Method for constructing metabolically modified microbial strains that produce useful compounds at high levels, and metabolically modified Escherichia coli strains.

Abstract

The present invention aims to provide a method for constructing metabolically modified microbial strains that produce useful compounds in high quantities, and to provide metabolically modified microbial strains that produce useful compounds in high quantities. [Solution] The present invention relates to a method for constructing a metabolically modified microbial strain that produces useful compounds at high levels, comprising: (A) a step of constructing a base strain by metabolically modifying a parent strain; (B) a step of preparing unit DNA cassettes in which a promoter for high expression and a terminator sequence are linked to the DNA encoding each enzyme of a group of enzymes that constitute the biosynthesis pathway of the useful compound functionally within the cell, and unit DNA cassettes in which a promoter for low expression (or without a promoter) and a terminator sequence are linked to the DNA encoding each enzyme, and then performing gene assembly using the OGAB method to construct a plasmid library; (C) the above The method for constructing metabolically modified microbial strains includes the steps of: introducing the constructed plasmid library into the base strain and measuring the production amount of the useful compound of each strain; analyzing the sequence information of the plasmids introduced into each strain obtained in steps (D) and (C); performing statistical analysis or machine learning that correlates the production amount of the useful compound of each strain obtained in steps (E) and (C) with the sequence information of the plasmids obtained in step (D) to identify useful genes that contribute to the production of the useful compound; and creating a strain in which the useful genes that contribute to the production of the useful compound identified in steps (F) and (E) have been recombinant.

Description

Technical Field

[0001] The present invention relates to a method for constructing a metabolically engineered microbial strain that highly produces useful compounds and a metabolically engineered Escherichia coli strain.

Background Art

[0002] Microorganisms have long been used for the production of various useful substances such as alcohol, amino acids, nucleic acids, organic acids, lipids, vitamins, antibiotics, etc. Microorganisms excellent in the production of specific useful substances have been isolated and industrially utilized. In recent years, the basic technology for the production of useful substances using microbial fermentation has attracted attention, and the related market is expected to expand rapidly.

[0003] Conventionally, in metabolic engineering approaches, various attempts have been made to improve microbial strains using molecular biological techniques and to enhance the productivity of target metabolites. However, existing methods for improving metabolically engineered microbial strains using molecular biological techniques have required a great deal of labor and time through trial and error. In recent years, with the elucidation of the genomic sequences of useful microorganisms, it has become possible to construct metabolic network models. Among them, the metabolic simulation method based on flux balance analysis (FBA) is a practical method that avoids the problem of complexity through bold simplification and is widely used in the field of metabolic engineering (McClosky, D. et al.: Mol. Sys. Biol., 9, 661 (2013), Japanese Patent Application Laid-Open No. 2008-527992). Using this FBA, it is possible to predict, to some extent, the genes necessary for improving the production efficiency of useful compounds and the unnecessary genes. However, since the results derived by this method are often still insufficient, a method for constructing a microbial strain that more efficiently improves the production efficiency of useful compounds is required.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] [Non-Patent Document 1] McClosky, D. et al.: Mol. Sys. Biol., 9, 661 (2013) [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a method for constructing metabolically modified microbial strains that produce useful compounds in high quantities, and to provide metabolically modified microbial strains that produce useful compounds in high quantities. [Means for solving the problem]

[0007] In this context, the inventors focused on the fact that many useful substances targeted by microbial fermentation can be produced using specific common metabolites as hub compounds. They believed that by having a lineup of strains capable of high production of such hub compounds, it would be possible to breed strains capable of high production of useful substances in a short period of time, and proceeded with their research. As a result, they constructed a base strain by subjecting the parent strain to metabolic modification based on metabolic flux balance analysis (FBA), and further succeeded in constructing a strain that produces high amounts of hub compounds by introducing a Combi-OGAB long-chain DNA library, which uses artificial DNA parts not subject to endogenous metabolic regulation, into the base strain. They also succeeded in selecting genes that contribute to high production of hub compounds by utilizing information analysis technology. Specifically, they targeted α-ketoglutaric acid (AKG) as the hub compound (useful compound), succeeded in developing a workflow for rapid breeding of this compound, and identified genes that contribute to high production of AKG. In other words, the gist of the present invention is as follows.

[0008] [1] A method for constructing a metabolically modified microbial strain that produces useful compounds at high levels, (A) A process of constructing a base strain by subjecting the parent plant to metabolic modification. (B) A step of preparing unit DNA cassettes in which a promoter for strong expression and a terminator sequence are ligated to the DNA encoding each enzyme of the enzyme group that constitutes the biosynthesis pathway of the above useful compounds functionally in cells, and unit DNA cassettes in which a promoter for low expression (or without a promoter) and a terminator sequence are ligated to the DNA encoding each enzyme, and then constructing a plasmid library by performing gene assembly using the OGAB method. (C) A step of introducing the plasmid library constructed above into the base strain and measuring the production amount of the above useful compound from each strain. (D)(C) A step to analyze the sequence information of plasmids introduced into each strain obtained in step (C), (E)(C) A step to identify useful genes that contribute to the production of the above useful compounds by performing statistical analysis or machine learning that correlates the production amount of the above useful compounds of each strain obtained in step (C) with the plasmid sequence information obtained in step (D), and (F)(E) A process to create a strain by recombining useful genes that contribute to the production of useful compounds identified in step (E). A method for constructing metabolically modified microbial strains, including those mentioned above. [2] A method for constructing a metabolically modified microbial strain as described in [1], wherein the series of steps (B) to (E) are repeated multiple times. [3] A method for constructing a metabolically modified microbial strain according to [1] or [2], wherein the metabolic modification in step (A) is based on metabolic flux balance analysis (FBA) for the production of the above-mentioned useful compound. [4] A method for constructing a metabolically modified microbial strain according to any one of [1] to [3], wherein the above useful compound is any of α-ketoglutaric acid, tyrosine, L-glutamic acid, pyruvate, UDP-glucose, succinic acid, acetic acid, farnesyl pyrophosphate, glutathione, formic acid, formaldehyde, L-methionine, glycine, glyoxylic acid, geranylgeranyl diphosphate, or acetyl-CoA. [5] A method for constructing a metabolically modified microbial strain according to any one of [1] to [4], wherein the metabolically modified microbial strain is one of Escherichia coli, yeast, microalgae, cyanobacteria (blue-green algae), actinomycetes, or coryneform bacteria. [6] A method for constructing a metabolically modified microbial strain according to any one of [1] to [5], wherein the above useful compound is α-ketoglutaric acid, and the enzyme group in step (B) comprises glk, pgi, pfkA, fbaA, tpiA, gapA, pgk, gpmA, eno, pykF, lpdA, aceE, aceF, gltA, acnB, icd, and ppc. A metabolically modified Escherichia coli strain with up-expression of aceF, ppc, and glk, constructed by the method described in [7][6]. [Effects of the Invention]

[0009] The present invention's method for constructing metabolically modified microbial strains makes it possible to efficiently construct strains that produce high levels of useful compounds, such as hub compounds, in microbial fermentation. By constructing a base strain by subjecting a parent strain to metabolic modification based on metabolic flux balance analysis (FBA), and then introducing a Combi-OGAB long-chain DNA library using artificial DNA parts that are not subject to endogenous metabolic regulation into the base strain, it is possible to construct a metabolically modified microbial strain that produces significantly more useful compounds compared to the parent strain. As shown in the examples, the present invention successfully constructed a strain that can produce high levels of α-ketoglutaric acid as a hub compound (useful compound). The present invention's method also makes it possible to construct high-production strains of other useful compounds. Furthermore, since the metabolically modified microbial strains constructed by the present invention can produce high levels of hub compounds in microbial fermentation, they can be utilized in the production of various substances using them as starting materials. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the flask culture results for BW25113 and the base strain. [Figure 2]Figure 2 is a diagram showing the genes that regulate the expression level by the Combi-OGAB method. [Figure 3] Figure 3 is a diagram showing the results of the culture test of the first-generation Combi-OGAB plasmid-introduced strain in a 96-well plate. [Figure 4-1] Figure 4-1 is a diagram showing the statistical analysis results of the first-generation Combi-OGAB plasmid-introduced strain. [Figure 4-2] Figure 4-2 is a diagram showing the statistical analysis results of the first-generation Combi-OGAB plasmid-introduced strain. ]> [Figure 4-3] Figure 4-3 is a diagram showing the statistical analysis results of the first-generation Combi-OGAB plasmid-introduced strain. [Figure 5] Figure 5 is a diagram showing the results of the culture test of the ppc and aceF overexpressing strains. [Figure 6] Figure 6 is a diagram showing the results of the culture test of the second-generation Combi-OGAB plasmid-introduced strain. [Figure 7-1] Figure 7-1 is a diagram showing the statistical analysis results of the second-generation Combi-OGAB plasmid-introduced strain. [Figure 7-2] Figure 7-2 is a diagram showing the statistical analysis results of the second-generation Combi-OGAB plasmid-introduced strain. [Figure 7-3] Figure 7-3 is a diagram showing the statistical analysis results of the second-generation Combi-OGAB plasmid-introduced strain. [Figure 8] Figure 8 is the sequence information of the Combi-OGAB plasmid introduced into the Cy1-A8 strain. [Figure 9] Figure 9 is the production test results of homocitric acid, GABA, and theanine.

Modes for Carrying Out the Invention

[0011] The present invention will now be described in detail. In this specification, unless otherwise specified, molecular biological techniques may be performed by methods known to those skilled in the art in general experimental manuals or similar methods. Furthermore, unless otherwise specified, terms used herein shall be interpreted in the sense commonly used in the art.

[0012] <Method for constructing metabolically modified microbial strains> The present invention relates to a method for constructing a metabolically modified microbial strain that produces useful compounds at high levels. Specifically, the present invention relates to a method for constructing a metabolically modified microbial strain that includes the following steps (A) to (F). (A) The process of constructing a base strain by subjecting the parent plant to metabolic modification. (B) A step to construct a plasmid library by preparing unit DNA cassettes in which a promoter for strong expression and a terminator sequence are ligated to the DNA encoding each enzyme of the enzyme group that constitutes the biosynthesis pathway of the above useful compounds functionally in cells, and by preparing unit DNA cassettes in which a promoter for low expression (or without a promoter) and a terminator sequence are ligated to the DNA encoding each enzyme, and then performing gene assembly using the OGAB method. (C) A step of introducing the plasmid library constructed above into the base strain and measuring the production amount of the above useful compound from each strain. (D)(C) Step to analyze the sequence information of plasmids introduced into each strain obtained in step (C). (E)(C) A step to identify useful genes that contribute to the production of the above useful compounds by performing statistical analysis or machine learning that correlates the production volume of the above useful compounds of each strain obtained in step (C) with the plasmid sequence information obtained in step (D). (F)(E) A process to create a strain by recombining useful genes that contribute to the production of useful compounds identified in step (E).

[0013] The above-mentioned useful compounds are not particularly limited as long as they are compounds produced by microbial fermentation, but examples include α-ketoglutaric acid, tyrosine, L-glutamic acid, pyruvate, UDP-glucose, succinic acid, acetic acid, farnesyl pyrophosphate, glutathione, formic acid, formaldehyde, L-methionine, glycine, glyoxylic acid, geranylgeranyl diphosphate, and acetyl-CoA.

[0014] The following describes in detail each step of the method for constructing the metabolically modified microbial strain of the present invention.

[0015] [Process (A)] This process involves metabolically modifying the parent plant to construct a base strain.

[0016] The parent strains mentioned above can be selected from microbial strains such as E. coli, yeast, microalgae, cyanobacteria (blue-green algae), actinomycetes, and coryneform bacteria. These parent strains may be naturally occurring strains or genetically modified strains.

[0017] The base strain mentioned above is a microbial strain used in step (B) and beyond to introduce a plasmid library constructed by gene assembly using the OGAB method (Tsuge, K., Matsui, K. & Itaya, M. One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res. 31, e133 (2003)). It refers to a microbial strain that has undergone metabolic modification to improve the production efficiency of useful compounds using methods such as the metabolic flux analysis described later. In other words, it is a strain in which the gene expression necessary to improve the production efficiency of useful compounds has been enhanced compared to the parent strain, and the gene expression that contributes little to or no to improving production efficiency has been suppressed (destroyed).

[0018] For the parent strains selected above, metabolic flux analysis (flux balance analysis) (FBA) is performed to predict the genes necessary to improve the production efficiency of useful compounds and the genes that contribute little to or no to improving production efficiency. Here, metabolic flux analysis (FBA) is one of the methods that has been frequently used in metabolic simulations in recent years. Based on genomic information and predictions, a metabolic pathway is constructed, and the increase (e.g., synthesis reactions and uptake transport) and decrease (decomposition / conversion reactions and efflux transport) of each node (metabolic molecule) are represented by a matrix. At this time, the equation is solved assuming that the metabolic system is in a steady state, that is, that metabolic intermediates in the model are maintained at a constant amount, and the obtained solution is restricted by adding constraints such as the direction and rate upper limit of the reaction. Based on this result, the phenomenon to be desired (e.g., enzyme inactivation or increase in biomass production) is given as the objective function, and the flux is predicted by finding a solution that satisfies it. Based on this prediction, the parent strains are modified to improve the production efficiency of useful compounds, that is, the gene expression necessary to improve the production efficiency of useful compounds is enhanced, and the expression of unnecessary genes is suppressed, thereby constructing a base strain. However, since predictions using FBA are not perfect, researchers should carefully review the analysis results and appropriately select which genes should be enhanced or suppressed.

[0019] If the target useful compound is α-ketoglutaric acid (AKG), then, based on the FBA results, it is possible to create a base strain such as the following: Specifically, create an E. coli strain lacking the acetate kinase gene (ackA) and phosphate acetyltransferase gene (pta), which are responsible for acetic acid biosynthesis; the glucose-6-phosphate 1-dehydrogenase gene (zwf), which is responsible for the reaction from glycolysis to the pentose phosphate pathway; and the gene encoding a protein involved in glucose uptake by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (ptsHI). Subsequently, plasmids for overexpression of the galP and glk genes can be introduced into this E. coli strain to create a base strain.

[0020] [Process (B)] This process involves preparing overexpression unit DNA cassettes, each containing a promoter and terminator sequence for strong expression linked to the DNA encoding each enzyme that constitutes the biosynthesis pathway of the target useful compound functionally within a cell, and non-overexpression unit DNA cassettes, each containing a promoter (or no promoter) and terminator sequence for low expression linked to the DNA encoding each enzyme, and constructing a combinatorial library using the Combi-OGAB method, which is an application of the OGAB method.

[0021] Because the central metabolic pathway within the base strain is strictly controlled by endogenous metabolic regulatory mechanisms, it is difficult to optimize the expression of genes in the useful compound production pathway as derived from FBA. Therefore, a Combi-OGAB long-chain DNA library is designed and constructed using artificial DNA parts that are not subject to endogenous metabolic regulation, and introduced into the base strain constructed in step (A) above. Furthermore, genes that contribute to high production of useful compounds can be selected using information analysis techniques.

[0022] The enzyme group described above is necessary for the biosynthesis pathway of the target useful compound and must function within the base strain described above. For example, if the target useful compound is α-ketoglutaric acid (AKG), the biosynthesis pathway and necessary enzyme group shown in Figure 2 can be cited. In Figure 2, glk, pgi, pfkA, fbaA, tpiA, gapA, pgk, gpmA, eno, pykF, lpdA, aceE, aceF, gltA, acnB, icd, and ppc are the enzymes necessary for the biosynthesis of AKG from glucose. The expression of these genes can be optimized using the Combi-OGAB method. Optimization involves the following two elements: finding the optimal combination of overexpressed genes from the above enzyme group that is effective in increasing the production of the useful compound, and finding the combination of genes that should not be overexpressed (or should be disrupted) that is effective in increasing the production of the useful compound.

[0023] In this process, overexpression unit DNA cassettes are prepared by ligating a strong expression promoter and a terminator sequence to the DNA encoding each enzyme, and non-overexpression unit DNA cassettes are prepared by ligating a low expression promoter (or no promoter) and a terminator sequence to the DNA encoding each enzyme. To verify the comprehensive overexpression combinations of genes for each enzyme in the metabolic pathway of useful compounds, two types of unit DNA cassettes, an overexpression DNA cassette and a non-overexpression DNA cassette, are created for each enzyme gene. The overexpression DNA cassette consists of a promoter sequence, a ribosome-binding sequence (RBS), a CDS, and a terminator sequence. For the non-overexpression cassette, one consisting of a CDS with the start codon removed and a terminator sequence, or one consisting of a low expression promoter sequence, a ribosome-binding sequence (RBS), a CDS, and a terminator sequence may be used, but it is preferable to use one consisting of a CDS with the start codon removed and a terminator sequence.

[0024] The origin of the CDS is not particularly limited, but is selected appropriately for each enzyme. Examples include sequences derived from E. coli, sequences derived from Klebsiella pneumoniae, and sequences in which mutations have been introduced to prevent allosteric inhibition. In addition, the promoter sequence and terminator sequence are selected appropriately for each enzyme. Overexpression unit DNA cassettes, in which the DNA encoding each enzyme is linked to a promoter and terminator sequence for strong expression, and non-overexpression unit DNA cassettes, in which the DNA encoding each enzyme is linked to a promoter (or no promoter) and terminator sequence for low expression, can be produced by artificial synthesis.

[0025] The unit DNA cassette is used after cloning a DNA fragment amplified by polymerase chain reaction (PCR) using primers that have restriction enzyme recognition sequences added to the base sequence of a template DNA to generate specific overhangs, or by chemosynthetic DNA fragments that have restriction enzyme recognition sequences pre-integrated to generate arbitrary overhangs at the ends, into a plasmid vector and confirming the base sequence. Each unit DNA is designed to be linked in a specific order to ultimately obtain the DNA fragment for microbial transformation.

[0026] Next, DNA fragments combining the accumulation vector with the unit DNA cassette of each enzyme gene in the enzyme group are ligated using the OGAB method. For the gene group involved in the metabolic pathway of the useful compound, overexpression and non-overexpression DNA cassettes for each enzyme are prepared. For example, let's explain using the case where the useful compound is α-ketoglutaric acid (AKG). Eighteen DNA fragments, each combining the accumulation vector with the unit DNA cassette of the genes of the 17 enzymes necessary for the biosynthesis pathway shown in Figure 2, are ligated using the OGAB method. The overexpression and non-overexpression DNA cassettes of all 17 enzyme genes involved in the AKG metabolic pathway are defined as the 1st to 17th unit DNA cassettes, respectively. The accumulation vector is defined as the 18th unit DNA cassette. The 1st to 18th unit DNA cassettes are consecutive in numerical order, and the 18th and 1st unit DNA cassettes are linked to form one insertion unit. At the end of each unit DNA cassette, there are 3' end overhangs of three bases, each specified for the unit DNA cassette number, on both the left and right sides of the fragment. This complementarity designates the linking partner. The structure of this overhang is not particularly restricted, including differences in the shape of 5' and 3' end overhangs, as long as it is not a batch structure (palindrome). However, the overhanging ends may be created by restriction enzyme digestion when preparing the unit DNA. If an enzyme capable of recognizing a specific sequence and creating an overhanging end of any sequence in its vicinity is used as the restriction enzyme, the overhanging ends of the unit DNA fragment will be different at each linking site, thus preserving the linking order. Examples of these restriction enzymes include, in addition to restriction enzymes commonly used in molecular biology, artificial restriction enzymes such as TALEN and ZNF, or CRISPR-related enzymes whose overhang ends can be purified, such as CRISPR-Cpf1. However, it is preferable to use Type II restriction enzymes such as AarI, AlwNI, BbsI, BbvI, BcoDI, BfuAI, BglI, BsaI, BsaXI, BsmAI, BsmBI, BsmFI, BspMI, BspQI, BtgZI, DraIII, FokI, PflMI, SfaNI, and SfiI. The multiple overhang sequences obtained by these restriction enzyme treatments must be unique sequences within a single plasmid.Furthermore, the species plasmids must have identical overhang sequences on the same strand and in the same order within the recombinant unit of the combinatorial library (in most cases, the unit DNA corresponds to that unit, but in some cases, the recombinant unit in some species plasmids may consist of multiple unit DNAs).

[0027] A single insertion unit containing the genes involved in the AKG metabolic pathway described above can be specifically constructed as follows: (18th unit DNA)-GTT-(1st unit DNA)-TGA-(2nd unit DNA)-CGA-(3rd unit DNA)-TGT-(4th unit DNA)-GAT-(5th unit DNA)-TTG-(6th unit DNA)-GTC-(7th unit DNA)-ATG-(8th unit DNA)-TGG-(9th unit DNA)-TAG-(10th unit DNA)-ACT-(11th unit DNA)-GTA-(12th unit DNA)-CTT-(13th unit DNA)-CAG-(14th unit DNA)-GAA-(15th unit DNA)-CTC-(16th unit DNA)-CAC-(17th unit DNA))-TCT-(18th unit DNA).

[0028] One or more of the unit DNAs that make up an inserted DNA unit must contain a valid replication origin in the host cell. Other unit DNAs, such as metabolic pathway clusters, parts or all of a continuous genome sequence, artificial genes, artificial gene circuits, etc., are elements that constitute a continuous base sequence, but there is no constraint that a single unit DNA must correspond to a biologically functional unit.

[0029] In constructing OGAB species plasmids, it is possible to create DNA fragments for microbial transformation by ligating the aforementioned unit DNAs in a mixture of unit DNAs adjusted to be approximately equimolar, using DNA ligase or the like. However, the starting material for gene accumulation is not limited to the aforementioned unit DNAs alone; any aggregate prepared by any accumulation method can be used as long as it ultimately has a structure that can be separated into the individual unit DNAs.

[0030] The method of ligating the unit DNA is not particularly limited, but it is preferable to carry it out in the presence of polyethylene glycol and a salt. A monovalent alkali metal salt is preferred as the salt. Specifically, it is more preferable to carry it out in a ligation reaction solution containing 10% polyethylene glycol 6000 and 250 mM sodium chloride. The concentration of each unit DNA in the reaction solution is not particularly limited, but it is preferable that each be at a concentration of 1 fmol / μL or more and equimolar. The enzyme, reaction temperature, and time for ligation are not particularly limited, but it is preferable to use T4 DNA polymerase at 37°C for 30 minutes or more.

[0031] The host microorganism in the DNA fragment for microbial transformation of the present invention is not particularly limited, as long as it has the ability to undergo spontaneous transformation. Examples of such microorganisms include those that have the ability to undergo spontaneous transformation by processing the DNA into single-stranded DNA before incorporating it. Specifically, examples include Escherichia coli, Bacillus species, Streptococcus species, Haemophilus species, and Neisseria species. Examples of Bacillus species include B. subtilis (Bacillus subtilis), B. megaterium (Giant Bacteria), and B. stearothermophilus (Moderate Hyperthermia). Among these, Escherichia coli and Bacillus subtilis are more preferred microorganisms due to their excellent spontaneous transformation and recombination abilities.

[0032] The method for making microbial cells competent can be selected from known methods suitable for each microorganism. Specifically, for example, in the case of Bacillus subtilis, it is preferable to use the method described in Anagnostopoulou, C. and Spizizen, JJ Bacteriol., 81, 741-746 (1961). Similarly, a known method suitable for each microorganism can be used for transformation. There are no particular restrictions on the volume of ligation product given to the competent cells. Preferably, it is 1 / 20 to the same volume as the competent cell culture medium, and more preferably half the volume. Known methods can also be used for purifying plasmids from the transformants.

[0033] The presence of the target insertion DNA in the plasmid obtained by the above method can be confirmed by the size pattern of the fragments generated by restriction enzyme digestion, PCR, or sequencing. Furthermore, if the insertion DNA has a function such as substance production, this can be confirmed by detecting that function.

[0034] For the preparation of seed plasmids used in combinatorial library construction, any method that is common for purifying circular plasmids can be used. However, it is preferable to use a method that does not risk contamination with DNA other than plasmid DNA, and specifically, cesium chloride-ethidium bromide density gradient ultracentrifugation is preferred.

[0035] Two plasmids are constructed using the OGAB method: Seed plasmid 1, which consists of DNA cassettes overexpressing each enzyme, and Seed plasmid 2, which consists of DNA cassettes not overexpressing each enzyme. The prepared seed plasmids are treated with restriction enzymes appropriate for each to degrade them into unit DNA, and a mixture of multiple types of unit DNA is prepared. The prepared seed plasmids are purified to a high degree of purity and then degraded into unit DNA.

[0036] The unit DNA mixture obtained in this process is purified to an extremely high degree of purity, so that no DNA fragments other than plasmid DNA are present. By cutting the prepared long-chain DNA with restriction enzymes and removing the restriction enzymes, a DNA fragment solution (unit DNA mixture) can be obtained in which the molar concentration ratio of all DNA fragments approaches 1.

[0037] A mixed solution of unit DNA cassettes derived from two types of seed plasmids is a DNA fragment solution (unit DNA mixture) in which the molar concentration ratio of all DNA fragments approaches 1. This DNA fragment is then reassembled using the OGAB method to prepare new DNA fragments, which are then transformed to construct a plasmid library. By using a DNA fragment solution (unit DNA mixture) in which the molar concentration ratio of all DNA fragments approaches 1 as the starting material for the gene assembly method (OGAB method), gene assembly can be performed more efficiently, and a plasmid library (Combi-OGAB plasmid library) can be constructed.

[0038] [Process (C)] This process involves introducing the plasmid library constructed above into the base strain and measuring the production volume of the useful compound from each strain.

[0039] The Combi-OGAB plasmid library obtained in step (B) is transformed into E. coli or other bacteria by conventionally known methods such as electroporation. The cells after electroporation are suspended in a culture medium and recovery culture is performed for about 1 hour under conditions such as 30°C and 150 rpm. The culture medium is spread onto a chloramphenicol-containing LB plate to obtain transformants.

[0040] From the Combi-OGAB plasmid library transformation plates obtained above, randomly selected colonies are taken, as well as colonies from plates of E. coli strains transformed with seed plasmids 1 and 2, respectively. These are then cultured for approximately 24 hours according to standard procedures, and further cultured in M9YE medium or similar at 37°C with stirring at approximately 1,000 rpm. After approximately 18 hours of culture, the useful compounds or their converters are measured in the culture supernatant. The amount of useful compounds in each sample can be measured using a biosensor.

[0041] [Process (D)] This step involves analyzing the sequence information of the plasmids introduced into each strain obtained in step (C).

[0042] In Combi-OGAB plasmids introduced into E. coli strains, melting curve analysis using real-time PCR is performed to determine whether the plasmids that produced high levels of useful compounds were overexpressed or not, or whether they were overexpressed DNA cassettes.

[0043] Specifically, to determine the accumulation cassette, primers are designed and used for each enzyme gene that sandwich the junction of the accumulated cassette, and primers that specifically anneal to the promoter of the overexpressed DNA cassette. PCR reactions are performed using the cultured bacterial suspension and seed plasmids 1 and 2 as PCR templates. The accumulation cassette of the Combi-OGAB plasmid introduced into the E. coli strain can be determined by comparing the melting curve profile of the enzyme gene obtained by PCR with the melting curve profile of the seed plasmid.

[0044] [Process (E)] This step involves performing statistical analysis or machine learning that correlates the production volume of the above-mentioned useful compounds from each strain obtained in step (C) with the sequence information of the plasmids obtained in step (D), in order to identify useful genes that contribute to the production of the above-mentioned useful compounds. In order to identify the genes among the enzyme group that contribute to the increase in the production volume of useful compounds, statistical analysis is performed using the data on the production volume of useful compounds and the sequence analysis data of the combinatorial plasmids. This makes it possible to find genes in which the overexpression or non-overexpression has a significant effect on improving the production volume of useful compounds.

[0045] [Process (F)] This step involves creating recombinant strains of useful genes that contribute to the production of the useful compounds identified in step (E). Plasmids for the single overexpression of the useful genes found to contribute to the production of the useful compounds, and plasmids for the simultaneous overexpression of all genes, are created and introduced into the base strain. It is then confirmed that the production of the useful compounds is improved in these strains.

[0046] In the method for constructing metabolically modified microbial strains of the present invention, it is preferable to repeat the series of steps (B) to (E) multiple times.

[0047] Specifically, as a second-generation combinatorial plasmid library, useful genes (genes that are preferably overexpressed) that have been found to contribute to the production of useful compounds will be expressed using only overexpression DNA cassettes, while the remaining genes will be expressed using both overexpression and non-overexpression DNA cassettes. In addition to overexpressing the useful genes identified in the first generation, this will allow for the search for genes that contribute to improving the production of useful compounds.

[0048] Similar to the first-generation combinatorial plasmid library, the created second-generation combinatorial plasmid library is introduced into a base strain, and statistical analysis or machine learning is performed to correlate the production of the above-mentioned useful compounds in each resulting strain with the plasmid sequence information, thereby further identifying useful genes that contribute to the production of the above-mentioned useful compounds. By repeating the same method, it is possible to introduce a third-generation combinatorial plasmid library into the base strain and obtain metabolically modified microbial strains with even higher production of useful compounds.

[0049] The present invention also includes, as a specific microbial strain constructed by the method for constructing metabolically modified microbial strains of the present invention described above, a metabolically modified Escherichia coli strain in which the expression of aceF, ppc, and glk is upregulated. This metabolically modified Escherichia coli strain is a strain in which the production of α-ketoglutaric acid is increased. [Examples]

[0050] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0051] <Experimental Method> 1. Flux Balance Simulation (FBA) for AKG High Production Genetic disruption targets promising for increasing AKG production from glucose were derived using FBA. The glucose consumption rate was fixed at 10 mmol / gDCW / h, and computer calculations were performed using linear programming with the E. coli genome-scale model iJO1366 (PMID:21988831, PMCID:PMC3261703, DOI:10.1038 / msb.2011.65) (Orth, JD, Thiele, I., Palsson, BO, 2010. What is flux balance analysis? Nat. Biotechnol. 28, 245-248. https: / / doi.org / 10.1038 / nbt.1614). Parameters were set so that E. coli could freely take in nitrogen in the form of ammonium ions, phosphorus in the form of phosphate ions, and sulfur in the form of sulfate ions, which are the minimum nutrients necessary for E. coli growth. In addition, the system was configured to allow the free uptake of other metal ions such as iron and magnesium. An environment was set up that would allow E. coli to grow using glucose as the sole carbon source. Furthermore, the amount of oxygen uptake was gradually increased from zero (anaerobic conditions) until the growth rate stopped increasing (perfectly aerobic conditions), allowing the amount of oxygen uptake to be varied. The solver used for the calculations was the open-source software GLPK (GNU Linear Programming Kit). To search for deficient and enhanced metabolic reaction enzymes, the objective function of the above linear programming was set to (1) maximization of E. coli growth and (2) maximization of the target compound, and calculations were performed for each. The predicted intracellular metabolic flux results were compared, and those in calculation (1) where a flux value exists but in calculation (2) where the flux value is 0 were considered candidates for deficient. Conversely, those in calculation (1) where there is no flux value or a flux value exists, but in calculation (2) where the flux value is increasing, were considered candidate reactions to be enhanced.

[0052] 2. Construction of AKG-based stocks based on FBA FBA identified ptsHI, ackA-pta, and zwf as disruption targets for high AKG production. These genes were disrupted from Escherichia coli strain BW25113 using the λ-red recombination method (Datsenko, KA, Wanner, BL, 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645. https: / / doi.org / 10.1073 / pnas.120163297). Briefly, the BW25113 strain was first transformed with the pKD46 plasmid containing the gene encoding the recombinase by electroporation to create the BW25113 / pKD46 strain.

[0053] DNA fragments for disrupting the ptsHI gene by substituting it with a kanamycin resistance gene were prepared by PCR using pKD13, which contains the kanamycin resistance gene, as a template and primers d-ptsHI F and d-ptsHI R (SEQ ID NOs: 1 and 2). The prepared DNA fragments were used to transform the BW25113 / pKD46 strain by electroporation to obtain transformants exhibiting kanamycin resistance (transformers in which ptsHI was replaced with the kanamycin resistance gene). Subsequently, to remove the kanamycin resistance marker, a flippase (FLP) expression plasmid was introduced by electroporation to obtain transformants exhibiting kanamycin sensitivity (BWΔptsHI / pKD46).

[0054] DNA fragments for disrupting the ackA-pta gene by substituting it with a kanamycin resistance gene were prepared by PCR using pKD13 as a template and primers d-ackA-pta F and d-ackA-pta R (SEQ ID NOs: 3 and 4). The prepared DNA fragments were transformed into the BWΔptsHI / pKD46 strain by electroporation to obtain transformants exhibiting kanamycin resistance (transformers in which ackA-pta was replaced with a kanamycin resistance gene). Subsequently, to remove the kanamycin resistance marker, a flippase (FLP) expression plasmid was introduced by electroporation to obtain transformants exhibiting kanamycin sensitivity (BWΔptsHIΔackA-pta / pKD46).

[0055] DNA fragments for disrupting the zwf gene by substituting it with a kanamycin resistance gene were prepared by PCR using pKD13 as a template and primers d-zwf F and d-zwf R (SEQ ID NOs. 5 and 6). The prepared DNA fragments were transformed into the BWΔptsHIΔackA-pta / pKD46 strain by electroporation to obtain a kanamycin-resistant transformant (a transformant in which zwf was replaced with a kanamycin resistance gene). Subsequently, a flippase (FLP) expression plasmid was introduced by electroporation to remove the kanamycin resistance marker, and a kanamycin-sensitive strain (BWΔptsHIΔackA-ptaΔzwf / pKD46) was obtained.

[0056] BWΔptsHIΔackA-ptaΔzwf / pKD46 was cultured overnight in 5 mL of antibiotic-free LB liquid medium, then seeded onto antibiotic-free LB agar medium. From the resulting colonies, ampicillin-sensitive transformants (BWΔptsHIΔackA-ptaΔzwf) with pKD46 detached were obtained. Furthermore, transformants obtained by introducing the galP-glk overexpression plasmid described later were used as the AKG-based strain in subsequent experiments.

[0057] 3. Plasmid construction Plasmid construction was performed using E. coli DH5α strain, and culture was carried out in LB medium (10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl). The galP-glk overexpression plasmid was prepared according to the procedure shown below.

[0058] The galP-glk overexpression plasmid was created by cloning the λpR promoter, galP-glk gene, and TrrnB terminator fragments in sequence onto the pETDuet-1 plasmid (Novagen) with the MCS and lacI gene regions removed. Each fragment was amplified by PCR using KOD one PCR Master Mix. The primers and templates used are described below.

[0059] The galP gene was obtained as a 1428 bp fragment using the genome of E. coli strain BW25113 as a template, with primers Pr-galP-F and galP-R-glk (sequence numbers 7 and 8). To conjugate the λpR promoter to the galP gene, the galP gene fragment obtained by PCR was used as a template for PCR with Pr-F1 and galP-R-glk (sequence numbers 9 and 8). Further PCR was performed using the obtained fragment as a template with Pr-F2 and galP-R-glk (sequence numbers 10 and 8) to obtain a 1570 bp fragment in which the galP gene was conjugated with the λpR promoter.

[0060] The glk gene was obtained as a 1006 bp fragment using the genome of E. coli strain BW25113 as a template and primers galP-glk-F and glk-R-TrrnB (sequences 11 and 12). The galP and glk fragments were configured to join to form an operon. The TrrnB terminator was obtained as a 102 bp fragment using the genome of E. coli strain BW25113 as a template and primers glk-TrrnB-F and TrrnB-R (sequences 13 and 14). The pETDuet plasmid, which forms the backbone, was obtained as a 2532 bp fragment using pETDuet-1 as a template and primers pET-F and pET-R (sequences 15 and 16).

[0061] After PCR, the five fragments were subjected to electrophoresis, and the fragments of the desired length were excised from the gel and recovered using the FastGene Gel / PCR Extraction kit (Genetics Japan). The recovered fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher) and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and those confirmed to have the correct sequence were used as plasmids for galP-glk overexpression.

[0062] The ppc overexpression plasmid (pCP-ppc) was prepared using the following procedure. pCP-ppc was created by cloning the ppc overexpression fragment obtained from seed plasmid 1 by PCR into a plasmid from which the MCS and lacI gene regions were removed from the pCOLADuet-1 plasmid (Novagen). The ppc overexpression fragment was amplified by PCR using KOD one PCR Master Mix with seed plasmid 1 as a template, using ppc_exp-F and ppc_exp-R (SEQ ID NOs. 17 and 18). The resulting PCR fragments were purified using the FastGene Gel / PCR Extraction kit. The purified PCR fragments and pCOLADuet-1 were then cleaved with restriction enzymes HpaI and PacI (New England Biolabs), respectively, and purified again using the FastGene Gel / PCR Extraction kit. PCR fragments cleaved with HpaI and PacI, along with pCOLADuet-1, were subjected to ligation reactions using Ligation high Ver.2 (TOYOBO) according to the attached manual, and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and plasmids with confirmed correct sequences were used for ppc overexpression.

[0063] The aceF overexpression plasmid (pCP-aceF) was prepared using the following procedure. pCP-aceF was created by cloning the aceF overexpression fragment obtained from seed plasmid 1 by PCR into the pCOLADuet-1 plasmid (Novagen) with the MCS and lacI gene regions removed. The aceF overexpression fragment was amplified by PCR using KOD one PCR Master Mix with seed plasmid 1 as a template, using aceF_exp-F and aceF_exp-R (SEQ ID NOs. 19 and 20). The resulting PCR fragments were purified using the FastGene Gel / PCR Extraction kit. The purified PCR fragments and pCOLADuet-1 were then cleaved with restriction enzymes HpaI and PacI (New England Biolabs), respectively, and purified using the FastGene Gel / PCR Extraction kit. PCR fragments cleaved with HpaI and PacI, along with pCOLADuet-1, were subjected to ligation reactions using Ligation high Ver.2 (TOYOBO) according to the attached manual, and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and plasmids with confirmed correct sequences were used for aceF overexpression.

[0064] The ppc-aceF overexpression plasmid (pCP-ppc-aceF) was prepared using the following procedure. pCP-ppc-aceF was created by cloning the ppc overexpression fragment and aceF overexpression fragment obtained by PCR from seed plasmid 1 into a plasmid from which the MCS and lacI gene regions of the pCOLADuet-1 plasmid (Novagen) had been removed. The ppc overexpression fragment was amplified by PCR using KOD one PCR Master Mix with seed plasmid 1 as a template, using duet-Pppc-F and PaceF_Tppc-R (sequence numbers 21 and 22). The aceF overexpression fragment was amplified by PCR using KOD one PCR Master Mix with seed plasmid 1 as a template, using Tppc_PaceF-F and duet-TaceF-R (sequence numbers 23 and 24). The vector was also amplified by PCR using pCOLADuet-1 as a template, using duet geneart-F and duet geneart-R (sequence numbers 25 and 26). The three obtained PCR fragments were purified using the FastGene Gel / PCR Extraction kit. The three purified PCR fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and the plasmid confirmed to have the correct sequence was used for ppc-aceF overexpression.

[0065] 4. Design of DNA cassette for OGAB method To verify the comprehensive overexpression combinations of 17 genes present in the metabolic pathway from glucose to AKG, two types of unit DNA cassettes were created for each of the 17 genes: an overexpression DNA cassette and a non-overexpression DNA cassette. The overexpression DNA cassette consists of a promoter sequence, a ribosome-binding sequence (RBS), a CDS, and a terminator sequence. For the non-overexpression cassette, one consisting of a CDS with the start codon removed and a terminator sequence, or one consisting of a low-expression promoter sequence, a ribosome-binding sequence (RBS), a CDS, and a terminator sequence, was used. In this study, the one consisting of a CDS with the start codon removed and a terminator sequence was used. The overexpression DNA cassettes for the 17 genes are shown as SEQ ID NOs. 112-128, and the non-overexpression DNA cassettes are shown as SEQ ID NOs. 129-145.

[0066] For CDS, sequences derived from E. coli were used for 15 genes: glk, pgi, pfkA, faA, tpiA, gapA, pgk, gpmA, eno, pykF, ppc, aceE, aceF, acnB, and icd. For lpdA, Kp_lpdA_E354K was used, which encodes an enzyme derived from Klebsiella pneumoniae, which is more active even under anaerobic conditions than the enzyme LpdA from E. coli, and into which E354K was introduced to prevent allosteric inhibition by NADH. Similarly, for gltA, gltA_R164L was used, which encodes an enzyme derived from E. coli GltA into which the R164L mutation was introduced to prevent allosteric inhibition by NADH.

[0067] For the promoter sequences, we used 17 constitutively expressed artificial promoters with different sequences, as reported by Jensen et al. (Jensen, PR, Hammer, K., 1998. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol. 64, 82-87.). For RBS, we basically used the native RBS sequences for each gene. For Kp_lpdA_E354K and gltA_R164L, we used artificial RBS sequences designed with translational strengths comparable to the native sequences using an RBS calculator (https: / / salislab.net / software / ). The terminator sequences used were 17 artificial terminators with different sequences, as reported by Chen et al. (Chen, YJ, Liu, P., Nielsen, AAK, Brophy, JAN, Clancy, K., Peterson, T., Voigt, CA, 2013. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Methods 10, 659-664. https: / / doi.org / 10.1038 / nmeth.2515).

[0068] The overexpression unit DNA (SEQ ID NOs. 112-128) and non-overexpression unit DNA (SEQ ID NOs. 129-145) for 17 genes—glk, pgi, pfkA, faA, tpiA, gapA, pgk, gpmA, eno, pykF, ppc, Kp_lpdA(E354K), aceE, aceF, gltA(R164L), acnB, and icd—were artificially synthesized (by Integrated DNA Technologies).

[0069] 5. Design of Unit DNA Cassette Overhang Sequences In this invention, 17 gene unit DNA cassettes are combined with the accumulation vector pGETS118 to ligate a total of 18 DNA fragments using the OGAB method. There are 17 genes involved in the AKG metabolic pathway in E. coli, and their overexpression and non-overexpression DNA cassettes are defined as the 1st to 17th unit DNA cassettes, respectively. The accumulation vector is defined as the 18th unit DNA cassette. The 1st to 18th unit DNA cassettes are arranged consecutively in numerical order, and the 18th and 1st unit DNA cassettes are linked to form a single insertion unit. At the end of each unit DNA cassette, there are 3' end overhangs of three bases, each specified for the unit DNA cassette number, on both the left and right sides of the fragment. This complementarity specifies the linking partner. Specifically, the structure is as follows. (18th unit DNA)-GTT-(1st unit DNA)-TGA-(2nd unit DNA)-CGA-(3rd unit DNA)-TGT-(4th unit DNA)-GAT-(5th unit DNA)-TTG-(6th unit DNA)-GTC-(7th unit DNA)-ATG-(8th unit DNA)-TGG-(9th unit DNA)-TAG-(10th unit DNA)-ACT-(11th unit DNA)-GTA-(12th unit DNA)-CTT-(13th unit DNA)-CAG-(14th unit DNA)-GAA-(15th unit DNA)-CTC-(16th unit DNA)-CAC-(17th unit DNA))-TCT-(18th unit DNA)

[0070] 6. Construction of Unit DNA Cassette Plasmids The amplified DNA fragments were separated from the plasmid vector and unit DNA by electrophoresis on a 0.7% low-melting-point agarose gel (2-Hydroxyeth yl Agarose Type VII, Sigma-Ace) in the presence of 1×TAE (Tris-Ace tate-EDTA Buffer) using a general-purpose agarose gel electrophoresis system (i-MyRun.N nucleic acid electrophoresis system, Cosmo Bio) under a voltage of 100V (approximately 8V / cm) for 30 minutes. The electrophoresis gel was stained with 100 ml of 1×TAE buffer containing 1 μg / ml ethidium bromide (Sigma-Ace) for 30 minutes and visualized by illumination with long-wavelength ultraviolet light (366 mn). The PCR product of the desired size was then cut out with a razor blade and collected in a 1.5 ml tube. The recovered low-melting-point agarose gel (approximately 300 mg) was mixed with 1×TAE buffer to a total volume of approximately 700 μl, and the gel was dissolved by incubating at 65°C for 10 min. Then, an equal volume of TE-saturated phenol (Nacalai Tesque) was added and mixed thoroughly to inactivate the restriction enzymes. The phenol phase and aqueous phase were separated by centrifugation (20,000 × g, 10 min), and the aqueous phase (approximately 900 μl) was collected in a new 1.5 ml tube. 500 μl of 1-butanol (Wako Pure Chemical Industries, Ltd.) was added to this, mixed thoroughly, and separated by centrifugation (20,000 × g, 1 min). This process of removing the water-saturated 1-butanol was repeated until the volume of the aqueous phase was 450 μl or less, thereby reducing the volume of the aqueous phase. To this, 50 μl of 3M potassium acetate-acetic acid buffer (pH 5.2) and 900 μl of ethanol were added, and the DNA was precipitated by centrifugation (20,000 × g, 10 min). This precipitate was then rinsed with 70% ethanol and dissolved in 20 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The recovered DNA was stored at -20°C until use.

[0071] The obtained DNA fragments were cloned into an E. coli plasmid vector by TA cloning using the method described below. To 8 μl of the DNA fragment, 1 μl of 10× Ex-Taq Buffer (included with TAKARA's PCR enzyme Ex-Taq), 0.5 μl of 100 mM dATP, and 0.5 μl of Ex-Taq were added, and the mixture was incubated at 65°C for 10 minutes to add an A overhang to the 3' end of the DNA fragment. To 1 μl of this DNA fragment solution, 1 μl of TAKARA's pMD19-Simple and 3 μl of sterile water were mixed, and then 5 μl of TAKARA Ligation (Mighty) Mix was added, and the mixture was incubated at 16°C for 30 m in. 5 μl of this ligation solution was added to 50 μl of chemically competent E. coli DH5α cells, incubated on ice for 15 min, then subjected to a heat shock at 42°C for 30 sec, left on ice for 2 min, then 200 μl of LB medium was added, incubated at 37°C for 1 hour, and then streaked onto an LB plate containing 1.5% agar with carbenicillin at a concentration of 100 μg / ml, and incubated overnight at 37°C to obtain plasmid transformants.

[0072] The obtained colonies were prepared using a PCR template DNA preparation reagent (Shika Genius DNA Preparation Reagent, Kanto Chemical). Specifically, 2.5 μl of a solution was prepared by mixing reagent a and reagent b from the reagent kit in a 1:10 ratio. A small amount of colony was taken from the plate using a toothpick and suspended in this solution. The solution was then treated at 72°C for 6 minutes, followed by treatment at 94°C for 3 minutes. To the obtained liquid, 2.5 μl of TAKARA Ex-Taq 10× enzyme, 2 μl of 2.5 mM dNTP solution, 0.25 μl of 10 pmol / μl M13F primer, 0.25 μl of 10 pmol / μl M13R primer, 17 μl of sterile water, and 0.5 μl of Ex-TaqHS were added. After incubation at 94°C for 5 minutes, DNA was amplified by 30 cycles of 98°C for 20 seconds, 55°C for 30 seconds, and 72°C for 1 minute. The base sequence of this PCR product was examined to check if it perfectly matched the desired sequence. Ultimately, the correct sequence was obtained from all clones.

[0073] E. coli transformants containing plasmids that clone DNA fragments with the desired sequence were cultured overnight in 2 ml of LB medium containing 100 μg / ml carbenicillin at 37°C and 120 spm. The resulting cells were purified using the QIAfilter Plasmid mini Kit (Qiagen) according to the manual. The obtained plasmids were cut with SfiI, and the overexpression DNA cassette and non-overexpression DNA cassette were recovered by size fractionation using electrophoresis. E. coli transformants containing plasmids that clone DNA fragments with the desired sequence were cultured overnight in 2 ml of LB medium containing 100 μg / ml carbenicillin at 37°C and 120 spm. The resulting cells were purified using the QIAfilter Plasmid mini Kit (Qiagen) according to the manual. Ten μl of the obtained plasmid was isolated, and 30 μl of sterile water, 5 μl of 10×NEB buffer #2, and 5 μl of SfiI restriction enzyme (New England Biolabs) were added. The mixture was reacted at 50°C for 2 hours to cleave 17 unit DNA cassettes from the plasmid vector. The plasmid vector and unit DNA cassette were separated by electrophoresis on a 0.7% low-melting-point agarose gel in the presence of 1×TAE buffer using a general-purpose agarose gel electrophoresis apparatus with a voltage of 50V (approximately 4V / cm) applied and run for 1 hour. This electrophoresis gel was stained for 30 minutes with 100 ml of 1×TAE buffer containing 1 μg / ml ethidium bromide (Sigma-Ace), visualized with long-wavelength ultraviolet light (366 nm), and the band of the desired length was cut out with a razor blade and collected in a 1.5 ml tube. The collected low-melting-point agarose gel (approximately 300 mg) was purified as described above and dissolved in 20 μl of TE. The unit DNA cassette prepared in this way was quantified using a SYBR Green II fluorescence plate reader with nucleic acid fluorescent dyes, using a calibration curve created based on the dilution series of commercially available Lambdaphage genome DNA (TOYOBO).

[0074] 7. Gene assembly for seed plasmid construction In this study, two plasmids were constructed using the OGAB method: Seed plasmid 1, which consists of a sequence of DNA cassettes overexpressing 17 genes, and Seed plasmid 2, which consists of a sequence of DNA cassettes not overexpressing 17 genes.

[0075] For the accumulation of seed plasmid 1, unit DNA cassettes of SEQ ID NOs. 112-128 were mixed with 1 μL of each unit DNA cassette of SEQ ID NOs. 129-145 were mixed with 1 μL of a 1 f mol / μL solution of the gene accumulation vector pGETS118 (SEQ ID NO. 146) for the accumulation of seed plasmid 2. A total of 18 μL of DNA mixture was prepared by adding 20 μL of 2× ligation buffer and 2 μL of T4 DNA ligase (Takara), and the mixture was incubated at 37°C for 4 hours. After confirming ligation by electrophoresis using 10 μL of the mixture, 10 μL of the ligation reaction solution was transferred to a new tube, 100 μL of Bacillus subtilis competent cells were added, and the mixture was rotated in a duck rotor at 37°C for 30 minutes. Subsequently, 300 μl of LB medium was added, and the cultures were rotated in a duck rotor at 30°C for 2 hours. After that, the culture was spread onto an LB plate containing 10 μg / ml tetracycline and incubated overnight at 30°C. More than 100 Bacillus subtilis colonies were obtained from both the overexpressed DNA cassette aggregate (seed plasmid 1) and the non-overexpressed DNA cassette aggregate (seed plasmid 2). PCR was performed on a colony suspension template using 17 primer sets (SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, SEQ ID NOs. 31 and 32, SEQ ID NOs. 33 and 34, SEQ ID NOs. 35 and 36, SEQ ID NOs. 37 and 38, SEQ ID NOs. 39 and 40, SEQ ID NOs. 41 and 42, SEQ ID NOs. 43 and 44, SEQ ID NOs. 45 and 46, SEQ ID NOs. 47 and 48, SEQ ID NOs. 49 and 50, SEQ ID NOs. 51 and 52, SEQ ID NOs. 53 and 54, SEQ ID NOs. 55 and 56, SEQ ID NOs. 57 and 58, SEQ ID NOs. 59 and 60) designed to sandwich the junction of the DNA cassette, and transformants in which the 17 DNA fragments were correctly accumulated in pGETS118 were selected.

[0076] 8. High-purity purification of seed plasmids High-purity plasmid DNA was obtained by cesium chloride / ethidium bromide density gradient ultracentrifugation. Specifically, 200 ml of LB medium with antibiotic (tetracycline) was prepared, and 100 ml was placed in each of 500 ml Erlenmeyer flasks and incubated overnight at 30°C. After sufficient growth, 100 μl of 1 M IPTG was added to each flask to increase the plasmid copy number, and the cultures were incubated for a further 3 to 12 hours. After incubation, 50 ml of the culture solution was dispensed into four 50 ml tubes and centrifuged at 5,000 rpm for 10 minutes. The supernatant was discarded, and the bacterial pellet was completely loosened by vortexing. A 10 mg / ml lysozyme-containing Sol. I solution (composed of 50 mM glucose, 25 mM Tris-Cl (pH 8.0), and 10 mM EDTA) was prepared, and 2.5 ml was added to each of the four bacterial tubes and thoroughly mixed. This was incubated at 37°C for 30 minutes. The mixture was centrifuged at 5,000 rpm for 10 minutes, the supernatant was removed by decanting, and 2.5 ml of fresh lysozyme-free Sol.I was added to each of the four tubes to uniformly suspend the pellet. Fresh Sol.II (composition: 0.2N NaOH, 1% (w / v) sodium dodecyl sulfate) was prepared and 5 ml was added to each of the four tubes, and the mixture was slowly mixed until clear. Sol.III (composition: 60 ml of 5M potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of water) was added to each tube at 3.75 ml and mixed with moderate force to ensure even dispersion of the turbid substance. The mixture was centrifuged at 5,000 rpm for 10 minutes, the supernatant was pipetted into four new 50 ml tubes. 5 ml of phenol-chloroform was added to each tube and mixed vigorously. The mixture was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was pipetteed into four new 50 ml screw-cap tubes (Falcon 2070). 25 ml of 100% ethanol was added to each tube and mixed. The mixture was then centrifuged at 5,000 rpm for 10 minutes, and the supernatant was removed. 2.5 ml of a solution (final concentration 10 μg / ml) prepared by adding 10 μl of 10 mg / ml RNase A solution to 10 ml of TE was added to each tube to dissolve the precipitate. The liquids from the four tubes were combined into one tube and incubated in a 37°C gas-phase incubator for 30 minutes.After incubation, 5 ml of phenol-chloroform was added and mixed well, then centrifuged at 5,000 rpm for 10 min. The supernatant was transferred to a new 50 ml tube, 1 ml of Sol. III was added, followed by 25 ml of 100% ethanol, and mixed. Then, the mixture was centrifuged at 5,000 rpm for 10 min and the supernatant was removed. 5.4 ml of TE was added to the precipitate and dissolved completely. Next, 6.50 g of accurately weighed cesium chloride was added and dissolved completely. Furthermore, 2.6 ml of 1.1 g / ml cesium chloride solution (a solution prepared by mixing 1.1 g of cesium chloride with 1 ml of TE buffer (not volume adjusted)) was added. Finally, 600 μl of 10 mg / ml ethidium bromide solution was added, mixed well, centrifuged, and the supernatant was collected. One ultracentrifuge tube (Beckman 362181) was prepared, and the supernatant was transferred to the ultracentrifuge tube. The weight was finely adjusted by adding 1.1 g / ml cesium chloride solution (specific gravity approximately 1.5 g / ml) so that the weight difference with the balance was within 20 mg. Centrifuge was performed for more than 15 hours in an ultracentrifuge apparatus (Beckman Coulter) under the following conditions: temperature 18°C, speed 50,000 rpm, acceleration maximum, deceleration maximum. After centrifugation, under ultraviolet (365nm) observation, a 1ml syringe fitted with a needle (21G × 5 / 8") was prepared and inserted into the band of the ccc-type plasmid. The plasmid solution was collected and transferred to a 15ml tube. 200μl of Sol.III was added, followed by water until the total volume reached 3ml. Then, 9ml of 100% ethanol was added. The mixture was centrifuged at 10,000rpm for 10min, and the supernatant was removed. 700μl of TE was added to the resulting precipitate in a 15ml tube, and the DNA was lysed. Transfer this to a 1.5 ml tube, add 600 μl of 1-butanol and mix, then centrifuge at 15,000 rpm for about 10 seconds to separate into two layers, discarding the upper butanol layer. Add another 600 μl of 1-butanol and mix, then centrifuge at 15,000 rpm for about 10 seconds to separate into two layers, discarding the upper butanol layer. Continue this process until the aqueous layer is 450 μl or less. Add 50 μl of Sol. III, and then add 900 μl of 100% ethanol. Centrifuge at 15,000 rpm for 10 minutes.The supernatant was discarded, and the precipitate was rinsed with 70% ethanol. The precipitate was dissolved in 22 μl of TE.

[0077] 9. Generation of unit DNA from seed plasmids The preparation of unit DNA cassettes from seed plasmids was performed as follows: Approximately 30 μg of seed plasmid, purified to high purity by ultracentrifugation, was separated and made up to 40 μl with sterile water. 5 μl of 10×NEBbuffer #2 and 5 μl of restriction enzyme SfiI (NEB) were added, and the reaction was carried out overnight at 50°C. 1 μl of the reaction mixture was subjected to electrophoresis to confirm that the DNA had been cleaved. Subsequently, the reaction mixtures of the two seed plasmids were combined, 450 μl of phenol-chloroform-isoamyl alcohol (25:24:1) (Nacalai Tesque) was added, and after mixing, the mixture was separated into a phenol phase and an aqueous phase by centrifugation (20,000×g, 10 min). The aqueous phase (approximately 900 μl) was collected in a new 1.5 ml tube. 500 μl of 1-butanol (Wako Pure Chemical Industries, Ltd.) was added to this solution, mixed thoroughly, and then separated by centrifugation (20,000 × g, 1 min). This process of removing the water-saturated 1-butanol was repeated until the volume of the aqueous phase was 450 μl or less, thereby reducing the volume of the aqueous phase. To this, 50 μl of 3M potassium acetate-acetic acid buffer (pH 5.2) and 900 μl of ethanol were added, and the DNA was precipitated by centrifugation (20,000 × g, 10 min). This precipitate was rinsed with 70% ethanol and dissolved in 20 μl of TE.

[0078] 10. Construction of a combinatorial library using the Combi-OGAB method The construction of a combinatorial library using the OGAB method (Combi-OGAB method) was performed as follows: A mixed solution of unit DNA cassettes derived from the two types of seed plasmids obtained in item 9 above was assembled using the gene accumulation method described in item 7 above, yielding approximately 1,000 transformants per plate. From the obtained transformants, 24 colonies were randomly selected, and PCR was performed on the colony suspension as a template using 17 primer sets (SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, SEQ ID NOs. 31 and 32, SEQ ID NOs. 33 and 34, SEQ ID NOs. 35 and 36, SEQ ID NOs. 37 and 38, SEQ ID NOs. 39 and 40, SEQ ID NOs. 41 and 42, SEQ ID NOs. 43 and 44, SEQ ID NOs. 45 and 46, SEQ ID NOs. 47 and 48, SEQ ID NOs. 49 and 50, SEQ ID NOs. 51 and 52, SEQ ID NOs. 53 and 54, SEQ ID NOs. 55 and 56, SEQ ID NOs. 57 and 58, SEQ ID NOs. 59 and 60) designed to sandwich the DNA cassette junctions. As a result, it was confirmed that 17 gene cassettes were randomly accumulated in all 24 clones.

[0079] 11. Introduction of the Combi-OGAB plasmid library into E. coli The Combi-OGAB plasmid library obtained in item 10 above was transformed into E. coli AKG chassis strain by electroporation (conditions). Electroporation was performed using GenePulser Xcell. TM The procedure was performed using a BIO-RAD system (1500V, 25μF, 200Ω). After electroporation, the bacterial cells were suspended in 1 mL of SOC medium (Toyobo) and cultured at 30°C and 150 rpm for 1 hour. The culture solution was spread onto LB plates containing 12.5 μg / ml chloramphenicol and incubated overnight at 30°C. Approximately 100 transformants were obtained per plate.

[0080] 12. Evaluation of Combi-OGAB plasmid library-introduced strains 88 colonies were randomly selected from the Combi-OGAB plasmid library transformation plates obtained in item 11 above, and 4 colonies each were selected from plates of E. coli AKG chassis strain transformed with seed plasmids 1 and 2, respectively. These were scraped with a toothpick and inoculated into 96-well deep-bottom plates (Corning, product number 3960) dispensed with 1 mL of LB medium supplemented with chloramphenicol and ampicillin. The 96-well plates were cultured at 37°C and stirred at 1000 rpm in (M·BR-022, Taitec). Culture for 24 hours. A new 96-well plate was then inoculated with 1 mL of M9YE medium supplemented with chloramphenicol and ampicillin (90 mM disodium hydrogen phosphate heptahydrate, 22 mM potassium dihydrogen phosphate, 8.5 mM sodium chloride, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.1% ammonium chloride, 0.01% thiamine, 1.0 g / L Bacto). TM Yeast extract (Becton, Dickinson and Company) and 20 g / L glucose were added, and the mixture was cultured at 37°C with stirring at 1,000 rpm. After 18 hours of incubation, 90 μl of distilled water and 10 μl of culture medium were mixed on a new 96-well plate, and the OD (600 nm) was measured using an Envision 2104 Multilabel Reader (Perkin Elmer). To measure the amount of glutamic acid and glucose in the culture supernatant after 18 hours of incubation, the 96-well plate was centrifuged at 1000 g for 10 min, and 300 μl of distilled water and 100 μl of culture supernatant were mixed on a new 96-well plate. The amount of glutamic acid and glucose in the sample from each well was measured using a biosensor (BF-9, Oji Instruments Co., Ltd.).

[0081] 13. Determination of gene cassettes on plasmids For 17 genes, melting curve analysis using real-time PCR was performed to determine whether overexpression or non-overexpression DNA cassettes were accumulated in the Combi-OGAB plasmid introduced into E. coli strains cultured in 96-well plates.

[0082] Specifically, for the determination of the accumulated cassettes, primers were designed for each of the 17 genes to sandwich the junction of the 17 accumulated cassettes, and primers to specifically anneal to the promoter of the overexpression DNA cassette (SEQ ID NOs. 61-63, 64-66, 67-69, 70-72, 73-75, 76-78, 79-81, 82-84, 85-87, 88-90, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, 109-111). KOD SYBR® qPCR Mix (TOYOBO Co., Ltd.) was used for PCR, and the PCR reaction solution was prepared according to the attached manual. As a PCR template, 1 μL of the bacterial suspension after culturing in a 96-well plate was added directly to the PCR reaction medium, and three types of primers were added at a final concentration of 0.3 pmol each. PCR reactions and melting curve analysis were performed using the LightCycler® 96 System (Roche). By performing PCR reactions in the same manner using seed plasmids 1 and 2 as templates, it was confirmed that differences in melting curve patterns occurred when overexpression DNA cassettes and non-overexpression DNA cassettes were accumulated for 17 genes. The accumulation cassette of the Combi-OGAB plasmid introduced into the E. coli strains contained in the cultured sample was determined by comparing the melting curve profiles of the 17 genes obtained by PCR with the melting curve profiles of the seed plasmids.

[0083] 14. Identification of gene cassettes that contribute to increased AKG production through statistical analysis. Statistical analysis was performed using glutamate production data obtained from 96-well plates and sequence analysis data of plasmids introduced into E. coli used for culture, in order to identify genes that improve AKG production.

[0084] We denote whether a given unit DNA cassette is overexpressed or not as P and A, respectively. Furthermore, the 17 unit DNA cassettes possessed by a given strain are described as a 17-dimensional vector v=(P,A,P,A,A,A,A,A,P,A,P,A,A,P,A,P,A,P). Here, v is called the cassette vector, and its i-th element indicates whether the i-th unit DNA cassette is overexpressed or not.

[0085] First, we combined the data of strains that had the same cassette vector from the cultured strains. Specifically, if multiple strains had the same cassette vector v', those strains were removed from the data, and a hypothetical strain with the same cassette vector v' was added instead. The glutamate production of the added strain was set to the average of the glutamate production of the removed strains.

[0086] Next, the Mann-Whitney U test was used to determine whether there was a statistically significant difference in glutamate production between strains where the i-th element of the cassette vector was P and strains where it was A. This test was performed for each element i (=1,2,3,...,17) of the cassette vector to determine the P-value for each i. Then, the False Discovery Rate (FDR) for each P-value was calculated using the Benjamini-Hochberg method, and the i values ​​for which FDR < 0.05 were identified. The i values ​​that satisfied this condition were 11 and 14. The genes accumulated in the 11th and 14th unit DNA cassettes were ppc and aceF, and these two genes were identified as genes that contribute to the improvement of AKG production.

[0087] 15. Design and evaluation of a second-generation Combi-OGAB plasmid library For the construction of the second-generation Combi-OGAB plasmid library, only overexpression DNA cassettes were used for the two genes ppc and aceF, while both overexpression and non-overexpression DNA cassettes were used for the remaining 15 genes. Seed plasmid 3, in which overexpression cassettes for ppc and aceF and non-overexpression cassettes for the remaining 15 genes were accumulated, was prepared using the method described in item 7 above. Subsequently, unit DNA cassettes were purified from seed plasmid 1 and seed plasmid 3 using the method described in item 9 above, and combinatorial libraries using Combi-OGAB were prepared using the method described in item 10 above. The obtained plasmid solutions were transformed into E. coli strains using the same method as in item 11 above, and culture tests were performed using the same methods as in items 12 and 13 above. Glutamine production was measured, and the accumulation cassette of the Combi-OGAB plasmid introduced into each evaluated E. coli strain was determined. The obtained data were statistically analyzed using the method described in item 14 above.

[0088] 16. Extraction of OGAB plasmid from the glutamine-producing strain (Cy1-A8 strain) The Combi-OGAB plasmid (pCy1-A8) was extracted from the E. coli strain (Cy1-A8 strain) that produced the highest amount of glutamate among the transformed E. coli strains evaluated in item 12 above. For plasmid extraction, the Cy1-A8 strain was cultured overnight at 37°C and 120 spm in 5 mL of LB medium at 12.5 μg / ml. From the resulting cells, the plasmid (pCy1-A8) was purified using the QIAfilter Plasmid mini Kit (Qiagen) according to the manual.

[0089] 17. Disruption of the gabT gene from BW25113WT and BWΔptsHIΔackA-ptaΔzwf The gabT gene was disrupted in E. coli strain BW25113 and AKG-based strains using the λ-red recombination method. Briefly, the pKD46 plasmid containing the gene encoding the recombinase was first transformed into strains BW25113 and BWΔptsHIΔackA-ptaΔzwf by electroporation, creating strains BW25113 / pKD46 and BWΔptsHIΔackA-ptaΔzwf / pKD46.

[0090] DNA fragments for disrupting the gabT gene by replacing it with a kanamycin resistance gene were prepared by PCR using pKD13, which contains the kanamycin resistance gene, as a template and primers d-gabTF and dgabTR (SEQ ID NOs. 147 and 148). The prepared DNA fragments were transformed into the BW25113 / pKD46 strain and the BWΔptsHIΔackA-ptaΔzwf / pKD46 strain by electroporation to obtain transformants exhibiting kanamycin resistance (transformers in which gabT is replaced with the kanamycin resistance gene). Subsequently, to remove the kanamycin resistance marker, a flippase (FLP) expression plasmid was introduced by electroporation to obtain transformants exhibiting kanamycin sensitivity (BWΔgabT / pKD46 and BWΔptsHIΔackA-ptaΔzwf strain ΔgabT / pKD46).

[0091] BWΔgabT / pKD46 and BWΔptsHIΔackA-ptaΔzwfΔgabT / pKD46 strains were cultured overnight in 5 mL of antibiotic-free LB liquid medium, and then seeded onto antibiotic-free LB agar medium. From the resulting colonies, ampicillin-sensitive transformants (BWΔgabT and BWΔptsHIΔackA-ptaΔzwfΔgabT strains) were obtained, from which pKD46 had been removed.

[0092] 18. Introduction of galP-glk overexpression plasmid and pCy1-A8 plasmid into BWΔptsHIΔackA-ptaΔzwfΔgabT The galP-glk overexpression strain prepared in item 3 was introduced into the BWΔptsHIΔackA-ptaΔzwfΔgabT strain by electroporation. The resulting transformant was then further introduced with pCy1-A8 by electroporation, and this transformed strain was used as the Cy1-A8ΔgabT strain in subsequent experiments.

[0093] 19. Creation of homocitric acid, GABA, and theanine production plasmids The nifV overexpression plasmid (pTrc-nifV) was prepared using the following procedure. pTrc-nifV was created by cloning the nifV gene (SEQ ID NO: 149) from Azotobacter vinelandii, obtained through artificial synthesis (Thermo Fisher Scientific), into the MCS of the pTrc99a plasmid (Pharmacia). The DNA fragment for cloning was amplified by PCR using the artificially synthesized nifV gene as a template with pTrc NifV F and pTrc NifV R (SEQ ID NOs: 154 and 155) using KOD one PCR Master Mix. Plasmid pTrc99a was cleaved with restriction enzymes NcoI and BamHI (New England Biolabs). The resulting PCR fragments and restriction enzyme-treated plasmids were purified using the FastGene Gel / PCR Extraction kit, and the purified fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and those with the correct sequence were designated as pTrc-nifV and used in subsequent experiments.

[0094] The gdhA and gadB overexpression plasmid (pTrc-gdhA-gadBm) was prepared using the following procedure. pTrc-gdhA-gadBm was created by cloning the gdhA gene (SEQ ID NO: 150) derived from E. coli obtained by PCR, and the gadB gene (SEQ ID NO: 151) derived from E. coli with the E89Q and Δ452-466 ​​mutations introduced, into the MCS of the pTrc99a plasmid. The gdhA gene was obtained using the genome of E. coli strain BW25113 as a template and the primers pTrc gdhA F and pTrc gdhA R (SEQ ID NOs: 156 and 157). Plasmid pTrc99a was digested with restriction enzymes NcoI and BamHI (New England Biolabs). The obtained PCR fragments and restriction enzyme-treated plasmids were purified using the FastGene Gel / PCR Extraction kit. The purified fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and those confirmed to have the correct sequence were designated as pTrc-gdhA and used in subsequent experiments. The gadB (E89Q, Δ452-466) gene was obtained using the genome of E. coli strain BW25113 as a template, with two primer sets: gdhA-gadBm F and gadB_E89Q R (sequences 158 and 159), and gadB_E89Q F and gdhA-gadBm R (sequences 160 and 161). The plasmid pTrc-gdhA was cleaved with the restriction enzyme BamHI (New England Biolabs). The two obtained PCR fragments and restriction enzyme-treated plasmids were purified using the FastGene Gel / PCR Extraction kit. The purified fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and the sequence confirmed to be correct was named pTrc-gdhA-gadBm and used in subsequent experiments.

[0095] The γ-glutamylmethylamide synthetase (GMAS) overexpression plasmid (pTrc-gmas) was prepared using the following procedure. pTrc-gmas was created by cloning the GMAS gene (SEQ ID NO: 152) derived from Pseudomonas syringae, optimized for E. coli codons and obtained through artificial synthesis (Thermo Fisher Scientific), into the MCS of the pTrc99a plasmid. The DNA fragments for cloning were amplified by PCR using the artificially synthesized GMAS gene as a template with pTrc gmas F and pTrc gmas R (SEQ ID NOs: 162 and 163) using KOD one PCR Master Mix. Plasmid pTrc99a was cleaved with restriction enzymes NcoI and BamHI (New England Biolabs). The resulting PCR fragments and restriction enzyme-treated plasmids were purified using the FastGene Gel / PCR Extraction kit. The purified fragments were assembled using GeneArt Seamless Cloning and Assembly Enzyme Mix and cloned into E. coli DH5α strain. After plasmid extraction, sequence verification was performed, and those with the correct sequence were designated as pTrc-gmas and used in subsequent experiments.

[0096] 20. Creation of homocitric acid, GABA, and theanine-producing strains pTrc-nifV was introduced into E. coli strains BW25113 and Cy1-A8 by electroporation. BW25113 / nifV and Cy1-A8 / nifV strains were obtained and used in homocitric acid production tests.

[0097] By introducing pTrc-gdhA-gadBm into Escherichia coli strains BW25113ΔgabT and Cy1-A8ΔgabT via electroporation, we obtained BW25113ΔgabT / gdhA-gadBm and Cy1-A8ΔgabT / gdhA-gadBm strains, which were then used in γ-aminobutyric acid (GABA) production experiments.

[0098] pTrc-gmas was introduced into E. coli strains BW25113 and Cy1-A8 by electroporation, resulting in BW25113 / gmas and Cy1-A8 / gmas strains, which were used in theanine production experiments.

[0099] 21. Homocitric acid, GABA, and theanine production experiment As a pre-culture, the BW25113 / nifV strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin, and the Cy1-A8 / nifV strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin and 12.5 μg / ml chloramphenicol, both at 37°C, 200 rpm, for 16 hours. Next, for homocitric acid production, 50 μL of BW25113 / nifV preculture was inoculated into 5 mL of TB medium containing 100 μg / ml ampicillin and 0.1 mM IPTG (12 g / L tryptone, 24 g / L yeast extract, 20 g / L glycerol, 2.3 g / L KH2HPO4, 12.5 g / L K2HPO4). 50 μL of Cy1-A8 / nifV strain preculture was inoculated into 5 mL of TB medium containing 12.5 μg / ml chloramphenicol, 100 μg / ml ampicillin and 0.1 mM IPTG, and incubated at 30°C and 200 rpm for 24 hours. The culture supernatant was analyzed using GC-MS.

[0100] As a pre-culture, the BW25113 / gdhA-gadBm strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin, and the Cy1-A8ΔgabT / gdhA-gadBm strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin and 12.5 μg / ml chloramphenicol, both at 37°C, 200 rpm, for 16 hours. Next, for GABA production, 100 μL of pre-culture solution of BW25113 / gdhA-gadBm was inoculated into 10 mL of M9YE medium containing 100 μg / ml ampicillin, and 100 μL of pre-culture solution of Cy1-A8ΔgabT / gdhA-gadBm was inoculated into 10 mL of M9YE medium containing 12.5 μg / ml chloramphenicol and 100 μg / ml ampicillin, and the cultures were incubated at 37°C and 150 rpm for 96 hours. The culture supernatant was analyzed using GC-MS.

[0101] As a pre-culture, the BW25113 / gmas strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin, and the Cy1-A8 / gmas strain was cultured in 5 mL of LB medium containing 100 μg / ml ampicillin and 12.5 μg / ml chloramphenicol, both at 37°C, 200 rpm, for 16 hours. Next, for theanine production, 50 μL of the BW25113 / gmas preculture was inoculated into 5 mL of TB medium (containing 100 μg / ml ampicillin, 0.1 mM IPTG, and 2 g / l ethylamine), and 50 μL of the Cy1-A8 / nifV strain preculture was inoculated into 5 mL of TB medium (containing 12.5 μg / ml chloramphenicol, 100 μg / ml ampicillin, 0.1 mM IPTG, and 2 g / l ethylamine (Tokyo Chemical Industries, Ltd.)). The cultures were incubated at 30°C and 200 rpm for 72 hours. The culture supernatant was analyzed using GC-MS.

[0102] 22. Analysis of GABA, homocitric acid, and theanine in the culture medium The concentrations of GABA, homocitric acid, and theanine were analyzed by gas chromatography-mass spectrometry (GC-MS) (Shimadzu). 5 μL of culture supernatant was mixed with 2 μL of 10 g / L ribitol as an internal standard and dried using CentriVap Benchtop Vacuum Concentrators (Labconco, Kansas City, MO, USA). Trimethylsilylation for GC-MS analysis was performed using the following procedure: 100 μL of 20 mg / mL O-methylhydroxylamine hydrochloride solution dissolved in pyridine was added to the dried sample and reacted for 90 minutes (1200 rpm, 30°C; M-BR-022UP; Taitec, Saitama, Japan). Then, 50 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added and reacted for 30 minutes (1200 rpm at 37°C; M-BR-022UP; Taitec). The samples were centrifuged at room temperature (3000g, 5 minutes), and the supernatant was used for analysis.

[0103] A GC-MS (GCMS-QP2010 Ultra; Shimadzu) was used with a DB-5ms column (15m length × 0.25mm id, film thickness of 0.25μm; Agilent). The settings for each parameter of the GC-MS were as follows: The temperature of the sample vaporization chamber was set to 230°C. The sample injection volume was set to 1 μL and the split ratio to 1:25. Helium was used as the carrier gas and the flow rate was set to 1.12 mL / min. The column temperature was maintained at 80°C for 2 minutes, then increased to 330°C at 15°C / min and maintained at 330°C for 6 minutes. The interface temperature and ion source temperature were set to 250°C and 200°C, respectively. Electron impact ionization (EI) was performed at 70 eV. The analysis was performed using Fast Automated Scan / SIM (FASTT) mode, with Scan mode (85-500 m / z) and Selected ion monitoring (SIM) mode (m / z 103 for ribitol) running in parallel.

[0104] <Experimental Results> 1. Creation of AKG production-enhancing strains based on FBA Flux balance analysis (FBA) was used to identify metabolic enzymes that should be deficient or enhanced to improve AKG production when glucose is used as a substrate. Table 1 shows the metabolic reactions that should be deficient or enhanced for high AKG production.

[0105] [Table 1]

[0106] In this invention, using Escherichia coli strain BW25113 as the host, we created the BWΔptsHIΔackA-ptaΔzwf strain, which lacks the acetate kinase gene (ackA) and phosphate acetyltransferase gene (pta), which are responsible for acetic acid biosynthesis, the glucose-6-phosphate 1-dehydrogenase gene (zwf), which is responsible for the reaction from glycolysis to the pentose phosphate pathway, and the gene encoding a protein involved in glucose uptake by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (ptsHI), among the metabolic reactions to be deleted and enhanced as shown in Table 1. Subsequently, we introduced a plasmid (pET-PR-galP-glk) for overexpression of the galP gene and the glk gene into the BWΔptsHIΔackA-ptaΔzwf strain, and named the resulting E. coli strain the AKG-based strain.

[0107] Fermentation tests for AKG production were conducted using strain BW25113 and an AKG-based strain. The bacterial cells used in the fermentation tests were first cultured overnight in 5 mL of LB medium. Subsequently, they were inoculated into 20 mL of M9YE medium to achieve an OD600 of 0.1, cultured at 37°C and 100 rpm, and samples were taken. After the culture period, the culture supernatant was analyzed using a biosensor and HPLC. Figure 1 shows the production amounts of AKG in the culture supernatant, as well as succinic acid and glutamic acid produced from AKG in a one-step reaction. It was confirmed that the AKG-based strain produced increased amounts of AKG and metabolites produced from AKG compared to strain BW25113.

[0108] 2. Creation of a combinatorial plasmid library Figure 2 shows 17 genes encoding the major enzymes responsible for metabolic reactions from glucose to AKG. To explore the optimal combination of overexpression for these 17 genes, a combinatorial plasmid library was constructed using the Ordered Gene Assembly in Bacillus subtilis method (OGAB). After creating the combinatorial plasmid library in Bacillus subtilis, 24 colonies were randomly selected from the colonies that grew on the plate, and PCR was performed on the colony suspension as a template using 17 primer sets designed to sandwich the DNA cassette junctions. As a result, it was confirmed that the 17 gene cassettes were randomly accumulated in all 24 clones.

[0109] 3. Evaluation of glutamate production in E. coli strains introduced with combinatorial plasmid libraries. A plasmid containing 17 overexpressed DNA cassettes (overexpressed AKG plasmid) was transformed into an AKG-based strain by electroporation. The resulting strain was named the overexpression control strain. A plasmid containing 17 non-overexpressed DNA cassettes (non-overexpressed AKG plasmid) was transformed into an AKG-based strain by electroporation. The resulting strain was named the non-overexpression control strain. In addition, a combinatorial plasmid library was also transformed into an AKG-based strain by electroporation.

[0110] Fermentation tests were conducted using 96-well plates with 88 strains randomly selected from the obtained transformant colonies. Simultaneously, four colonies each of the overexpression control strain and the non-overexpression control strain were cultured. The results of the culture tests are shown in Figure 3. The results of the culture tests showed that the glutamate production of the non-overexpression control strain and the overexpression control strain was 100 mg / L and 1330 mg / L, respectively. On the other hand, the glutamate production of the combinatorial library-introduced strains varied from 97 mg / L to 2180 mg / L, and strains with improved productivity were obtained compared to the overexpression control strain that overexpressed all 17 genes.

[0111] 4. Sequence verification of the introduced plasmid Melting curve analysis using a quantitative PCR instrument was used to determine the gene cassettes accumulated in the combinatorial plasmids introduced into the E. coli strains used for culture. In the 88 strains into which the combinatorial plasmids were introduced, it was investigated which of the 17 genes were overexpressed and which were not. Of the 88 strains into which the combinatorial plasmids were introduced, 84 strains had combinatorial plasmids with the 17 gene cassettes correctly accumulated. Four strains had combinatorial plasmids with some cassettes missing. Of the 84 correctly accumulated combinatorial plasmids, 79 sequences were different, and 5 sequences showed duplication.

[0112] 5. Identification of genes contributing to increased AKG production through statistical analysis. To identify the genes contributing to increased AKG production among the 17 genes, statistical analysis was performed using glutamate production data from 96 strains and combinatorial plasmid sequence analysis data. The results of the statistical analysis are shown in Figure 4. It was revealed that overexpression of ppc and aceF significantly affected the improvement in glutamate production among the 17 genes.

[0113] Therefore, we created plasmids for the individual overexpression of ppc and aceF (pCP-ppc and pCP-aceF), and a plasmid for the simultaneous overexpression of both genes (pCP-ppc-aceF). The created plasmids were introduced into the BW25113 strain. Culture tests were performed using the BW25113 strain, a ppc overexpression strain, an aceF overexpression strain, and a ppc-aceF overexpression strain. The results of the culture tests are shown in Figure 5. Glutamate production increased in strains overexpressing aceF and ppc.

[0114] 6. Preparation and evaluation of a second-generation combinatorial plasmid library In the second-generation combinatorial plasmid library, we used only overexpression DNA cassettes for the two genes ppc and aceF, and both overexpression and non-overexpression DNA cassettes for the remaining 15 genes. This allowed us to search for genes that contribute to increased glutamate production in addition to overexpression of ppc and aceF.

[0115] Similar to the first-generation combinatorial plasmid library, the prepared second-generation combinatorial plasmid library was introduced into base strains. After transformation, 88 randomly selected colonies were cultured in 96-well plates. The results of the culture test are shown in Figure 6. In the strains introduced with the second-generation combinatorial plasmid library, glutamine production was increased in more strains than in the strains introduced with the first-generation combinatorial plasmid library. Next, as with the introduction of the first-generation combinatorial plasmid library, the gene cassettes accumulated in the combinatorial plasmids introduced into each E. coli strain used for culture were determined. Of the 88 strains into which the combinatorial plasmid was introduced, 65 strains had combinatorial plasmids in which 17 gene cassettes were correctly accumulated. In 23 strains, combinatorial plasmids with some cassettes missing were introduced. Of the 65 correctly accumulated combinatorial plasmids, 64 were different sequences, and one sequence was duplicated.

[0116] To identify genes contributing to increased AKG production, statistical analysis was performed using glutamate production data from 96 strains and combinatorial plasmid sequence analysis data. The results of the statistical analysis are shown in Figure 7. It was shown that overexpression of glk, in addition to overexpression of aceF and ppc, significantly contributes to increased glutamate production.

[0117] 7. Results of homocitric acid, GABA, and theanine production tests in AKG-enhanced strains introduced with Combi-OGAB plasmid. Using E. coli BW25113 and the E. coli strain (Cy1-A8) that produced the highest amount of glutamate among the transformed E. coli from the Combi-OGAB plasmid library evaluated in item 12, production tests were conducted for useful compounds (homocitric acid, GABA, theanine) that can be produced from AKG. The sequence information of the Combi-OGAB plasmid introduced into the Cy1-A8 strain is shown in Figure 8, and 12 of the 17 targeted genes (glk, pgi, pfkA, tpiA, pgk, gpmA, eno, pykF, ppc, aceF, gltA, icd) are overexpressed in the Cy1-A8 strain.

[0118] Wild-type Escherichia coli does not produce homocitrate, but by introducing the homocitrate synthase gene, it can produce homocitrate through the condensation of AKG and acetyl-CoA. In this study, the nifV gene (SEQ ID NO: 149) derived from Azotobacter vinelandii (Zheng L, White RH, Dean DR: Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase. J. Bacteriol. 1997, 179(18):5963-5966.) was introduced into strains BW25113 and Cy1-A8, and the amount of homocitrate produced was evaluated. The homocitric acid production of the BW25113 / nifV strain and the Cy1-A8 / nifV strain was 422 mg / L and 775 mg / L, respectively, with the Cy1-A8 / nifV strain producing 1.8 times more homocitric acid than the BW25113 / nifV strain (Figure 9-a).

[0119] In Escherichia coli, GABA is produced from AKG through the action of glutamate dehydrogenase (gdhA) and glutamate decarboxylase (gadB), and is degraded by GABA transaminase (GabT). Therefore, in this study, we first disrupted the GabT gene in strains BW25113 and Cy1-A8. Furthermore, since it is known that the activity of glutamate decarboxylase (gadB) in Escherichia coli becomes very low above pH 6 (Thu Ho NA, Hou CY, Kim WH, Kang TJ: Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness. J. Biosci. Bioeng. 2013, 115(2):154-158.), we overexpressed gadB (SEQ ID NO: 151) with two mutations, E89Q and Δ452-466, which show activity even above pH 6. The GABA production of the BW25113ΔgabT / gdhA-gadBm strain and the Cy1-A8ΔgabT / gdhA-gadBm strain was 0.72 g / L and 1.53 g / L, respectively, with the Cy1-A8ΔgabT / gdhA-gadBm strain producing 2.1 times more GABA than the BWBW25113ΔgabT / gdhA-gadBm strain (Figure 9-b).

[0120] Wild-type Escherichia coli does not produce theanine, but by introducing the γ-glutamylmethylamide synthase gene, it becomes possible to produce theanine through the condensation of glutamic acid and ethylamine. In this study, the gmas gene (SEQ ID NO: 152) (WO2018190398A1) derived from Pseudomonas syringae was introduced into the BW25113 and Cy1-A8 strains, and theanine production was evaluated. The theanine production of the BW25113 / gmas strain and the Cy1-A8 / gmas strain was 0.93 g / L and 1.87 g / L, respectively, with the Cy1-A8 / gmas strain showing 2.0 times the theanine production of the BW25113 / gmas strain (Figure 9-c).

[0121] As described above, it was confirmed that by using an AKG production-enhanced strain obtained by introducing the Combi-OGAB plasmid as a host, strains capable of producing high levels of various useful compounds (homocitrate, GABA, theanine, etc.) that can be produced from AKG can be easily prepared. From these results, it became clear that the metabolically modified microbial strains constructed by the method of the present invention can produce high levels of hub compounds in microbial fermentation such as AKG, and can therefore be utilized in the production of various substances using these as starting materials. [Industrial applicability]

[0122] The present invention's method for constructing metabolically modified microbial strains makes it possible to efficiently construct strains that produce high levels of useful compounds, such as hub compounds, in microbial fermentation. By constructing a base strain by subjecting a parent strain to metabolic modification based on metabolic flux balance analysis (FBA), and then introducing a Combi-OGAB long-chain DNA library using artificial DNA parts that are not subject to endogenous metabolic regulation into the base strain, it is possible to construct a metabolically modified microbial strain that produces significantly more useful compounds compared to the parent strain. As shown in the examples, the present invention successfully constructed a strain that can produce high levels of α-ketoglutaric acid as a hub compound (useful compound). The present invention's method also makes it possible to construct high-production strains of other useful compounds. Furthermore, since the metabolically modified microbial strains constructed by the present invention can produce high levels of hub compounds in microbial fermentation, they can be utilized in the production of various substances using them as starting materials.

Claims

[Claim 1] The invention described herein.

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