),  VV20469 (Putative pyruvate dehydrogenase E1 component,  alpha subunit),  VV20470 (Putative pyruvate dehydrogenase E1 component,  beta subunit),  VV20471 (Putative dihydrolipoamide acetyltransferase),  VV20478 (Alanine racemase 2),  VV20488 (Putative short-hanin alcohol dehydrogenase),  VV20489 (3-hydroxyisobutyrate dehydrogenase),  VV20490 (Enoyl-CoA hydrataseisomerase family),  VV20491 (Enoyl-CoA hydrataseisomerase family),  VV20493 (NAD-dependent aldehyde dehydrogenase),  VV20494 (Acetyl-CoA acetyltransferase),  VV20496 (Acyl-CoA dehydrogenase),  VV20497 (Acetyl-CoA carboxylase,  carboxyltransferase component),  VV20498 (Enoyl-CoA hydratasecarnithine racemase),  VV20499 (Putative hydroxymethylglutaryl-CoA lyase),  VV20500 (# N / A),  VV20514 (# N / A),  VV20515 (Mannose-6-phosphate isomerase),  VV20531 (Deoxycytidylate deaminase),  VV20532 (Peptidase T),  VV20543 (GTP cyclohydrolase I),  VV20552 (Transaldolase B),  VV20553 (Transketolase 1),  VV20558 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV20560 (Polyprenyltransferase (cytochrome oxidase assembly factor)),  VV20561 (Uncharacterized protein required for cytochrome oxidase assembly),  VV20565 (Cytochrome C oxidase,  subunit III),  VV20566 (Cytochrome C oxidase assembly factor CtaG),  VV20567 (Cytochrome C oxidase,  subunit I),  VV20568 (Cytochrome C oxidase,  subunit II),  VV20569 (Phosphomannomutase),  VV20712 (GMP reductase),  VV20721 (Periplasmic nitrate reductase),  VV20730 (Putative N-acetylneuraminate lyase),  VV20734 (Putative N-acetylmannosamine-6-phosphate epimerase),  VV20735 (Putative N-acetylmannosamine kinase),  VV20736 (N-acetylglucosamine-6-phosphate deacetylase),  VV20741 (Acetyl-CoA acetyltransferase),  VV20742 (Acetoacetyl-CoA reductase),  VV20752 (Autoinducer 2 sensor kinasephosphatase LuxQ),  VV20768 (Adenylosuccinate synthase),  VV20789 (Cytosine deaminase),  VV20833 (3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase),  VV20835 (Vulnibactin-specific isochorismate synthase),  VV20854 (Tryptophanase),  VV20869 (NAD-dependent aldehyde dehydrogenase),  VV20878 (Tyrosyl-tRNA synthetase),  VV20903 (Alpha-amylase),  VV20904 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV20905 (2-keto-3-deoxygluconate kinase),  VV20914 (2-deoxy-D-gluconate 3-dehydrogenase),  VV20966 (Oxygen-insensitive NAD (P) H nitroreductase),  VV20996 (Methionine synthase II (cobalamin-independent)),  VV21024 (Predicted tagatose 6-phosphate kinase),  VV21030 (Diaminopimelate decarboxylase),  VV21050 (6-phospho-beta-glucosidase,  Family 4 glycosyl hydrolase),  VV21062 (Bifunctional PLP-dependent enzyme with beta-cystathionase and maltose regulon repressor activities),  VV21064 (D-mannonate dehydratase),  VV21069 (Mannonate oxidoreductase),  VV21070 (Uronate isomerase),  VV21071 (Sugar kinase,  ribokinase family),  VV21072 (2-keto-3-deoxy-6-phosphogluconate aldolase),  VV21084 (3-hexulose-6-phosphate synthase SgbH),  VV21085 (Putative hexulose-6-phosphate isomerase SgbU),  VV21093 (Dehydrogenase with different specificities (related to short-chain alcohol dehydrogenases)),  VV21094 (Galactose-1-phosphate uridylyltransferase),  VV21095 (UDP-glucose 4-epimerase),  VV21118 (Putative proline dehydrogenase),  VV21122 (Pyridoxamine-phosphate oxidase),  VV21136 (Putative acetyltransferase (isoleucine patch superfamily)),  VV21142 (Putative PTS system sucrose-specific IIBC component),  VV21180 (GTP cyclohydrolase II),  VV21200 (Glucosamine-6-phosphate isomerase),  VV21204 (Gl utathione synthase),  VV21235 (Ornithine decarboxylase,  inducible),  VV21237 (Putative pyridoxine kinase),  VV21250 (Maltodextrin phosphorylase),  VV21251 (4-alpha-glucanotransferase),  VV21266 (NAD-dependent aldehyde dehydrogenase),  VV21287 (Glycosyl hydrolase family 1),  VV21318 (Pseudouridylate synthase,  23S RNA-specific),  VV21327 (Beta-galactosidase LacZ),  VV21330 (Alpha-galactosidase),  VV21348 (Mannose-6-phosphate isomerase),  VV21349 (PTS system,  fructose-specific IIABC component),  VV21352 (PTS system fructose-specific component IIB),  VV21353 (PTS system,  fructose-specific IIABC component),  VV21356 (PTS system,  fructose-specific IIBC component),  VV21357 (PTS system,  fructose-specific IIA component),  VV21373 (Uridine phosphorylase),  VV21395 (Probable taurine catabolism dioxygenase),  VV21412 (Diacylglycerol kinase),  VV21426 (3, 4-dihydroxy-2-butanone 4-phosphate synthase (DHBP synthase)),  VV21432 (Putative membrane-associated phospholipid phosphatase),  VV21433 (Phosphomethylpyrimidine kinase),  VV21457 (Lactate dehydrogenase),  VV21473 (Catalase KatE),  VV21484 (2-amino-3-ketobutyrate coenzyme A ligase),  VV21485 (L-threonine 3-dehydrogenase),  VV21520 (Carbonic anhydrase),  VV21540 (Purine-nucleoside phosphorylase),  VV21596 (Dihydroorotase),  VV21599 (NH (3) -dependent NAD (+) synthetase),  VV21615 (Coproporphyrinogen III oxidase),  VV21622 (Alpha-amylase),  VV21635 (Spermidine synthase),  VV21637 (3-hydroxyisobutyrate dehydrogenase),  VV21651 (Hexapeptide-repeat containing-acetyltransferase),  VV21663 (4-aminobutyrate aminotransferase),  VV21664 (2-aminoethylphosphonate: pyruvate aminotransferase),  VV21677 (L-serine deaminase),  VV21687 (NAD-dependent aldehyde dehydrogenase),  Method based on a gene family consisting of VV21688 (Choline dehydrogenase). The method of any one of claims 1 to 3, wherein the application of the linear programming in step (c) reflects all the nutrient conditions necessary for cell growth. The method of claim 6, wherein the nutrient is (S) -Lactate, (S) -Malate, 2-Oxoglutarate, 2-Phospho-D-glycerate, 3-Phospho-D-glycerate, Acetate, Adenosine, alpha, alpha-Trehalose , alpha-D-Glucose, Citrate, Cytidine, Cytosine, D-alanine, Deoxyadenosine, Deoxycytidine, Deoxyguanosine, Deoxyuridine, D-Fructose, D-Gluconate, D-Glutamate, D-Mannitol, Fumarate, Glycerol, Glycine, Glycolate, , Isocitrate, Isomaltose, L-Alanine, L-Arginine, L-Asparagine, L-Aspartate, L-Cysteine, L-Glutamate, L-Glutamine, L-Histidine, L-Homoserine, L-Isoleucine, L-Leucine, L -Lysine, L-Methionine, L-Ornithine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Maltose, Melibiose, N-Acetyl-D-glucosamine , NH3, Nitrate, Nitrite, O2, phosphate, Putrescine, sn-Glycerol 3-phosphate, sodium, Spermidine, Succinate, Sucrose, sulfate, Thymidine, Uracil, Urea, Uridine, Xanthine . The method according to any one of claims 1 to 3, wherein when the combination of the genes is made in the step (h), the enzymes involved in the nonessential reaction among the genes constituting the second drug target gene group are encoded. Wherein said genes are configured to belong to the same combination. The method of any one of claims 1 to 3, wherein when making a combination of genes in step (h), the genes selected from the second drug target gene group are configured to belong to the same combination. The method according to any one of claims 1 to 3, wherein the drug target enzyme candidates selected by the chokepoint analysis are VV10053, VV10060, VV10061, VV10136, VV10145, VV10156, VV10157, VV10176, VV10177, VV10179, VV10187, VV10209, VV10236, VV10246, VV10248, VV10249, VV10254, VV10256, VV10265, VV10272, VV10288, VV10289, VV10291, VV10314, VV10315, VV10316, VV10319, VV10321, VV10322, VV10323, VV10325, V10 V10 VV10427, VV10430, VV10450, VV10465, VV10484, VV10487, VV10494, VV10495, VV10504, VV10507, VV10508, VV10526, VV10543, VV10544, VV10545, VV10558, VV10559, VV10565, VV10566, V105 V105, V105 VV10583, VV10591, VV10610, VV10613, VV10623, VV10625, VV10638, VV10639, VV10647, VV10648, VV10649, VV10654, VV10655, VV10656, VV10657, VV10662, VV10665, VV10678, VV10665, V10726, V8826, V8826, V7, V6, V7, V6, V7, V7,6,6, V6,6,6,6,6,6,6,6,6,6,6,6 VV10728, VV10774, VV10779, VV10796, VV10797, VV10799, VV10803, VV10804, VV10808, VV10814, VV10819, VV10828, VV10830, VV10831, VV10852, VV10854, VV10889, VV10902, VV10907, VV10909, VV10933, VV10935, VV10940, VV10963, VV10964, VV10978, VV10981, VV10982, V110 110, V110, V110, V110, V110 and V110 VV11032, VV11047, VV11053, VV11054, VV11057, VV11077, VV11083, VV11100, VV11102, VV11120, VV11121, VV11122, VV11123, VV11127, VV11141, VV11153, VV11163, VV11164, VV11165, V11 V, V11 VV11226, VV11227, VV11234, VV11235, VV11236, VV11237, VV11257, VV11270, VV11276, VV11284, VV11291, VV11299, VV11306, VV11307, VV11311, VV11312, VV11313, VV11314, VV113V, V11 V, V11 VV11371, VV11372, VV11373, VV11374, VV11382, VV11383, VV11386, VV11396, VV11402, VV11403, VV11404, VV11423, VV11424, VV11425, VV11428, VV11461, VV11464, VV11465, VV11474, V115 V15 VV11546, VV11552, VV11568, VV11569, VV11575, VV11576, VV11579, VV11582, VV11583, VV11584, VV11593, V V11594, VV11606, VV11608, VV11621, VV11622, VV11627, VV11630, VV11631, VV11632, VV11637, VV11642, VV11643, VV11644, VV11653, VV11654, VV11664, VV11678, VV11683, VV11691, V117 V, V11691, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V116, V11, V11, V11, 6, 6, 6, 6, 10, and together VV11728, VV11730, VV11766, VV11767, VV11770, VV11772, VV11787, VV11790, VV11799, VV11810, VV11838, VV11846, VV11855, VV11865, VV11866, VV11870, VV11872, VV11873, VV11876, V118 V118 VV11975, VV11976, VV11978, VV11981, VV11986, VV11988, VV11989, VV11992, VV11993, VV11994, VV11997, VV12002, VV12016, VV12022, VV12064, VV12074, VV12075, VV12086, VV12088, V12 V12, V12 V12, V12, V12, V12 VV12173, VV12219, VV12220, VV12227, VV12234, VV12248, VV12254, VV12257, VV12260, VV12265, VV12341, VV12349, VV12355, VV12356, VV12357, VV12370, VV12371, VV12378, VV12379 V123 V123 V123 VV12560, VV12599, VV12614, VV12641, VV12654, VV12682, VV12683, VV12699, VV12702, VV12730, VV12731, VV 12732, VV12754, VV12755, VV12765, VV12768, VV12771, VV12772, VV12783, VV12785, VV12786, VV12787, VV12788, VV12797, VV12799, VV12810, VV12813, VV12824, VV12826, VV12843, V12128 V12 VV12914, VV12915, VV12916, VV12917, VV12918, VV12919, VV12920, VV12924, VV12928, VV12940, VV12942, VV12943, VV12944, VV12945, VV12946, VV12977, VV12983, VV13005, VV13006 V130 VV13028, VV13040, VV13050, VV13052, VV13060, VV13066, VV13067, VV13068, VV13069, VV13100, VV13111, VV13115, VV13140, VV13153, VV13168, VV13169, VV13170, VV13172, VV1373, V174 V2 VV20186, VV20190, VV20198, VV20200, VV20214, VV20218, VV20237, VV20256, VV20280, VV20330, VV20334, VV20349, VV20367, VV20397, VV20398, VV20407, VV20455, VV20456, VV20455 V2 VV20493, VV20496, VV20497, VV20498, VV20499, VV20500, VV20532, VV20543, VV20552, VV20553, VV20558, VV2 0569, VV20721, VV20742, VV20752, VV20768, VV20789, VV20833, VV20835, VV20854, VV20869, VV20878, VV20904, VV20914, VV20996, VV21024, VV21050, VV21062, VV21064, VV21069, VV210 V2 VV21118, VV21136, VV21142, VV21180, VV21204, VV21237, VV21250, VV21251, VV21287, VV21318, VV21327, VV21330, VV21349, VV21352, VV21353, VV21356, VV21357, VV21373, VV21395, V214214, V21421, V214 And an enzyme encoded by a gene selected from the group consisting of VV21540, VV21596, VV21599, VV21635, VV21651, VV21663, VV21664 and VV21677. According to any one of claims 1 to 3, wherein the drug target enzyme candidates selected by applying the linear programming method is VV10060, VV10061, VV10136, VV10256, VV10291, VV10314, VV10315, VV10316, VV10319, VV10321, VV10322, VV10323, VV10366, VV10427, VV10430, VV10495, VV10504, VV10508, VV10558, VV10577, VV10578, VV10580, VV10581, VV10582, VV10583, VV10591, VV10613, VV10623, VV10625, VV10649, VV10625, V106, V106 V10 VV10854, VV10889, VV10909, VV10981, VV10982, VV11057, VV11083, VV11127, VV11165, VV11195, VV11197, VV11200, VV11234, VV11235, VV11236, VV11284, VV11311, VV11312, VV11353, V113 V11 VV11558, VV11568, VV11582, VV11583, VV11608, VV11621, VV11622, VV11627, VV11642, VV11643, VV11790, VV11810, VV11846, VV11865, VV11866, VV11876, VV11896, VV11897, VV V1212, V1 V1, V1 VV11993, VV11994, VV12098, VV12126, VV12127, VV12131, VV12132, VV12173, VV12234, V V12265, VV12560, VV12614, VV12654, VV12699, VV12799, VV12810, VV12813, VV12824, VV12983, VV13002, VV13005, VV13006, VV13007, VV13009, VV13010, VV13011, VV13052, VV13168, VV13168, VV13168, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V130, V1202, V120, V12, V120, V1302, V1302, and V1302 And an enzyme encoded by a gene selected from the group consisting of VV20280, VV20349, VV20488, VV20490, VV20491, VV20498, VV20543, VV20558, VV20752, VV20833, VV20835, VV21180, VV21426, VV21432 and VV21599. The essential metabolite obtained in step (f) is 1,4-dihydroxy-2-naphthoate, 1-Deoxy-D-xylulose 5-phosphate, 2,3- Dihydrodipicolinate, 2-Amino-4-hydroxy-6- (erythro-1,2,3-trihydroxypropyl) -dihydropteridine triphosphate, 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, 2-Demethylmenaquinone, 2 -Oxobutanoate, 2-Oxoglutarate, 3-Dehydroshikimate, 3-Methyl-2-oxobutanoic acid, 4-Aminobenzoate, 5,10-Methylenetetrahydrofolate, 5-Phospho-alpha-D-ribose 1-diphosphate, Acetyl- [acyl-carrier protein ], Acetyl-CoA, Acyl-carrier protein, all-trans-Octaprenyl diphosphate, AMP, ATP, beta-Alanine, beta-D-Fructose 6-phosphate, CDP-diacylglycerol, Chorismate, CO2, coenzyme A (CoA), CTP , D-alanine, dATP, dCTP, D-Erythrose 4-phosphate, D-Glucose 1-phosphate, D-Glutamate, D-Glyceraldehyde 3-phosphate, dGTP, Dihydrofolate, Dodecanoyl- [acyl-carrier protein], D-Ribose 5-phosphate, D-Ribulose 5-phosphate, dTMP, dTTP, Flavin adenine dinucleotide (FAD) , Flavin mononucleotide (FMN), Fumarate, GDP, Glycerone phosphate, Glycine, Glycogen, GMP, GTP, Hexadecanoyl- [acyl-carrier protein], Hexadecenoyl- [acyl-carrier protein], Isopentenyl diphosphate, L-Alanine, L-Arginine , L-Asparagine, L-Aspartate, L-Aspartate 4-semialdehyde, L-Cysteine, L-Glutamate, L-Glutamine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L- Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Malonyl- [acyl-carrier protein], menaquinol, menaquinone, meso-2,6-Diaminopimelate, NADH, NADPH , NH3, Nicotinamide adenine dinucleotide (NAD +), Nicotinamide adenine dinucleotide phosphate (NADP +), Nicotinate D-ribonucleotide, Octadecanoyl- [acyl-carrier protein], Octadecenoyl- [acyl-carrier protein], O-Phospho-4-hydroxy-L -threonine, Pentadecanoyl- [acyl-carrier protein], Phosphatidylethanolamine, Phosphatidylglycerol, Phosphatidylserine, Phosphoenolpyruvate, Propanoyl- [acyl-carrier protein], Propanoyl-CoA, Pyridoxine, Pyridoxine 5'-phosphate ate, Pyruvate, S-Adenosyl-L-methionine, Sedoheptulose 7-phosphate, sn-Glycerol 3-phosphate, Succinyl-CoA, Tetradecanoyl- [acyl-carrier protein], Tetrahydrofolate, Thioredoxin, UDP, UDPglucose, UDP-N-acetyl -D-glucosamine, UMP and UTP method characterized in that it is selected from the group consisting of. The essential metabolite of claim 1, wherein the essential metabolite, which is obtained in step (g), is not homologous to a human protein, is selected from 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, D-Glutamate, 2,3-Dihydrodipicolinate, 1-Deoxy-D-xylulose 5-phosphate, 4-Aminobenzoate. The method according to any one of claims 1 to 3, wherein the primary drug target enzyme obtained in step (d) is VV10060, VV10061, VV10136, VV10256, VV10291, VV10314, VV10315, VV10316, VV10319, VV10321, VV10322, VV10323, VV10427, VV10430, VV10495, VV10504, VV10508, VV10558, VV10577, VV10578, VV10580, VV10581, VV10582, VV10583, VV10591, VV10613, VV10623, VV10625, VV10649, VV10662, V106, V106, V106, V108, V106, V106, V108, V106, V106, V108, V108, V108, V10, V10, V10, V, V10, V, V10, V, V10, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V, V VV10889, VV10981, VV10982, VV11057, VV11083, VV11127, VV11165, VV11195, VV11197, VV11200, VV11234, VV11235, VV11236, VV11284, VV11311, VV11312, VV11382, VV11383, VV11423, V115, V115, V115, V11, V11, V11, V11, V11, V3, V3, V3, V3, V3, V3, V3, V3, V3, V3, V3, V2, V3, V2, V3, V3, V3, V3, V3, V3, V3, V3, V3, V3, V2, V2, V2, v2, 5, 5, 5, 5, VV11621, VV11622, VV11627, VV11642, VV11643, VV11790, VV11810, VV11846, VV11865, VV11866, VV11876, VV11896, VV11897, VV11912, VV11916, VV11975, VV11976, VV11981, VV11986, V11912 VV12132, VV12173, VV12234, VV12265, VV12560, VV12614, VV12654, VV12699, VV12799, VV12810, VV12813, VV13005, V V13006, VV13007, VV13009, VV13010, VV13011, VV13052, VV13168, VV13169, VV13170, VV13172, VV13173, VV20065, VV20214, VV20280, VV20349, VV20488, VV20490, VV20491, VV20498, V20 V20 And an enzyme encoded by a gene selected from the group consisting of VV21426, VV21432 and VV21599. The method of claim 2, wherein the second drug target enzyme obtained in step (e) is an enzyme encoded by the VV10323 gene. The method of claim 2, wherein the combination made in step (h) comprises VV10323, VV11644, and VV11691 (combination ID: II-1); VV10323, VV11175 and VV10580 (combination ID: II-2); VV10323 and VV10567 (combination ID: II-3); VV10323, VV11866 and VV11568 (Combination ID: II-4); VV10323 and VV11691 (Combination ID: II-5); VV11644, VV11691, VV11175 and VV10580 (combination ID: III-1); VV11644, VV11691 and VV10567 (Combination ID: III-2); VV11644, VV11691, VV11866 and VV11568 (Combination ID: III-3); VV11644, VV11691 (Combination ID: III-4); VV11175, VV10580 and VV10567 (combination ID: III-5); VV11175, VV10580, VV11866 and VV11568 (Combination ID: III-6); VV11175, VV10580 and VV11691 (Combination ID: III-7); VV10567, VV11866 and VV11568 (Combination ID: III-8); VV10567 and VV11691 (Combination ID: III-9); And a gene combination selected from the group consisting of VV11866, VV11568 and VV11691 (Combination ID: III-10). The first, VV10323, VV11644,, Vibrio (Vibrio) into the gene of a drug target microorganism is selected from the group consisting of VV11691, VV11175, VV10580, VV10567, VV11866 VV11568 and obtained by the method of claim 2. Comprising the steps of, Vibrio (Vibrio) a screening method of a drug target enzyme or gene of the microorganism of the genus: comprising the steps of: (a) establish the metabolic network model of the microorganism of the genus Vibrio (Vibrio); (b) the established Vibrio (Vibrio) spp Selecting chokepoints, the only enzymes that consume or produce a particular metabolite in the metabolic network, as a primary drug target enzyme candidate group; (c) among the primary drug target enzyme candidate groups selected in step (b) No homology with human proteins, no isozymes, microorganisms of the Vibrio genus Selecting those involved in the one or more enzyme reaction schemes constituting the metabolic network as a second drug target enzyme group and selecting genes encoding the second drug target enzymes as a first drug target gene group; (d) Vibrio genus microorganism constructed in step (a) When blocking all enzyme reactions that consume certain metabolites on the metabolic network, determining the specific metabolite as essential metabolite (I) when the growth rate of the cell is zero; (e) further selecting essential metabolites where all of the essential metabolites (I) determined in (d) above consume each of the essential metabolites only if they do not have homology with human proteins, Selecting genes encoding enzymes used in an enzyme reaction consuming the selected essential metabolites (II) as a second drug target gene group; (f) making various combinations of genes selected from the group consisting of the first drug target gene group selected in step (c) and the second drug target gene group selected in step (e); And (g) For the reaction schemes in which the enzymes encoding the genes making up the combinations made in step (f) are involved, the mean distance on the metabolic network is calculated and the genes making up the combinations of cases where the mean distance is far from Selecting the enzymes encoded by the genes or encoded by the drug target enzymes. Comprising the steps of, Vibrio (Vibrio) a screening method of a drug target enzyme or gene of the microorganism of the genus: comprising the steps of: (a) establish the metabolic network model of the microorganism of the genus Vibrio (Vibrio); (b) the Vibrio (Vibrio) spp Selecting a first enzyme target enzyme candidate for a deleted enzyme reaction in a case where cell growth does not occur when the linear programming method is applied to the enzyme reactions constituting the metabolic network one by one; (c) among the first drug target enzyme candidates selected in step (b) Human proteins and there is no homology between, does not have the isozyme (isozyme), the Vibrio (Vibrio) spp Selecting those involved in the one or more enzyme reaction schemes constituting the metabolic network as a second drug target enzyme group and selecting genes encoding the second drug target enzymes as a first drug target gene group; (d) a Vibrio (Vibrio) spp built in the step (a) When blocking all enzyme reactions that consume certain metabolites on the metabolic network, determining the specific metabolite as essential metabolite (I) when the growth rate of the cell is zero; (e) further selecting essential metabolites where all of the essential metabolites (I) determined in (d) above consume each of the essential metabolites only if they do not have homology with human proteins, Selecting genes encoding enzymes used in an enzyme reaction consuming the selected essential metabolites (II) as a second drug target gene group; (f) making various combinations of genes selected from the group consisting of the first drug target gene group selected in step (c) and the second drug target gene group selected in step (e); And (g) For the reaction schemes in which the enzymes encoding the genes making up the combinations made in step (f) are involved, the mean distance on the metabolic network is calculated and the genes making up the combinations of cases where the mean distance is far from Selecting the enzymes encoded by the genes or encoded by the drug target enzymes. Claim 18 or claim 19, wherein said Vibrio (Vibrio) into the microorganism Vibrio nipi carcass (Vibrio vulnificus ).