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 sp. And having improved L-isoleucine production ability, and a method for producing L-isoleucine using the same, the microorganism having improved L-isoleucine production ability by weakening or deactivating the activity of acetohydroxy acid synthetases I and III or simultaneously enhancing the activity of acetohydroxy acid synthetase II, and the method for producing L-isoleucine by using the microorganism belonging to the genus Escherichia sp. Therefore, the biosynthetic pathway of the L-isoleucine is enhanced, the generation of byproducts is weakened, and compared with the parent strain, the production yield of the L-isoleucine can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to an Escherichia microorganism having improved L-isoleucine-producing ability and a method for producing L-isoleucine using the same. BACKGROUND

[0002] L-isoleucine is an essential amino acid that cannot be synthesized in the human or animal body and must be supplied from the outside, and is generally produced by fermentation using microorganisms such as bacteria or yeast.

[0003] L-isoleucine shares a major biosynthetic pathway with L-valine and L-leucine, which are branched-chain amino acids. In particular, L-isoleucine and L-valine have very similar chemical structures and chemical properties such as isoelectric points and solubilities, and thus many process steps and costs are required to produce a high-purity single amino acid such as L-isoleucine, and thus it is difficult to recover them at a high yield. In order to reduce the production cost of L-isoleucine, it is important to develop a strain that produces L-isoleucine in large amounts while producing L-valine in small amounts.

[0004] The production of L-isoleucine can use a wild-type strain obtained in a natural state or a variant strain modified to improve the L-isoleucine-producing ability thereof. The biosynthesis of L-isoleucine using a microorganism produces L-threonine using pyruvic acid and oxaloacetic acid as precursors, and then sequentially synthesizes L-isoleucine.

[0005] In recent years, in order to improve the production efficiency of L-isoleucine, gene recombination techniques have been applied to microorganisms such as Escherichia, Corynebacterium, and Brevibacterium, which are commonly used for the production of L-amino acids and other useful substances, and thus various recombinant strains or variant strains having excellent L-isoleucine-producing ability and methods for producing L-isoleucine using the same have been developed. In particular, attempts have been made to directly mutate genes of enzymes, transcription factors, transport proteins, etc. involved in the biosynthetic pathway of L-isoleucine or to induce mutations in promoters that regulate the expression thereof, thereby increasing the production amount of L-isoleucine. However, there are many types of proteins such as enzymes, transcription factors, and transport proteins that are directly or indirectly related to the production of L-isoleucine, and thus it is actually necessary to conduct a large amount of research on whether the L-isoleucine-producing ability is increased according to changes in the activity of such proteins.

[0006] PRIOR ART DOCUMENT

[0007] PATENT DOCUMENT

[0008] Korean Patent No. 10-1747542 SUMMARY

[0009] An object of the present application is to provide an Escherichia microorganism having improved L-isoleucine-producing ability.

[0010] In addition, an object of the present application is to provide a method for producing L-isoleucine using the above-described Escherichia microorganism.

[0011] One embodiment of the present application provides an Escherichia microorganism in which the activity of acetohydroxy acid synthase (AHAS) III or the activities of acetohydroxy acid synthase I and III are attenuated or inactivated, and thus the L-isoleucine-producing ability is improved.

[0012] "Acetohydroxy acid synthase (AHAS)" used in the present application is the first enzyme that participates in the biosynthesis of L-isoleucine, L-valine, L-leucine, and the like, and uses pyruvic acid and alpha-ketobutyric acid as substrates. When acetohydroxy acid synthase has high substrate specificity for pyruvic acid, it participates in the polymerization of pyruvic acid to produce 2-acetolactate, which is a precursor of L-valine and L-leucine, and when it has high substrate specificity for alpha-ketobutyric acid, it participates in the polymerization of pyruvic acid and alpha-ketobutyric acid to produce 2-aceto-hydroxy-butyrate, which is a precursor of L-isoleucine.

[0013] As the acetohydroxy acid synthase, acetohydroxy acid synthase I (AHAS I), acetohydroxy acid synthase II (AHAS II), and acetohydroxy acid synthase III (AHAS III) are known, and each of the acetohydroxy acid synthases is composed of a large subunit and a small subunit. The acetohydroxy acid synthase I in the present application can be a polypeptide having acetohydroxy acid synthase activity by an ilvB gene encoding the large subunit and an ilvN gene encoding the small subunit, the acetohydroxy acid synthase II can be a polypeptide having acetohydroxy acid synthase activity by an ilvG gene encoding the large subunit and an ilvM gene encoding the small subunit, and the acetohydroxy acid synthase III can be a polypeptide having acetohydroxy acid synthase activity by an ilvH gene encoding the large subunit and an ilvI gene, but is not limited thereto.

[0014] The nucleic acid sequence and the protein sequence information of the above-described three kinds of acetohydroxy acid synthases can be obtained from a publicly known sequence database (for example, GenBank, UniProt).

[0015] "Attenuation" used in the present application means that the expression amount of a gene encoding a protein such as an enzyme, a transcription factor, a transport protein, etc. as a target is reduced compared to an original microorganism, i.e. a wild type strain or a strain before modification. Such attenuation of the target protein includes the following cases: a case where the activity of the protein itself is reduced compared to the activity of the protein possessed by the original microorganism by nucleotide modification within an intrinsic gene encoding the target protein (e.g. substitution, insertion, deletion of a part of nucleotides within the target gene, or a combination thereof), a case where the degree of the overall protein activity in the cell is low compared to the wild type strain or the strain before modification due to modification of a non-coding region such as a promoter (e.g. modification of all or a part of nucleotides within the promoter sequence, replacement with a weak promoter), which hinders the expression of the target gene or hinders translation, etc., and further a case where a combination thereof is included.

[0016] The above-mentioned nucleotide modification means that the entire or a part of the nucleotide sequence is substituted, inserted, deleted or a combination thereof, and is different from the original nucleotide sequence. The above-mentioned promoter modification means that the entire or a part of the nucleotide sequence of the promoter is substituted, inserted, deleted or a combination thereof, and is different from the original promoter sequence, thereby resulting in reduction of the expression level or attenuation of the activity of the target gene. Further, the above-mentioned promoter modification includes replacement with a promoter which has a lower expression level or lower activity of the target gene compared to the promoter of the original gene. Herein, substitution means a change in which a base, a nucleotide, a polynucleotide or a nucleic acid is replaced with another base, nucleotide, polynucleotide or nucleic acid. Insertion means a change in which another base, nucleotide, polynucleotide or nucleic acid is added. Deletion means a change in which a base, nucleotide, polynucleotide or nucleic acid is removed.

[0017] According to an embodiment of the present application, the attenuation of the acetohydroxy acid synthase I can be nucleotide modification, promoter modification, or a combination thereof of a gene (ilvB and / or ilvN) encoding the acetohydroxy acid synthase I.

[0018] Further, according to an embodiment of the present application, the attenuation of the acetohydroxy acid synthase III can be nucleotide modification, promoter modification, or a combination thereof of a gene (ilvH and / or ilvI) encoding the acetohydroxy acid synthase III.

[0019] "Deactivation" used in the present application means a case where a gene encoding a protein such as an enzyme, a transcription factor, a transport protein, etc. is not expressed at all compared to an original microorganism, i.e. a wild type strain or a strain before modification, or a case where even if expressed, the protein has no activity.

[0020] According to an embodiment of the present application, the acetylhydroxy acid synthase I and III can be inherent to Escherichia coli.

[0021] The large subunit of the acetylhydroxy acid synthase I can be encoded by the base sequence of SEQ ID NO: 1 or consist of the amino acid sequence of SEQ ID NO: 2, and the small subunit of the acetylhydroxy acid synthase I can be encoded by the base sequence of SEQ ID NO: 3 or consist of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0022] The large subunit of the acetylhydroxy acid synthase III can be encoded by the base sequence of SEQ ID NO: 9 or consist of the amino acid sequence of SEQ ID NO: 10, and the small subunit of the acetylhydroxy acid synthase III can be encoded by the base sequence of SEQ ID NO: 11 or consist of the amino acid sequence of SEQ ID NO: 12, but is not limited thereto.

[0023] The Escherichia microorganism according to the present application weakens or inactivates the activity of acetylhydroxy acid synthase III or weakens or inactivates the activity of acetylhydroxy acid synthases I and III, thereby having relatively low affinity for pyruvic acid, reducing the production of L-valine, and maintaining the activity of acetylhydroxy acid synthase II, and thus can maintain and increase the production of L-isoleucine.

[0024] Accordingly, the Escherichia microorganism of the present application can include the activity of acetylhydroxy acid synthase II.

[0025] In addition, in order to improve the production of L-isoleucine, the Escherichia microorganism according to the present application can weaken or inactivate the activity of acetylhydroxy acid synthases I and III while enhancing the activity of acetylhydroxy acid synthase II.

[0026] According to an embodiment of the present application, the Escherichia microorganism can further enhance the activity of acetylhydroxy acid synthase II.

[0027] The "activity enhancement" used in the present application means that the expression amount of a gene encoding a protein, such as an enzyme, a transcription factor, a transport protein, etc., as a target is increased compared with that of an original microorganism, i.e., a wild-type strain or a strain before modification. Such activity enhancement of the target protein includes the following cases: a case where the activity of the protein itself is increased compared with that of the protein possessed by the original microorganism by modification of a nucleotide within an intrinsic gene encoding the target protein (e.g., substitution, insertion, deletion, or a combination thereof of a part of nucleotides within the gene), a case where the copy number of the gene is increased, a case where the expression of the gene is increased or translation is increased due to modification of a non-coding region such as a promoter (e.g., modification of all or a part of nucleotides within the promoter sequence, replacement with a strong promoter), a case where the degree of overall protein activity in the cell is high compared with that of the wild-type strain or the strain before modification, and the like, and a case where a combination thereof is included.

[0028] The above-mentioned nucleotide modification means that the entire or a part of the nucleotide sequence is substituted, inserted, deleted, or a combination thereof, and is different from the original nucleotide sequence. The above-mentioned promoter modification means that the entire or a part of the nucleotide sequence of the promoter is substituted, inserted, deleted, or a combination thereof, and is different from the original promoter sequence, thereby resulting in an increase in the expression level or activity enhancement of the target gene. In addition, the above-mentioned promoter modification includes replacement with a promoter that is stronger in the expression level or activity of the target gene than the promoter of the original gene. Here, substitution means a change in which a base, a nucleotide, a polynucleotide, or a nucleic acid is replaced with another base, nucleotide, polynucleotide, or nucleic acid. Insertion means a change in which another base, nucleotide, polynucleotide, or nucleic acid is added. Deletion means a change in which a base, nucleotide, polynucleotide, or nucleic acid is removed.

[0029] In addition, the activity enhancement of the target protein includes a case where a foreign gene that is not possessed by the original microorganism is introduced, in which case the nucleotide of the foreign gene can be modified.

[0030] According to an embodiment of the present application, the activity enhancement of the acetohydroxy acid synthase II can be nucleotide modification, an increase in the copy number, promoter modification, introduction, or a combination thereof of a gene (ilvG and / or ilvM) encoding the acetohydroxy acid synthase II.

[0031] According to an embodiment of the present application, the acetohydroxy acid synthase II can be derived from Escherichia coli.

[0032] The large subunit of the above-described acetohydroxy acid synthase II can be encoded by the base sequence of SEQ ID NO: 5 or consist of the amino acid sequence of SEQ ID NO: 6, and the small subunit of the acetohydroxy acid synthase II can be encoded by the base sequence of SEQ ID NO: 7 or consist of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

[0033] The respective base sequences or amino acid sequences of the three acetohydroxy acid synthases according to the present application can consist of or must include a sequence 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 NOs: 1 to 12, and can have the original function. Here, the "homology" or "identity" refers to the rate (%) of agreement between two sequences when analyzed by aligning the base sequence or amino acid sequence to be the reference with any other base sequence or amino acid sequence in the most corresponding manner.

[0034] The "production capacity is improved" used in the present application means that the productivity of L-isoleucine is increased compared to the variation object (parent strain). The above-described parent strain refers to a wild type or a variation strain that is directly the object of variation or is transformed by a recombinant vector, etc. In the present application, the parent strain can be a wild type Escherichia microorganism or strain having no L-isoleucine production capacity or having L-isoleucine production capacity or an Escherichia microorganism or strain that is varied from the wild type.

[0035] According to an embodiment of the present application, the above-described Escherichia can be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, or Escherichia vulneris, but is not limited thereto.

[0036] As an example, the above-described Escherichia can be Escherichia coli.

[0037] The Escherichia microorganism according to the present application can have improved L-isoleucine production capacity due to the activity / inactivation of acetohydroxy acid synthase III, the weakened / inactivation of the activities of acetohydroxy acid synthases I and III, or the weakened / inactivation of the activities of acetohydroxy acid synthases I and III, while the activity of acetohydroxy acid synthase II is enhanced.

[0038] Specifically, the Escherichia microorganism having improved L-isoleucine production ability shows increased L-isoleucine production ability compared to the parent strain, and particularly, the L-isoleucine production amount can be increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or can be increased by 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, but is not limited thereto. As an example, the Escherichia microorganism in which the activity of the acetohydroxy acid synthase III or the acetohydroxy acid synthase I and III is weakened or inactivated, or in which the activity of the acetohydroxy acid synthase II is simultaneously enhanced, can have an L-isoleucine production amount increased by more than 5%, specifically, by 5 to 50% (preferably, 10 to 40%) compared to the parent strain.

[0039] The composition comprising the Escherichia microorganism according to the present application can be used as a composition for L-isoleucine production.

[0040] The Escherichia microorganism according to the specific example of the present application can be obtained by a gene inactivation method or a recombinant vector in order to delete a gene encoding acetohydroxy acid synthase III or acetohydroxy acid synthase I and III, or to introduce a gene encoding acetohydroxy acid synthase II, with the parent strain as an object.

[0041] The gene inactivation method can be performed by a known method. For example, there are a CaCl2method (Cohen, S. N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), a Hanahan method (Cohen, S. N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)), and an electroporation method (Dower, W. J. et al., Nucleic. Acids Res., 16:6127-6145 (1988)), but are not limited thereto.

[0042] The "vector" used in the present application means all types of nucleic acid sequence transport structures used as a means for transferring and expressing a gene of interest to a variation object (host cell). The above vector can mean that a nucleic acid sequence carried is inserted into a host cell gene to be expressed and / or independently expressed, unless otherwise specified. Such a vector includes necessary regulatory elements operably linked for insertion of a gene expression product, "operably linked" means that a gene of interest and its regulatory sequence are functionally combined with each other and are linked in a manner capable of gene expression, and "regulatory elements" include a promoter for implementing transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation.

[0043] The vector used in the present application is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art can be used. As one example of the above vector, plasmids, cosmids, viruses, and bacteriophages in a natural state or a recombinant state can be cited. For example, as a bacteriophage vector or a cosmid vector, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λtlO, λtl 1, Charon 4A, Charon 21A, and the like can be cited, and as a plasmid vector, pBR series, pUC series, pBluescript II series, pGEM series, pTZ series, pCL series, and pET series, and the like can be cited, but are not limited thereto.

[0044] The above vector can be typically constructed as a vector for cloning or a vector for expression. The vector for expression can use a conventional vector used in the art for expressing a foreign gene or a protein in a plant, an animal, or a microorganism, and can be constructed by various methods known in the art.

[0045] The "recombinant vector" used in the present application can be constructed using a prokaryotic cell or a eukaryotic cell as a host, can replicate independently of the genome of the host cell, or can be integrated into the genome itself. The host cell can replicate the vector and can include a replication origin that is a specific base sequence for initiating replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, it generally includes a strong promoter that can initiate transcription (e.g., a pL lambda promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the replication origin included in the vector that initiates replication in the eukaryotic cell includes an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adenovirus replication origin, an AAV replication origin, and a BBV replication origin, but is not limited thereto. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, a HSV tk promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0046] The above-described recombinant vector can include a selection marker for screening a transformant (host cell) transformed with the vector, and only cells expressing the selection marker can survive in a medium treated with the selection marker, and thus the transformed cells can be screened. As representative examples, the selection marker includes ampicillin, kanamycin, streptomycin, chloramphenicol, etc., but is not limited thereto.

[0047] A transformant can be produced by inserting the above-described recombinant vector into a host cell, and the transformant can be obtained by introducing the recombinant vector into a suitable host cell. The host cell is a cell that can stably and continuously clone or express the above-described expression vector, and any host cell known in the art can be used.

[0048] As host cells for the transformation of prokaryotic cells for the production of recombinant microorganisms, Escherichia coli JM109, E. coli BL21, E. coli RRl, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli XLl-Blue, and the like can be used, but are not limited thereto. Corynebacterium, Bacillus subtilis, Bacillus thuringiensis, and the like can be used, but are not limited thereto. Salmonella typhimurium, Serratia marcescens, Pseudomonas, and the like can be used, but are not limited thereto.

[0049] As host cells for the transformation of eukaryotic cells for the production of recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO Kl, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, and the like can be used, but are not limited thereto.

[0050] The "transformation" used in the present application refers to a phenomenon in which foreign DNA is introduced into a host cell to artificially cause a genetic change, and the "transformant" refers to a host cell into which foreign DNA is introduced and which stably maintains expression of a target gene.

[0051] In the above transformation, a suitable vector introduction technique is selected according to the host cell, and thus a target gene or a recombinant vector including the same can be expressed in the host cell. For example, the vector introduction can be performed by electroporation, heat-shock, CaPO4 precipitation, CaCl2 precipitation, microinjection, PEG method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. The transformed gene can be included as long as it can be expressed in the host cell, and is not limited to being inserted into the chromosome of the host cell or being located outside the chromosome.

[0052] The transformant includes a cell transfected, transformed or infected with the recombinant vector according to the present application in vivo or in vitro, and can be used as the same term as the recombinant host cell, the recombinant cell or the recombinant microorganism.

[0053] The gene inserted into the recombinant vector for transformation according to the present application can be replaced into the host cell such as the microorganism of the genus Escherichia by crossing due to homologous recombination.

[0054] According to an embodiment of the present application, the host cell can be the microorganism of the genus Escherichia. For example, it can be Escherichia coli, but is not limited thereto.

[0055] Another aspect of the present application provides a method for producing L-isoleucine, including the steps of culturing the microorganism of the genus Escherichia in a culture medium, and recovering L-isoleucine from the microorganism of the genus Escherichia or the culture medium in which the microorganism of the genus Escherichia is cultured.

[0056] The culture can be performed according to a suitable culture medium and culture conditions known in the art, and the culture medium and culture conditions can be easily adjusted by those skilled in the art. Specifically, the culture medium can be a liquid medium, but is not limited thereto. The culture method can include, for example, batch culture, continuous culture, fed-batch culture, or a combination culture thereof, but is not limited thereto.

[0057] According to an embodiment of the present application, the culture medium must satisfy the requirements of a specific strain in a suitable manner, and can be appropriately modified by those skilled in the art. With respect to the culture medium for the strain of the genus Escherichia, a well-known document (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981) can be referred to, but is not limited thereto.

[0058] According to an embodiment of the present application, various carbon sources, nitrogen sources, and trace element components can be included in the culture medium. As carbon sources that can be used, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid can be included. These substances can be used alone or in a mixture, but are not limited thereto. As nitrogen sources that can be used, 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 can be included. The nitrogen sources can also be used alone or in a mixture, but are not limited thereto. As a source of phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or a corresponding sodium-containing salt can be included, but is not limited thereto. In addition, the culture medium can contain metal salts such as magnesium sulfate or ferrous sulfate required for growth, but is not limited thereto. In addition thereto, essential growth substances such as amino acids and vitamins can be included. In addition, precursors suitable for the culture medium can be used. The above-described culture medium or individual components can be added to the culture solution in batches or continuously during the culture process by an appropriate method, but are not limited thereto.

[0059] According to an embodiment of the present application, during the culture process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the microbial culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, during the culture process, an antifoaming agent such as fatty acid polyethylene glycol ester can be used to inhibit the generation of bubbles. Further, in order to maintain the aerobic state of the culture solution, oxygen or an oxygen-containing gas such as air can be injected into the culture solution. The temperature of the culture solution can generally be 20 to 45°C, for example, 25 to 40°C. The culture time can continue until the desired production amount of the useful substance is obtained, for example, 10 to 160 hours.

[0060] According to an embodiment of the present application, in the above-described step of recovering L-isoleucine from the cultured transformant or the culture medium of the cultured transformant, the produced L-isoleucine can be collected or recovered from the culture medium according to the culture method and using a suitable method known in the art. For example, methods such as 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 the like can be used, but are not limited thereto.

[0061] According to an embodiment of the present application, in the above-described step of recovering L-isoleucine, the culture medium is centrifuged at a low speed to remove biomass, and the obtained supernatant can be separated by ion exchange chromatography.

[0062] According to an embodiment of the present application, the step of recovering L-isoleucine can include a process of purifying L-isoleucine.

[0063] The Escherichia microorganism according to the present application has weakened or inactivated activity of acetylhydroxy acid synthase III or acetylhydroxy acid synthase I and III, or simultaneously has enhanced activity of acetylhydroxy acid synthase II, so that the biosynthesis pathway of L-isoleucine is enhanced, and the production of by-products is weakened, and the production yield of L-isoleucine can be improved compared to the parent strain. DETAILED DESCRIPTION

[0064] Hereinafter, the present application will be described in more detail. However, such description is only illustratively presented to help understanding of the present application, and the scope of the present application is not limited to such illustrative description.

[0065] Example 1. Preparation of a variant strain having weakened activity of AHAS I

[0066] In order to prepare a strain having weakened activity of AHAS I, Escherichia coli (also referred to as E. coli) DS44 (accession number KCTC 11602BP) producing L-isoleucine was used, and the ilvBN gene (SEQ ID NO: 1 and 3) encoding AHAS I was deleted by a one step inactivation method (Warner et al., PNAS, 6:6640-6645 (2000).

[0067] First, a fragment was obtained by performing PCR using the primer pair of lvBN_F and ilvBN_R and the pKD13 plasmid (GenBank AY048744) as a template. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into E. coli DS44, the prepared PCR fragment was injected by electroporation, and a kanamycin-resistant colony was screened. The screened transformant was subjected to PCR using the primer pair of ilvBN_CF and ilvBN_CR, and whether the ilvBN gene was deleted was confirmed. DS44 ΔilvBN into which the disruption DNA was inserted generated a product of about 2.6 kb as expected, and DS44 generated a product of about 3.5 kb.

[0068] Using the strain in which the ilvBN gene was confirmed to be deleted, a process of deleting the antibiotic resistance marker gene was performed. In this process, the ilvBN gene deletion strain was introduced with the pCP20 plasmid (Cherepanov and Wackerneagel, 1995; Datsenko and Wanner, 2000), and FLP recombination was induced. Then, the ilvBN gene deletion strain was cultured in LB plates with or without antibiotics, and it was confirmed that the antibiotic resistance marker gene was deleted.

[0069] Here, the PCR reaction was performed as follows: the total reaction volume was 50 μl, and after 1 cycle of 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes, and then 72°C for 2 minutes.

[0070] The primer sequences used therein are shown in Table 1 below.

[0071] [Table 1]

[0072]

[0073] Example 2. Preparation of a variant strain in which the activity of AHAS II is weakened

[0074] To prepare a strain in which the activity of AHAS II is weakened, Escherichia coli DS44 (Accession No. KCTC 11602BP) producing L-isoleucine was used, and the ilvGM gene (SEQ ID NO: 5 and 7) encoding AHAS II was deleted by a one step inactivation method (Warner et al., PNAS, 6:6640-6645 (2000).

[0075] First, a fragment was obtained by performing a PCR reaction using the primer pair of ilvGM_F and ilvGM_R and the pKD13 plasmid (GenBank AY048744) as a template. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into E. coli DS44, the prepared PCR fragment was injected by electroporation, and a colony having kanamycin resistance was selected. The selected transformant was subjected to a PCR reaction using the primer pair of ilvGM_CF and ilvGM_CR, and whether the ilvGM gene was deleted was confirmed. DS44 ΔilvGM in which the disruption DNA was inserted generated a product of about 2.6 kb as expected, and DS44 generated a product of about 3.3 kb.

[0076] Using the strain confirmed to lack the ilvGM genes, a process of deleting the antibiotic resistance marker gene was performed. In this process, the ilvGM gene-lacking strain was introduced with the pCP20 plasmid, and FLP recombination was induced. Then, the ilvGM gene-lacking strain was cultured in LB plates with or without antibiotics, and it was confirmed that the antibiotic resistance marker gene was deleted.

[0077] Here, the PCR reaction was performed as follows: the total reaction volume was 50 μl, and after 1 cycle of 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes, and then 72°C for 2 minutes.

[0078] The primer sequences used herein are shown in Table 2 below.

[0079] [Table 2]

[0080]

[0081] Example 3. Preparation of a variant strain with weakened activity of AHAS III

[0082] To prepare a strain with weakened activity of AHAS III, Escherichia coli DS44 (Accession No. KCTC 11602BP) producing L-isoleucine was used, and the ilvIH gene (SEQ ID NO: 9 and 11) encoding AHAS III was deleted by a one step inactivation method (Warner et al., PNAS, 6:6640-6645 (2000).

[0083] First, a fragment was obtained by performing a PCR reaction using the primer pair of ilvIH_F and ilvIH_R and the pKD13 plasmid (GenBank AY048744) as a template. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into E. coli DS44, the prepared PCR fragment was injected by electroporation, and a colony having kanamycin resistance was selected. The selected transformant was subjected to a PCR reaction using the primer pair of ilvIH_CF and ilvIH_CR, and whether the ilvIH gene was deleted or not was confirmed. DS44 ΔilvIH into which the disruption DNA was inserted generated a product of about 2.5 kb as expected, and DS44 generated a product of about 3.6 kb.

[0084] Using strains confirmed to lack the ilvIH gene, a process was implemented to remove the antibiotic resistance marker gene. During this process, the pCP20 plasmid was introduced into the ilvIH gene-deficient strains, inducing FLP recombination. Then, the ilvIH gene-deficient strains were cultured in LB plates with or without antibiotics to confirm the removal of the antibiotic resistance marker gene.

[0085] 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.

[0086] The primer sequences used are shown in Table 3 below.

[0087] Table 3

[0088]

[0089] Example 1. Evaluation of L-Isoleucine Production Capacity

[0090] The L-isoleucine production capacity of the Escherichia coli variants prepared in Examples 1 to 3 was evaluated by comparison with the parent strain.

[0091] The injection medium for isoleucine production, as shown in Table 4 below, was used in a 5L fermenter. 2 L of culture medium and 342 mL of supplemental culture medium were added, and the culture was carried out at a temperature of 30℃, a stirring speed of 500 rpm, and an aeration rate of 1 vvm. After the culture was completed, the concentrations of L-valine and AABA (L-α-aminobutyric acid) generated as byproducts along with L-isoleucine in the culture medium were determined by HPLC (Agilent), and the results are shown in Table 5 below.

[0092] Table 4

[0093] injection medium supplemental medium glucose 80g / l 550g / l corn steep liquor 20g / l - ammonium sulfate 20g / l 1g / l phosphoric acid 15g / l 1g / l fumaric acid 1g / l - sodium glutamate 7g / l - sodium citrate 1g / l - choline-HCl 1g / l - thiamine-HCl 5㎎ / l - pyridoxine-HCl 10㎎ / e - nicotinic acid 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 -

[0094] Table 5

[0095]

[0096] As shown in Table 5 above, when the activity of AHASⅡ was weakened (DS44ΔilvGM), compared with the parent strain, the production capacity of L-isoleucine decreased by about 44.2%, while the production capacity of L-valine and AABA increased by about two times.

[0097] In contrast, when the activity of AHAS I was weakened (DS44ΔilvBN) or the activity of AHAS III was weakened (DS44ΔilvIH), the L-isoleucine production ability was increased, while the production abilities of L-valine and AABA were decreased, compared to the parent strain. Specifically, the L-isoleucine production ability of DS44ΔilvBN and DS44ΔilvIH was increased by about 11.9% and 7.9%, respectively, compared to that of DS44, the L-valine / L-isoleucine (V / I) ratio was decreased by about 41.6% and 29.8%, respectively, and the AABA / L-isoleucine (AABA / I) ratio was decreased by about 19% and 20.6%, respectively.

[0098] These results indicate that the weakening of the activity of AHAS I or AHAS III induces an increase in the L-isoleucine production ability in the L-isoleucine biosynthetic pathway.

[0099] Example 4. Preparation of a mutant strain in which the activities of AHAS II and III are weakened

[0100] To prepare a strain in which the activities of AHAS II and III are weakened, DS44ΔilvGM of Example 2 was used instead of E. coli DS44, and the same method as in Example 3 was used, except for this. As a result, DS44ΔilvGMΔilvIH in which the ilvGM and ilvIH genes were deleted was prepared.

[0101] Example 5. Preparation of a mutant strain in which the activities of AHAS I and III are weakened

[0102] To prepare a strain in which the activities of AHAS I and III are weakened, DS44ΔilvBN of Example 1 was used instead of E. coli DS44, and the same method as in Example 3 was used, except for this. As a result, DS44ΔilvBNΔilvIH in which the ilvBN and ilvIH genes were deleted was prepared.

[0103] Example 6. Preparation of a mutant strain in which the activities of AHAS I and II are weakened

[0104] To prepare a strain in which the activities of AHAS I and II are weakened, DS44ΔilvBN of Example 1 was used instead of E. coli DS44, and the same method as in Example 2 was used, except for this. As a result, DS44ΔilvBNΔilvGM in which the ilvBN and ilvGM genes were deleted was prepared.

[0105] Experimental Example 2. Evaluation of L-isoleucine production ability

[0106] The L-isoleucine production ability of the E. coli mutant strains prepared in Examples 4 to 6 was evaluated, compared to the parent strain.

[0107] The parent strain or the mutant strain was cultured in the same manner as in Experimental Example 1 above and the concentrations of L-isoleucine, L-valine and AABA in the culture medium were measured, and the results are shown in Table 6 below.

[0108] [Table 6]

[0109]

[0110] As shown in Table 6 above, when the activities of AHAS II and III were weakened (DS44 ΔilvGM ΔilvIH) or the activities of AHAS I and II were weakened (DS44 ΔilvBN ΔilvGM) compared to the parent strain, the L-isoleucine production ability decreased, but, on the contrary, when the activities of AHAS I and III were weakened (DS44 ΔilvBN ΔilvIH), the L-isoleucine production ability increased, and at the same time, the L-valine and AABA production abilities decreased. Specifically, the L-isoleucine production amount of DS44 ΔilvBN ΔilvIH was increased by about 14.9% compared to that of DS44, the L-valine / L-isoleucine (V / I) ratio was decreased by about 75.5%, and the AABA / L-isoleucine (AABA / I) ratio was decreased by about 69.9%.

[0111] Such results indicate that AHAS II has the most excellent suitability for L-isoleucine biosynthesis compared to AHAS I or AHAS III.

[0112] Example 7. Preparation of a mutant strain in which the activities of AHAS I and III are weakened and the activity of AHAS II is enhanced

[0113] In order to prepare a strain in which the activities of acetylhydroxy acid synthase (AHAS) I and III are weakened and the activity of AHAS II is enhanced, Escherichia coli DS44 (Accession No. KCTC 11602BP) producing L-isoleucine was used and the ilvBN and ilvIH genes encoding AHAS I and III, respectively, were deleted by one step inactivation (Warner et al., PNAS, 6:6640-6645 (2000)) while the ilvGM gene encoding AHAS II was introduced.

[0114] First, a fragment was obtained by performing PCR using the primer set of ilvIH_F and Ptrc+pKD13_R and the pKD13 plasmid (GenBank AY048744) as a template. A fragment was obtained by performing PCR using the primer set of pTRC+ilvG_F and ilvIH+ilvM_R and the DS44 genomic DNA as a template. A DNA fragment was obtained by performing PCR using the two fragments. After introducing the red recombinase plasmid pKD46 (GenBank AY048746) into the DS44ΔilvBN prepared in Example 1, the prepared DNA fragment was injected by electroporation, and a kanamycin-resistant colony was selected. The selected transformant was subjected to PCR using the primer set of ilvIH_CF and ilvIH_CR, and the absence or presence of the ilvIH gene deletion and the introduction of the ilvGM gene were confirmed. DS44ΔilvBNΔilvIH::Ptrc-ilvGM produced a product of about 4.5 kb as expected, while DS44 produced a product of about 3.6 kb.

[0115] The process of eliminating the antibiotic resistance marker gene was performed using the strain in which the ilvBN and ilvIH genes were confirmed to be deleted and the ilvGM gene was introduced. In this process, the strain in which the ilvBN and ilvIH genes were deleted and the ilvGM gene was introduced was introduced with the pCP20 plasmid, and FLP recombination was induced. Then, the strain in which the ilvBN and ilvIH genes were deleted and the ilvGM gene was introduced was cultured in an LB plate with or without the addition of an antibiotic, and it was confirmed that the antibiotic resistance marker gene was eliminated.

[0116] Here, the PCR reaction was performed as follows: the total reaction volume was 50 μl, and after 1 cycle of 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes, and then 72°C for 2 minutes.

[0117] The primer sequences used therein are shown in Table 7 below.

[0118] [Table 7]

[0119]

[0120] Experimental Example 3. Evaluation of L-isoleucine production ability

[0121] The L-isoleucine production ability of the E. coli variant strains prepared in Examples 5 and 7 was evaluated in comparison with the parent strain.

[0122] The parent strain or the mutant strain was cultured in the same manner as in Experimental Example 1 above and the concentrations of L-isoleucine, L-valine and AABA in the culture medium were measured, and the results are shown in Table 8 below.

[0123] [Table 8]

[0124]

[0125] As shown in Table 8 above, when the strain in which the ilvBN and ilvIH genes were deleted and only AHAS II was expressed (DS44ΔilvBNΔilvIH) was further enhanced in the expression of AHAS II (DS44ΔilvBNΔilvIH::Ptrc-ilvGM), the L-isoleucine production ability was further increased and the L-valine and AABA production abilities were further decreased compared to the parent strain. Specifically, the L-isoleucine production amount of DS44ΔilvBNΔilvIH::Ptrc-ilvGM was increased by about 20.7% and the L-valine / L-isoleucine (V / I) ratio was decreased by 77.2% compared to DS44ΔilvBNΔilvIH.

[0126] So far, the present application has been described around its preferred embodiments. It will be understood by those skilled in the art to which the present application pertains that the present application can be implemented in modified forms without departing from the essential characteristics of the present application. Accordingly, the disclosed embodiments should be considered in a descriptive sense only and not as a restrictive sense. The scope of the present application is shown in the claims rather than the above description, and all differences within the equivalent range of the scope thereof should be construed as being included in the present application.

[0127] [Deposit Information]

[0128] Name of Depository: Korean Collection for Type Cultures (KCTC)

[0129] Accession No.: KCTC 11602BP

[0130] Date of Deposit: 20091120

[0131] Classification Name of Biological Material: Escherichia coli

Claims

1. An Escherichia coli microorganism whose L-isoleucine production capacity is enhanced by weakening or inactivating the activity of acetylhydroxy acid synthase III or by weakening or inactivating the activities of acetylhydroxy acid synthase I and III.

2. The Escherichia coli microorganism according to claim 1, wherein, The weakening of the activity of acetylhydroxy acid synthase I is achieved through nucleotide modifications, promoter modifications, or a combination thereof, of the gene encoding acetylhydroxy acid synthase I. The weakening of the activity of acetylhydroxy acid synthase III is achieved through nucleotide modification, promoter modification, or a combination thereof of the gene encoding acetylhydroxy acid synthase III.

3. The Escherichia coli microorganism according to claim 1, wherein, The activity of the Escherichia coli microorganisms was further enhanced.

4. The Escherichia coli microorganism according to claim 3, wherein, The enhanced activity of acetylhydroxy acid synthase II is achieved through nucleotide modification, copy number increase, promoter modification, introduction, or a combination thereof of the gene encoding acetylhydroxy acid synthase II.

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

6. 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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