Escherichia sp. Microorganism having improved L-isoleucine production capacity, and method for producing L-isoleucine using same

Abstract

The present invention relates to a microorganism belonging to the genus Escherichia, which has improved L-isoleucine production capacity, and a method for producing L-isoleucine using the same, and more particularly, to a microorganism belonging to the genus Escherichia, which enhances the biosynthetic pathway of L-isoleucine by weakening or inactivating the activity of a threonine operon leader peptide and threonine dehydrogenase, and which has improved L-isoleucine production capacity, and to a method for producing L-isoleucine using the same. Meanwhile, generation of by-products is weakened, and compared with a parent strain, the production yield of the L-isoleucine can be improved.

Description

Technical Field

[0001] This invention relates to Escherichia coli microorganisms that enhance L-isoleucine production capacity and to a method for producing L-isoleucine using these microorganisms. Background Technology

[0002] L-Isoleucine is an essential amino acid that cannot be synthesized in the human or animal body and must be obtained from external sources. Under normal circumstances, it is produced by fermentation using microorganisms such as bacteria or yeast.

[0003] L-Isoleucine shares a major biosynthetic pathway with L-valine and L-leucine, both branched-chain amino acids. In particular, L-isoleucine and L-valine have very similar chemical structures, isoelectric points, and solubilities, making the production of high-purity single amino acids like L-isoleucine difficult due to numerous steps and costs, resulting in low-yield recovery. Therefore, it is important to discover or develop strains that can produce L-isoleucine in large quantities while simultaneously producing L-valine in small quantities to reduce the production cost of L-isoleucine.

[0004] L-Isoleucine production can be achieved using wild-type strains obtained under natural conditions or modified mutant strains to enhance their L-isoleucine production capacity. Microbial biosynthesis of L-isoleucine utilizes pyruvic acid and oxaloacetic acid as precursors to produce L-threonine, followed by the sequential synthesis of L-isoleucine.

[0005] In recent years, to improve the production efficiency of L-isoleucine, gene recombination technology has been applied to microorganisms such as *Escherichia coli* and *Corynebacterium*, which are widely used in the production of L-amino acids and other useful substances. This has led to the development of various recombinant strains or variants with excellent L-isoleucine production capabilities, as well as methods for L-isoleucine production using these strains. In particular, attempts have been made to directly induce mutations in genes involved in the L-isoleucine biosynthesis pathway, such as enzymes, transcription factors, and transport proteins, or to induce mutations in promoters that regulate their expression, thereby increasing L-isoleucine production. However, the number of enzymes, transcription factors, transport proteins, and other proteins directly or indirectly related to L-isoleucine production ranges from dozens to hundreds. Therefore, whether changes in the activity of these proteins increase L-isoleucine production capacity still requires extensive research.

[0006] Existing technical documents

[0007] Patent documents

[0008] Korean Patent No. 10-1747542 Summary of the Invention

[0009] The purpose of this invention is to provide Escherichia coli microorganisms with improved L-isoleucine production capacity.

[0010] In addition, the present invention aims to provide a method for producing L-isoleucine using the above-mentioned Escherichia coli microorganisms.

[0011] One aspect of the present invention provides Escherichia coli microorganisms in which the activity of the threonine operon leader peptide and threonine dehydrogenase is weakened or inactivated, while the L-isoleucine production capacity is enhanced.

[0012] The "threonine operon leader peptide" used in this invention constitutes the threonine operon, which participates in the regulation of threonine biosynthesis. The threonine operon leader peptide in this invention can be a polypeptide encoded by the thrL gene and possessing threonine operon leader peptide activity, but is not limited to this.

[0013] The threonine dehydrogenase used in this invention catalyzes the NAD+-dependent oxidation of L-threonine to produce 2-amino-3-ketobutyrate. The threonine dehydrogenase in this invention can be a polypeptide encoded by the tdh gene and possessing threonine dehydrogenase activity, but is not limited to this.

[0014] The nucleic acid and protein sequence information of the aforementioned threonine operon leader peptide and threonine dehydrogenase can be obtained from well-known sequence databases (e.g., GenBank, UniProt).

[0015] In this invention, "activity attenuation" refers to a reduction in the expression level of genes encoding target proteins such as enzymes, transcription factors, and transporters compared to the original microorganism, i.e., the wild-type strain or the unmodified strain. Such attenuation includes the following: reductions in protein activity compared to the original microorganism due to nucleotide modifications of the encoding gene (e.g., substitution, insertion, deletion, or combinations thereof within the target gene); and reductions in overall intracellular protein activity compared to the wild-type strain or the unmodified strain due to modifications of non-coding regions of non-coding genes, such as promoters (e.g., modification of all or part of the promoter sequence, replacement with a weak promoter), which inhibit the expression of the target gene or hinder translation. This also includes combinations of these conditions.

[0016] The aforementioned nucleotide modifications refer to changes in the nucleotide sequence due to substitution, insertion, deletion, or a combination thereof, resulting in a difference from the original nucleotide sequence. The aforementioned promoter modifications refer to changes in the promoter sequence due to substitution, insertion, deletion, or a combination thereof, resulting in a difference from the original promoter sequence, thereby reducing the expression level or weakening the activity of the target gene. Furthermore, the aforementioned promoter modifications include replacement with a promoter that results in a weaker expression level or activity of the target gene compared to the original gene's promoter. Substitution refers to the change in which a base, nucleotide, polynucleotide, or nucleic acid is replaced with another base, nucleotide, polynucleotide, or nucleic acid. Insertion refers to the change in which another base, nucleotide, polynucleotide, or nucleic acid is added. Deletion refers to the change in which a base, nucleotide, polynucleotide, or nucleic acid is removed.

[0017] According to a specific embodiment of the present invention, the weakening of the activity of the aforementioned threonine operon leader peptide may be due to nucleotide modification, promoter modification, or a combination thereof of the gene encoding the threonine operon leader peptide.

[0018] According to a specific embodiment of the present invention, the weakening of the activity of the above-mentioned threonine dehydrogenase may be due to nucleotide modification, promoter modification, or a combination thereof of the gene encoding the threonine dehydrogenase.

[0019] In this invention, "inactivation" refers to the following situations: the expression of genes encoding proteins such as enzymes, transcription factors, and transport proteins is completely absent compared to the original microorganism, i.e., the wild-type strain or the strain before modification, or even if expressed, the genes are inactive.

[0020] According to a specific embodiment of the present invention, the aforementioned threonine operon leader peptide may be encoded by the thrL gene derived from Escherichia coli.

[0021] The aforementioned threonine operon leader peptide may be encoded by the base sequence of SEQ ID NO:1 or composed of the amino acid sequence of SEQ ID NO:2, but is not limited thereto.

[0022] According to a specific embodiment of the present invention, the above-mentioned threonine dehydrogenase may be encoded by the tdh gene derived from Escherichia coli.

[0023] The aforementioned threonine dehydrogenase may be encoded by the base sequence of SEQ ID NO:3 or composed of the amino acid sequence of SEQ ID NO:4, but is not limited thereto.

[0024] The base sequences or amino acid sequences of the threonine operon leader peptide and threonine dehydrogenase according to the present invention may consist of, or must contain, sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the base sequences or amino acid sequences of SEQ ID NO: 1 to 4, and may have the original function. Here, "homology" or "identity" refers to the percentage (%) of similarity between two sequences when the base sequence or amino acid sequence that will serve as the benchmark is aligned with any other base sequence or amino acid sequence in a manner that maximizes correspondence.

[0025] In this invention, "increased production capacity" refers to an increase in the production rate of L-isoleucine compared to the mutated target (parental strain). The aforementioned parental strain refers to a wild-type or mutant strain that becomes the target of mutation, including those directly becoming the target or those transformed through recombinant vectors, etc. In this invention, the parental strain can be a wild-type Escherichia microorganism or strain that lacks or possesses L-isoleucine production capacity, or a wild-type mutated Escherichia microorganism or strain.

[0026] According to a specific embodiment of the present invention, the aforementioned Escherichia genus may be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, or Escherichia vulneris, etc., but is not limited to these.

[0027] As an example, the aforementioned Escherichia genus could be Escherichia coli.

[0028] The Escherichia coli microorganisms according to the present invention can improve L-isoleucine production capacity due to the weakening or inactivation of the threonine operon leader peptide and threonine dehydrogenase activity.

[0029] Specifically, Escherichia coli strains exhibited enhanced L-isoleucine production capacity compared to parental strains or strains with weakened activity of the threonine operon precursor peptide and threonine dehydrogenase. In particular, compared to parental strains, L-isoleucine production could be increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 65%. 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or an increase of 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, but not limited to these. As an example, in Escherichia coli strains with weakened or inactivated threonine operon precursor peptide and threonine dehydrogenase activity, L-isoleucine production can be increased by more than 5% compared to the parent strain, specifically by 5% to 50% (preferably 10% to 40%).

[0030] Compositions containing Escherichia coli microorganisms according to the present invention can be used as compositions for the production of L-isoleucine.

[0031] According to a specific example of the present invention, in order to delete the genes encoding the threonine operon leader peptide and threonine dehydrogenase in a parent strain of Escherichia coli, gene inactivation methods or recombinant vectors can be used.

[0032] The aforementioned gene inactivation methods can be implemented using known methods. For example, there are the CaCl2 method (Cohen, S. Netal., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), the Hanahan method (Cohen, S. Netal., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)), and the electroporation method (Dower, W. J et al., Nucleic. Acids Res., 16:6127-6145 (1988)), but it is not limited to these methods.

[0033] As used in this invention, "vector" refers to all types of nucleic acid sequence transport structures used as a means of delivering and expressing a target gene to a variant object (host cell). Unless otherwise stated, the above-described vector can refer to a vector that allows the carried nucleic acid sequence to be inserted into a host cell gene for expression and / or expressed independently. Such a vector includes operably linked necessary regulatory elements for expressing the gene insert. "Operably linked" means that the target gene and its regulatory sequence are functionally linked to each other in a manner that enables gene expression. "Regulatory elements" include promoters for carrying out transcription, arbitrary operon sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation.

[0034] The vectors used in this invention are not particularly limited as long as they can replicate in host cells, and any vector known in the art can be used. Examples of the aforementioned vectors include plasmids, granules, viruses, and bacteriophages in their natural or recombinant states. For example, as bacteriophage vectors or granule vectors, there are pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., and as plasmid vectors, there are pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET lines, etc., but the invention is not limited to these.

[0035] The vectors described above can be constructed representatively as vectors for cloning or vectors for expression. Vectors for expression can be conventional vectors used in the art for expressing exogenous genes or proteins in plants, animals, or microorganisms, and can be constructed using various methods known in the art.

[0036] The "recombinant vector" used in this invention can be constructed using prokaryotic or eukaryotic cells as hosts, and can replicate independently of the host cell's genome, or can be stitched into the genome itself. The host cell is capable of replicating the vector and may include a replication origin consisting of a specific base sequence that initiates replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, it typically includes a strong promoter that enables transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When a eukaryotic cell is used as the host, the replication origin initiated in the eukaryotic cell containing the vector includes, but is not limited to, f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin. In addition, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) can be used, and they usually have a polyadenylated sequence as the transcription termination sequence.

[0037] The recombinant vectors described above may include selection markers, which are used to screen transformants (host cells) transformed using the vector. In the culture medium treated with the selection markers, only cells expressing the selection markers can survive, thus enabling the screening of transformed cells. Representative examples of selection markers include ampicillin, kanamycin, streptomycin, and chloramphenicol, but the study is not limited to these.

[0038] Transformants can be created by inserting the recombinant vector into host cells. These transformants can be obtained by introducing the recombinant vector into suitable host cells. The host cell can be a cell capable of stably and continuously cloning or expressing the expression vector, or any host cell known in the art can be used.

[0039] When transforming prokaryotic cells to create recombinant microorganisms, various intestinal bacteria and strains can be used as host cells, such as *Escherichia coli* (e.g., *E. coli* JM109, *E. coli* BL21, *E. coli* RR1, *E. coli* LE392, *E. coli* B, *E. coli* X 1776, *E. coli* W3110, *E. coli* XL1-Blue, *Corynebacterium*, *Bacillus*, *Bacillus thuringiensis*, *Salmonella*, *Serratia marcescens*, and *Pseudomonas*, but are not limited to these.

[0040] When transforming eukaryotic cells to create recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells can be used as host cells, such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc., but are not limited to these.

[0041] In this invention, "transformation" refers to the phenomenon of artificially inducing genetic changes by introducing exogenous DNA into host cells, and "transformant" refers to a host cell in which exogenous DNA has been introduced and the expression of the target gene is stably maintained.

[0042] In the above transformation, a suitable vector delivery technique is selected based on the host cell, thereby enabling the expression of the target gene or a recombinant vector containing it within the host cell. For example, vector delivery can be performed via electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or combinations thereof, but is not limited to these methods. The transformed gene can be included as long as it can be expressed within the host cell, without limitation on whether it is inserted into or located extrachromosomally within the host cell.

[0043] The aforementioned transformants include cells transfected, transformed, or infected in vivo or in vitro using the recombinant vector according to the invention, and may be used interchangeably with recombinant host cells, recombinant cells, or recombinant microorganisms.

[0044] Genes inserted into the recombinant vector for transformation of the present invention can be replaced into host cells such as Escherichia coli microorganisms due to homologous recombination crossover.

[0045] According to a specific embodiment of the present invention, the host cell may be a microorganism of the genus Escherichia. For example, it may be Escherichia coli, but is not limited thereto.

[0046] Another aspect of the present invention provides a method for producing L-isoleucine, comprising the steps of: culturing the aforementioned Escherichia coli microorganisms in a culture medium; and recovering L-isoleucine from the aforementioned Escherichia coli microorganisms or the culture medium used to culture the Escherichia coli microorganisms.

[0047] The above-described culture can be carried out using suitable culture media and conditions known in the art, and those skilled in the art can easily adjust the culture media and conditions for use. Specifically, the culture media can be liquid culture media, but is not limited thereto. Culture methods can include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but are not limited thereto.

[0048] According to a specific embodiment of the invention, the culture medium described above must be suitably adapted to meet the requirements of a particular strain and can be appropriately modified by those skilled in the art. For information on culture media for Escherichia strains, reference can be made to well-known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but it is not limited thereto.

[0049] According to a specific embodiment of the invention, the culture medium may contain various carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used alone or in mixtures, but are not limited thereto. Usable nitrogen sources may include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used alone or in mixtures, but are not limited thereto. Usable phosphorus sources may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts, but are not limited thereto. Furthermore, the culture medium may contain metal salts such as magnesium sulfate or ferrous sulfate required for growth, but is not limited thereto. In addition, it may contain essential growth substances such as amino acids and vitamins. Furthermore, precursors suitable for the culture medium may be used. The aforementioned culture medium or individual components may be added to the culture medium in batches or continuously during the culture process in an appropriate manner, but are not limited thereto.

[0050] According to one specific embodiment of the invention, during the cultivation process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microbial culture medium in an appropriate manner to adjust the pH of the culture medium. Furthermore, during the cultivation process, antifoaming agents such as polyethylene glycol esters of fatty acids can be used to suppress bubble formation. Further, to maintain an aerobic state in the culture medium, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium. The temperature of the culture medium is typically between 20°C and 45°C, for example, between 25°C and 40°C. The cultivation time can continue until the desired production of the useful substance is achieved, for example, between 10 and 160 hours.

[0051] According to a specific embodiment of the present invention, in the step of recovering L-isoleucine from the cultured transformant or the culture medium for the transformant described above, the produced L-isoleucine can be collected or recovered from the culture medium according to the culture method and using suitable methods known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion) and other methods can be used, but are not limited thereto.

[0052] According to a specific embodiment of the present invention, in the above-described step of recovering L-isoleucine, the culture medium is centrifuged at low speed to remove biomass, and the resulting supernatant can be separated by ion exchange chromatography.

[0053] According to a specific embodiment of the present invention, the step of recovering L-isoleucine described above may include a step of purifying L-isoleucine.

[0054] The Escherichia coli microorganisms of the present invention enhance the L-isoleucine biosynthesis pathway by weakening or inactivating the activity of the threonine operon precursor peptide and threonine dehydrogenase, while weakening the generation of byproducts, thereby improving the L-isoleucine production yield compared with the parent strain. Detailed Implementation

[0055] The present invention will now be described in more detail. However, such description is merely illustrative and is intended to aid in understanding the invention, and the scope of the invention is not limited to such illustrative description.

[0056] Example 1. Preparation of mutant strains with weakened activity of threonine operon leader peptide

[0057] To produce a strain with weakened activity of the threonine operon leader peptide, Escherichia coli DS44 (accession number KCTC11602BP), which produces L-isoleucine, was used, and the thrL gene (SEQ ID NO:1) encoding the threonine operon leader peptide was deleted by a one-step inactivation method (Warner et al., PNAS, 6:6640-6645 (2000)).

[0058] First, PCR was performed using the primer pair thrL_F and thrL_R with pKD13 plasmid (GenBank AY048744) as a template to obtain the fragment. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into E. coli DS44, the prepared PCR fragment was injected via electroporation, and colonies resistant to kanamycin were screened. The screened transformants were then subjected to PCR using the primer pair thrL_CF and thrL_CR to confirm the deletion of the thrL gene. DS44ΔthrL with inserted disruptive DNA produced approximately 2.1 kb of product as expected, while DS44 produced approximately 0.8 kb of product.

[0059] Using strains confirmed to lack the thrL gene, a process was performed to remove the antibiotic resistance marker gene. In this process, the pCP20 plasmid was introduced into the thrL gene-deficient strains (Cherepanov and Wackerneagel, 1995; Datsenko and Wanner, 2000), inducing FLP recombination. Then, the thrL gene-deficient strains were cultured in LB plates with or without antibiotics, confirming the removal of the antibiotic resistance marker gene.

[0060] Here, the PCR reaction was performed as follows: the total reaction volume was 50 μl, and the reaction was cycled once at 94 °C for 1 minute, then cycled 30 times at 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 2 minutes, and then at 72 °C for 2 minutes.

[0061] The primer sequences used in Example 1 are shown in Table 1 below.

[0062] Table 1

[0063]

[0064] Example 2. Preparation of mutant strains with weakened threonine dehydrogenase activity

[0065] To produce a strain with weakened threonine dehydrogenase activity, Escherichia coli DS44 (accession number KCTC11602BP), which produces L-isoleucine, was used, and the tdh gene (SEQ ID NO:3) encoding threonine dehydrogenase was deleted by a one-step inactivation method (Warner et al., PNAS, 6:6640-6645 (2000)).

[0066] First, PCR was performed using the primer pair tdh_F and tdh_R with pKD13 plasmid (GenBank AY048744) as a template to obtain the fragment. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into E. coli DS44, the prepared PCR fragment was injected via electroporation, and colonies resistant to kanamycin were screened. The screened transformants were then subjected to PCR using the primer pair tdh_CF and tdh_CR to confirm the deletion of the tdh gene. The introduction of DS44Δtdh with destructive DNA produced a product of approximately 2.6 kb as expected, while DS44 produced a product of approximately 3.3 kb.

[0067] Using strains confirmed to lack the tdh gene, a process was performed to remove the antibiotic resistance marker gene. During this process, the pCP20 plasmid was introduced into the tdh gene-deficient strains, inducing FLP recombination. Then, the tdh gene-deficient strains were cultured in LB plates with or without antibiotics to confirm the removal of the antibiotic resistance marker gene.

[0068] Here, the PCR reaction was performed as follows: the total reaction volume was 50 μl, and the reaction was cycled once at 94°C for 1 minute, then cycled 30 times at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes, and finally at 72°C for 2 minutes.

[0069] The primer sequences used in Example 2 are shown in Table 2 below.

[0070] Table 2

[0071]

[0072] Example 3. Preparation of mutant strains with weakened activity of threonine operon leader peptide and threonine dehydrogenase

[0073] To produce a strain with weakened threonine operon leader peptide and threonine dehydrogenase activity, DS44ΔthrL from Example 1 was used instead of E. coli DS44, except that the procedure was the same as in Example 2. The final product was a DS44ΔthrLΔtdh strain with the thrL and tdh genes deleted.

[0074] Example 1. Evaluation of L-Isoleucine Production Capacity

[0075] The L-isoleucine production capacity of the Escherichia coli variants prepared in Examples 1 to 3 was evaluated by comparison with the parent strain.

[0076] The isoleucine production medium (Table 3 below) was used with an initial liquid volume of 2 L and an additional liquid volume of 342 mL, and each strain was cultured at 30 °C, a stirring speed of 500 rpm, and an aeration rate of 1 vvm. After culture, the concentrations of L-valine and AABA (L-α-aminobutyric acid), which were generated as byproducts along with L-isoleucine, in the culture medium were determined by HPLC (Agilent), and the results are shown in Table 4 below.

[0077] Table 3

[0078] Element Initial culture medium concentration Additional culture medium concentration glucose 80g / l 550g / l Corn soaking solution 20g / l - ammonium sulfate 20g / l 1g / l Phosphoric acid 15g / l 1g / l fumaric acid 1g / l - Monosodium glutamate 7g / l - Sodium citrate 1g / l - Choline-HCl 1g / l - Thiamine-HCl 5㎎ / l - Pyridoxine-HCl 10㎎ / l - niacin 5㎎ / l - Biotin 5㎎ / l - Calcium chloride 5㎎ / l - cobalt chloride 5㎎ / l - Ferrous sulfate 20㎎ / l - manganese sulfate 5㎎ / l - Zinc sulfate 5㎎ / l - Copper sulfate 5㎎ / l - Sodium hydroxide 10g / l -

[0079] Table 4

[0080]

[0081] As shown in Table 4 above, compared with the parental strains, when the activity of the threonine operon leader peptide or threonine dehydrogenase was weakened (DS44ΔthrL and DS44Δtdh), only the production capacity of L-isoleucine was increased. Specifically, compared with DS44, the L-isoleucine production of DS44ΔthrL and DS44Δtdh increased by approximately 4.4% and 3.4%, respectively, while the L-valine / L-isoleucine (V / I) ratio and the AABA / L-isoleucine (AABA / I) ratio were at similar levels.

[0082] Furthermore, compared to the parent strain, the weakened activities of the threonine operon leader peptide and threonine dehydrogenase (DS44ΔthrLΔtdh) showed an increased L-isoleucine production capacity while a decreased L-valine and AABA production capacity. Specifically, the L-isoleucine production of DS44ΔthrLΔtdh increased by approximately 28.3% compared to DS44, and by approximately 22.8% and 24.0% compared to DS44ΔthrL and DS44Δtdh, respectively. The L-valine / L-isoleucine (V / I) ratio decreased by approximately 49.7% compared to DS44, and the AABA / L-isoleucine (AABA / I) ratio decreased by approximately 35% compared to DS44.

[0083] These results indicate that weakening the activity of both the threonine operon leader peptide and threonine dehydrogenase significantly improves L-isoleucine yield and reduces byproduct production.

[0084] So far, the invention has been studied around its preferred embodiments. Those skilled in the art will understand that the invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustratively rather than restrictively. The scope of the invention is shown in the claims rather than in the foregoing description and should be interpreted as including all differences within its equivalent scope.

[0085] [Collection Information]

[0086] Name of the depository: Korean Center for Type Cultures (KCTC)

[0087] Collection Number: KCTC11602BP

[0088] Date of preservation: 20091120

[0089] Classification and nomenclature of biological materials: Escherichia coli.

Claims

1. An Escherichia coli microorganism whose L-isoleucine production capacity is enhanced by weakening or inactivating the activity of its threonine operon leader peptide and threonine dehydrogenase.

2. The Escherichia coli microorganism according to claim 1, wherein, The weakening of the activity of the threonine operon leader peptide is achieved through nucleotide modification, promoter modification, or a combination thereof of the gene encoding the threonine operon leader peptide.

3. The Escherichia coli microorganism according to claim 1, wherein, The weakening of the activity of the threonine dehydrogenase is achieved through nucleotide modification, promoter modification, or a combination thereof of the gene encoding the threonine dehydrogenase.

4. The Escherichia coli microorganism according to claim 1, wherein, The Escherichia coli mentioned is Escherichia coli.

5. A method for producing L-isoleucine, comprising the following steps: The step of culturing the Escherichia spp. microorganisms of claim 1 in a culture medium; as well as The step of recovering L-isoleucine from the Escherichia coli microorganism or the culture medium for culturing Escherichia coli microorganisms.

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