WO2008075855A1 - Escherichia coli variety producing l-threonine and method for fabricating the same - Google Patents

Escherichia coli variety producing l-threonine and method for fabricating the same Download PDF

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Publication number
WO2008075855A1
WO2008075855A1 PCT/KR2007/006531 KR2007006531W WO2008075855A1 WO 2008075855 A1 WO2008075855 A1 WO 2008075855A1 KR 2007006531 W KR2007006531 W KR 2007006531W WO 2008075855 A1 WO2008075855 A1 WO 2008075855A1
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threonine
rpos
vector
escherichia coli
pccbac
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PCT/KR2007/006531
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French (fr)
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Eun Sung Koh
Soo An Shin
Jin Ho Lee
Keun Chul Lee
Young Hoon Park
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Cj Cheiljedang Corporation
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Publication of WO2008075855A1 publication Critical patent/WO2008075855A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to a transformed microorganism having improved threonine productivity. More particularly, the present invention relates to a threonine- producing microorganism transformed with a vector comprising a gene encoding an rpoS protein, the amino acid sequence of which has leucine at position 33.
  • L-threonine is known to be an essential amino acid, which has been widely used as an animal feed and food additive, as well as a component of medical aqueous solutions and other raw material for medicinal products, and is rich in animal proteins but short in vegetable proteins. Thus, L-threonine tends to be low in vegetarian diets. L- threonine comprises 5.3% of egg proteins and 4% of milk proteins, and is present in a form of phosphate ester in proteins such as casein.
  • L-threonine is synthesized from aspartic acid via homoserine or from glycine by threonine aldolase, and metabolized to glycine, ⁇ -ketobutyric acid, 2-aminoacetoacetic acid or the like.
  • L-threonine is mainly produced by a fermentation method, using Escherichia coli or
  • Corynebacterium developed by artificial mutation or genetic recombination developed by artificial mutation or genetic recombination.
  • the known technique using artificial mutants is exemplified by Japanese Patent Publication No. 10037/81, in which disclosed is a method of producing L-threonine using a strain belonging to the genus Escherichia coli, which has a nutritional requirement for di- aminopimelic acid and methionine and has resistance to the feedback inhibition of threonine synthesis by its biosynthetic system, and exemplified by a method of producing L-threonine using a strain belonging to the genus C.
  • glutamicum which is resistant to S-(2-aminoethyl)-L-cysteine and ⁇ -amino- ⁇ -hydroxyvalerate and has a nutritional requirement for methionine (Kangogaku Zasshi, et al, Agric. Biol. Chem., 36:1611, 1972).
  • Escherichia coli proliferation is delicately controlled in response to transition from log phase to stationary phase, and environmental factors such as high/ low temperature, high osmotic pressure, oxygen stress, and nutrient starvation, by the expression of the related genes and proteins, for example, ftsQAZ, dps, poxB, treA, otsBA, proU, and katG genes.
  • a sigma factor of RNA polymerase, rpoS(o ) functions to regulate the expression of the genes.
  • rpoS The in vivo level of rpoS is regulated at the levels of transcription, translation, proteolysis or the like (Regine Hengge-Aronis, Micro. Molecul. Biol. Rev., 66: 373-395, 2002). Further, in the fermentation using L-threonine-producing Escherichia coli, the expression of rpoS is considered as a very important factor during culture in accordance with environmental stresses and transition to stationary phase. Disclosure of Invention Technical Problem
  • the present inventors have made an effort to develop a strain producing high levels of L-threonine by optimal expression of rpoS in L- threonine-producing Escherichia coli. They found that a mutant having an artificial chromosome with a gene encoding an rpoS protein, of which an amino acid sequence has leucine at position 33, can produce L-threonine in a high yield, thereby completing the present invention.
  • the present invention provides a threonine-producing microorganism, characterized in that the microorganism is transformed with a vector comprising a gene encoding a mutant rpoS protein (sigma factor, ⁇ s ) involved in stress response), the amino acid sequence of which has leucine at position 33.
  • FIG. 1 illustrates the construction of an artificial chromosome recombinant vector pccBac-mrpoS, comprising a gene encoding a mutant rpoS protein, the amino acid sequence of which has leucine at position 33. Best Mode for Carrying Out the Invention
  • the present invention provides an artificial chromosome recombinant vector, comprising a base sequence encoding an rpoS protein, the amino acid sequence of which has leucine at position 33.
  • the base sequence may be represented by SEQ ID NO. 5
  • an amino acid sequence of the rpoS protein having leucine at position 33 may be represented by SEQ ID NO. 6.
  • the present invention provides an artificial chromosome recombinant vector comprising a base sequence that has 95% or more homology with the base sequence of SEQ ID NO. 5 and encodes a protein having the same activity as the rpoS protein, the amino acid sequence of which has leucine at position 33.
  • Escherichia coli used for producing L-threonine generally has the rpoS protein containing various types of amino acids at position 33.
  • the wild type Escherichia coli MG 1655 has the rpoS protein, the amino acid sequence of which has glutamine (abbreviated to "GIn” or "Q") at position 33
  • other Escherichia coli has the rpoS protein, the base sequence of which has an amber stop codon (TAG) or a gene encoding glutamic acid (GAG) or leucine (CTG) instead of glutamine at position 33.
  • TAG amber stop codon
  • GAG GAG
  • CCG leucine
  • the "amber stop codon” means a stop codon with the base sequence TAG on the coding strand in a DNA molecule corresponding to UAG on the mRNA read by this DNA molecule.
  • the translation reinitiates at position 54, resulting in a smaller rpoS protein consisting of 277 amino acids with a molecular weight of 30 kDa (T.Atlung, et al, MoI. Genet. Genomics, 266:873-881, 2002).
  • Escherichia coli containing the mutant rpoS protein shows reduced activity, as compared to those containing wild type rpoS, and thus the enzyme activity of catalase (decomposing hydrogen peroxide) is reduced (P.R.Subbarayan, M.Sarkar, MoI. Genet. Genomics, 270:533-538, 2004).
  • the present invention is characterized in that an artificial chromosome is used as a recombinant vector instead of using known multi-copy plasmids, since the artificial chromosome is very stable without antibiotics, and maintained in a microorganism as a single copy to avoid side effects due to over- expression of specific genes.
  • the bacterial artificial chromosome of the present invention may be a vector capable of independent replication in a host cell, that is, a self replication vector.
  • the vector when the vector is introduced into the host cell, the vector may be integrated into the host cell genome and be replicated with the integrated chromosome.
  • the artificial chromosome may include pBeloBac and pccBac, and preferably pccBac (Copy Control Bacterial Artificial Vector: hereinafter, abbreviated to "pccBac”).
  • a mutant rpoS gene containing the base sequence of SEQ ID NO. 5, which encodes the rpoS protein having leucine at position 33 is introduced into the artificial chromosome pccBac to complete a recombinant vector, which is designated as "pccBac-mrpoS" (Fig. 1).
  • the DNA sequence should be operably linked to a suitable promoter.
  • the promoter may be any DNA sequence exhibiting transcriptional activity in a selected host cell, and may be derived from genes encoding proteins that are either homologous or heterologous to the host cell.
  • examples of the promoter, which regulates the transcription of the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6 in a microbial host may include the lac operon promoter ofEscherichia coli (E. col ⁇ ) and the dagA promoter of agarose gene of Streptomyces coelicolor.
  • the present invention provides a method for fabricating the artificial chromosome recombinant vector, pccBac-mrpoS.
  • the recombinant vector of the present invention may be fabricated by using various recombination techniques known in the art, for example, DNA recombination technique comprising the steps of (a) introducing the gene fragment comprising the base sequence of SEQ ID NO. 5 or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6 into a cloning vector; (b) performing PCR using the cloning vector as a template and primers of SEQ ID NOs. 3 and 4; and (c) ligating the PCR product to the artificial chromosome vector.
  • DNA recombination technique comprising the steps of (a) introducing the gene fragment comprising the base sequence of SEQ ID NO. 5 or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6 into a cloning vector; (b) performing PCR using the cloning vector as a template and primers of
  • Any recombination technique can be employed, as long as it can fabricate the recombinant vector by introducing DNA comprising the base sequence encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, into the artificial chromosome, but is not limited thereto. Any known method may be employed to fabricate the recombinant vector of the present invention.
  • the recombinant vector of the present invention can be fabricated by a mutagenic technique suitable for replacement of gene or allele.
  • the recombinant vector pccBac-mrpoS of the present invention can be fabricated using the DNA recombination technique and site- directed mutagenesis, comprising the steps of (a) preparing a wild type rpoS gene fragment from Escherichia coli chromosome; (b) introducing the gene fragment into the cloning vector; (c) performing PCR using the cloning vector as a template to induce site directed mutation; and (d) ligating the mutated PCR product to the artificial chromosome vector.
  • PCR amplification for the induction of site directed mutation may be preferably performed with the primers of SEQ ID NOs. 3 and 4.
  • any cloning vector may be used, preferably pCR2.1-TOPO.
  • the resulting vector was designated as "TOPO2.1-rpoS”.
  • the artificial chromosome may be preferably pccBac.
  • the resulting recombinant vector was designated as "pccBac- mrpoS”.
  • the wild type rpoS gene fragment derived from Escherichia coli chromosome in step (a) may be prepared by the steps of (D) extracting chromosomal DNA from Escherichia coli to prepare the DNA fragment comprising the rpoS open reading frame and promoter; and (D) performing PCR to amplify the DNA fragment, and PCR may be performed using the primers of SEQ ID NOs. 1 and 2. Further, in step (c), PCR amplification for the induction of site directed mutation may be performed using the primers of SEQ ID NOs. 3 and 4.
  • the Escherichia coli may be a known strain MC4100, but is not limited thereto. Any Escherichia coli can be used, as long as it contains the DNA fragment comprising the rpoS open reading frame and promoter.
  • the present invention provides a transformed microorganism prepared by introducing the recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6, into a microbial host.
  • the present invention provides a method for preparing the transformed microorganism, comprising the steps of (a) preparing the recombinant vector pccBac-mrpoS; and (b) introducing the vector into the host cell.
  • Examples of the microbial host to be used in the present invention may include microorganisms belonging to gram negative bacteria, and preferably microorganisms belonging to the species Escherichia coli.
  • L-threonine producing strains may be used as the transformed mutant strain harboring the pccBac vector, in which the mutant rpoS is introduced to the artificial chromosome.
  • Examples thereof include microorganisms belonging to Escherichia coli, which is capable of producing L-threonine and has resistance to a L- methionine analogue, methionine auxotroph, resistance to L-threonine analogue, isoleucine-leaky auxotroph, and resistance to an L-lysine analog and ⁇ -aminobutyric acid; and mutant microorganisms, in which an endogeneous phosphoenol pyruvate carboxylase (PEPCase) gene (ppc) and genes in the threonine operon and additionally, at least one copy of ppc gene and thrA, thrB, thrc inthe threonine operon are inserted in the chromosomal DNA.
  • Examples of the L-methionine analogue may include at least one selected from the group consisting of D, L-methionine, norleucine, ⁇ - methylmethionine, and L-methionine-D,L-sulphoximine. Further, examples of the L- threonine analogue may include at least one selected from the group consisting of ⁇ - amino- ⁇ -hydroxy valerate and D,L-threonine hydroxamate. Examples of the L-lysine analogue may include at least one selected from the group consisting of S- (2-aminoethyl)-L-cysteine and ⁇ -methyl-L-lysine.
  • mutant microorganisms may include microorganisms, in which is inactivated the pckA gene that is involved in the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the biosynthesis of L-methionine; and microorganisms, in which is inactivated the galR gene that inhibits the expression of the galP gene involved in glucose transfer.
  • OOA oxaloacetate
  • PEP phosphoenolpyruvate
  • the present invention provides a microorganism transformed with the recombinant vector pccBac-mrpoS by using Escherichia coli KCCM 10541 as a host cell (accession number: KCCM10815P, hereinafter designated as "CA030016").
  • the present invention provides a method for producing
  • the cultivation of the transformed microorganism may be conducted in suitable media and under culture conditions known in the art.
  • the culturing procedures can be readily adjusted by those skilled in the art. Examples of the culturing procedures include batch type, continuous type and fed-batch type manners, but are not limited thereto.
  • Various culturing procedures are disclosed in literature, for example, "Biochemical Engineering” (James M. Lee, Prentice-Hall International Editions, pp 138-176, 1991).
  • the production method of L-threonine of the present invention may comprise the steps of (a) culturing the microorganism transformed with the artificial chromosome recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence that has 95% or more homology with SEQ ID NO.
  • the transformed microorganism may be preferably accession number: KCCMl 0815P.
  • the media used in the culture method should preferably meet the requirements of a specific strain.
  • culture media contain various carbon sources, nitrogen sources and minerals.
  • the carbon sources useful in the present invention include carbohydrates such as glucose, fructose, sucrose, lactose, maltose, starch, and cellulose, lipids such as soybean oil, regular sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohol such as glycerol and ethanol, and organic acids such as acetic acid.
  • These carbon sources may be used alone or in combination.
  • nitrogen sources useful in the present invention include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soy bean, and inorganic nitrogen sources such as urea (CO(NH ) ), ammonium sulfate ((NH ) SO ), ammonium chloride (NH Cl),
  • ammonium phosphate (NH ) HPO ), ammonium carbonate ((NH ) CO ) and ammonium nitrate (NH NO ).
  • nitrogen sources may be used alone or in combination.
  • phosphorus sources such as potassium dihydrogen phosphate (KH PO ), dipotassium hydrogen phosphate (K HPO ) or corresponding sodium-containing salts may be added.
  • the media may contain metal salts such as magnesium sulfate or ferrous sulfate.
  • the media may be supplemented with amino acids, vitamins, and appropriate precursors. These media or precursors may be added to cultures by a batch type or continuous type method.
  • ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be properly added so as to adjust the pH of the cultures.
  • Defoaming agents such as fatty acid polyglycol ester may be properly added so as to reduce the formation of foams in cultures.
  • oxygen or oxygen-containing gas e.g., air
  • the cultures are maintained at 20 to 45 0 C and preferably at 25 to 4O 0 C.
  • the cultivation may be continued until a desired amount of L-methionine is obtained, and preferably for 10 to 160 hrs.
  • the isolation of L-threonine from the culture broth can be performed by the conventional method known in the art. Examples thereof may include centrifugation, filtration, ion-exchange chromatography, and crystallization. For example, the cultures are subjected to low- speed centrifugation to remove the biomass, and the supernatant was separated by ion-exchange chromatography.
  • the L- threonine-producing mutant CA030016 of the present invention (accession number: KCCM10815P) that is expressed by introducing the mutant rpoS gene, the amino acid sequence of which has leucine at position 33, into the artificial chromosome is a strain capable of producing L-threonine in a high yield.
  • the present invention provides a method for preparing an L-threonine containing feed supplements by mixing L-threonine that is prepared and isolated according to the method of the present invention and conventional feed supplements, and a feed supplement prepared by the same.
  • the method for preparing a feed supplement of the present invention comprises the steps of (a) culturing the microorganism transformed with the artificial chromosome recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence that has 95% or more homology with SEQ ID NO.
  • the transformed microorganism may be preferably accession number: KCCM10815P.
  • feed refers to any natural or artificial dietary, one meal, or ingredients of one meal that is eaten, consumed, and digested by animals, and examples thereof may include concentrated feeds, bulky feeds and/or specialized feeds.
  • inventions as used herein belongs to the category of feed additives under the Control of Livestock and Fish Feed Act (Korea), and refers to substances that are added to the feed for the purpose of nutrient supplement and weight loss prevention, improvement of dietary fiber digestion, milk quality, and pregnancy rate, and prevention of reproductive disorder and heat stress.
  • the livestock of the present invention refers to livestock being suitable for domestication and increasing farm income, defined by Article 2-1 of the Livestock Industry Act (Korea) and each Section, Article 2 of the administrative regulation (Korea), and examples thereof include cow, horse, mule, donkey, goat, sheep, lamb, deer, swine, rabbit, dog, and poultry such as chicken, turkey, duck, ostrich, goose, and covey, but are not limited thereto.
  • the feed supplement according to the present invention can be processed in a liquid or solid form.
  • Example 1 Preparation of recombinant L-threonine producing strain by artificial chromosome vector carrying wild type rpoS gene
  • Chromosomal DNA was extracted from Escherichia coli MC4100 strain using a
  • Genomic-tip system Qiagen
  • polymerase chain reaction hereinafter, abbreviated to "PCR” was performed using the chromosomal DNA as a template to amplify a DNA fragment of 2357 bp containing the rpoS open reading frame (hereinafter, abbreviated to "ORF” and promoter.
  • the used primer set was as represented by SEQ ID NOs. 1 and 2
  • the used reaction solution was a PCR HL premix kit (BIONEER Co., hereinafter the same).
  • PCR conditions included 30 cycles of de- naturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min 30 sec.
  • the PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of the desired size was excised out from the gel.
  • the rpoS gene fragment obtained from PCR was ligated into a pCR2.1-TOPO vector using a TA cloning kit (Invitrogen Co., hereinafter the same). Then, Escherichia coli Top 10 (Invitrogen Co., hereinafter the same) was transformed with the vector, and smeared on an LB (Lurina-Bertani) solid medium (Bacto tryptone: 10 g/L, Bacto Yeast extract: 5 g/L, sodium chloride: 10 g/L) containing ampicillin (100 mg/L), followed by incubation at 37°C overnight. The rpoS base sequence was confirmed by DNA sequencing analysis (data not shown).
  • the TOPO2.1-rpoS plasmid was treated with a restriction enzyme EcoRI, and then excised from the 0.8% agarose gel to give a DNA fragment of about 2.4 kb, which was designated as "wild type rpoSIEcoRl gene fragment".
  • Example 2 Preparation of recombinant L-threonine producing strain by artificial chromosome vector carrying amber rpoS gene
  • Chromosomal DNA was extracted from the threonine producing strain, FTR2533 using a Genomic-tip system (Qiagen), and PCR was performed using the chromosomal DNA as a template to amplify DNA fragment of 2357 bp containing the rpoS ORF and promoter.
  • the used primer set was as represented by SEQ ID NOs. 1 and 2
  • the used reaction solution was a PCR HL premix kit. PCR conditions included 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min 30 sec.
  • the PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of the desired size was excised out from the gel.
  • the rpoS gene fragment obtained from PCR was ligated into a pCR2.1-TOPO vector using a TA cloning kit. Then, Escherichia coli Top 10 was transformed with the vector, and smeared on an LB solid medium containing ampicillin (100 mg/L), followed by incubation at 37°C overnight. The base sequence of amber rpoS was confirmed by DNA sequencing analysis (data not shown).
  • recombinant vector pccBac-arpoS 3 ml of a liquid LB medium containing chloramphenicol, followed by incubation overnight. Then, plasmid DNA was recovered using a plasmid miniprep kit to confirm the size of the recombinant vector (data not shown), which was designated as "recombinant vector pccBac-arpoS".
  • PCR conditions included 18 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 1 min and extension at 68°C for 6 min 30 sec. Then, Escherichia coli Top 10 was transformed with 12 uL of each PCR product, and smeared on a solid medium containing ampicillin, followed by incubation at 37°C overnight.
  • plasmid DNA was recovered using a plasmid miniprep kit, and the rpoS base sequence was confirmed by DNA sequencing analysis (data not shown).
  • the TOPO2.1-rpoS plasmid having an amino acid sequence with leucine at position 33 was treated with a restriction enzyme EcoRI, and then excised from the 0.8% agarose gel to give a DNA fragment of about 2.4 kb, which was designated as "mutant rpoSIEcoRl gene fragment".
  • Example 4 Comparison of L-threonine productivity of strains transformed with artificial chromosome vector carrying rpoS gene
  • Example 4-1 The transformed strains prepared in Example 4-1 were cultured in Erlenmeyer flasks containing threonine titer medium of Table 1, and the L-threonine productivity was compared.
  • the productivity was increased by 2.8 g/L and 2.4g/L, as compared to the strain harboring the wild type rpoS gene and the KCCM 10541 strain used as a control, respectively (Table 2), which indicates that the mutant rpoS gene with amino acid sequence having leucine at position 33 was introduced to increase the L- threonine productivity.
  • the transformed strain was designated as "CA030016", and deposited at the Korean Culture Center of Microorganisms (KCCM) under an accession number of KCCM10815P.
  • the microorganism of the present invention can produce L-threonine in a high yield, thereby being used in medicinal, pharmaceutical, and feed industries, in particular, as an animal feed.

Abstract

The present invention relates to a transformed microorganism having improved threonine productivity. More particularly, the present invention relates to a threonine-producing microorganism transformed with a vector comprising a gene encoding an rpoS protein, the amino acid sequence of which has leucine at position 33. The microorganism of the present invention can produce L-threonine in a high yield, thereby being used in medicinal, pharmaceutical, and feed industries, in particular, as an animal feed.

Description

Description
ESCHERICHIA COLI VARIETY PRODUCING L-THREONINE AND METHOD FOR FABRICATING THE SAME
Technical Field
[1] The present invention relates to a transformed microorganism having improved threonine productivity. More particularly, the present invention relates to a threonine- producing microorganism transformed with a vector comprising a gene encoding an rpoS protein, the amino acid sequence of which has leucine at position 33. Background Art
[2] L-threonine is known to be an essential amino acid, which has been widely used as an animal feed and food additive, as well as a component of medical aqueous solutions and other raw material for medicinal products, and is rich in animal proteins but short in vegetable proteins. Thus, L-threonine tends to be low in vegetarian diets. L- threonine comprises 5.3% of egg proteins and 4% of milk proteins, and is present in a form of phosphate ester in proteins such as casein. In yeast and bacteria, L-threonine is synthesized from aspartic acid via homoserine or from glycine by threonine aldolase, and metabolized to glycine, α-ketobutyric acid, 2-aminoacetoacetic acid or the like.
[3] L-threonine is mainly produced by a fermentation method, using Escherichia coli or
Corynebacterium developed by artificial mutation or genetic recombination. The known technique using artificial mutants is exemplified by Japanese Patent Publication No. 10037/81, in which disclosed is a method of producing L-threonine using a strain belonging to the genus Escherichia coli, which has a nutritional requirement for di- aminopimelic acid and methionine and has resistance to the feedback inhibition of threonine synthesis by its biosynthetic system, and exemplified by a method of producing L-threonine using a strain belonging to the genus C. glutamicum, which is resistant to S-(2-aminoethyl)-L-cysteine and α-amino-β-hydroxyvalerate and has a nutritional requirement for methionine (Kangogaku Zasshi, et al, Agric. Biol. Chem., 36:1611, 1972).
[4] The known fermentation technique using genetic recombinant strain is exemplified by Japanese Patent Laid-open No. 05-227977, in which disclosed is a method of producing L-threonine, utilizing a recombinant strain of Escherichia coli, of which the threonine operon containing a gene encoding aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase is amplified with a plasmid, and exemplified by a method of amplifying the threonine operon derived from Escherichia coli in a threonine-producing strain, Brevibacterium flavum (TURBA E, et al, Agric. Biol. Chem. 53:2269-2271, 1989). [5] Generally, Escherichia coli proliferation is delicately controlled in response to transition from log phase to stationary phase, and environmental factors such as high/ low temperature, high osmotic pressure, oxygen stress, and nutrient starvation, by the expression of the related genes and proteins, for example, ftsQAZ, dps, poxB, treA, otsBA, proU, and katG genes. In Escherichia coli, a sigma factor of RNA polymerase, rpoS(o ) functions to regulate the expression of the genes. The in vivo level of rpoS is regulated at the levels of transcription, translation, proteolysis or the like (Regine Hengge-Aronis, Micro. Molecul. Biol. Rev., 66: 373-395, 2002). Further, in the fermentation using L-threonine-producing Escherichia coli, the expression of rpoS is considered as a very important factor during culture in accordance with environmental stresses and transition to stationary phase. Disclosure of Invention Technical Problem
[6] Accordingly, the present inventors have made an effort to develop a strain producing high levels of L-threonine by optimal expression of rpoS in L- threonine-producing Escherichia coli. They found that a mutant having an artificial chromosome with a gene encoding an rpoS protein, of which an amino acid sequence has leucine at position 33, can produce L-threonine in a high yield, thereby completing the present invention. Technical Solution
[7] It is an object of the present invention to provide a threonine-producing microorganism transformed with a vector comprising a gene encoding an rpoS protein, the amino acid sequence of which has leucine at position 33.
Advantageous Effects
[8] The present invention provides a threonine-producing microorganism, characterized in that the microorganism is transformed with a vector comprising a gene encoding a mutant rpoS protein (sigma factor, σs) involved in stress response), the amino acid sequence of which has leucine at position 33. Brief Description of the Drawings
[9] Fig. 1 illustrates the construction of an artificial chromosome recombinant vector pccBac-mrpoS, comprising a gene encoding a mutant rpoS protein, the amino acid sequence of which has leucine at position 33. Best Mode for Carrying Out the Invention
[10] In one embodiment, the present invention provides an artificial chromosome recombinant vector, comprising a base sequence encoding an rpoS protein, the amino acid sequence of which has leucine at position 33. Preferably, the base sequence may be represented by SEQ ID NO. 5, and an amino acid sequence of the rpoS protein having leucine at position 33 may be represented by SEQ ID NO. 6.
[11] Further, the present invention provides an artificial chromosome recombinant vector comprising a base sequence that has 95% or more homology with the base sequence of SEQ ID NO. 5 and encodes a protein having the same activity as the rpoS protein, the amino acid sequence of which has leucine at position 33.
[12] Escherichia coli used for producing L-threonine generally has the rpoS protein containing various types of amino acids at position 33. For example, it has been reported that the wild type Escherichia coli MG 1655 has the rpoS protein, the amino acid sequence of which has glutamine (abbreviated to "GIn" or "Q") at position 33, and other Escherichia coli has the rpoS protein, the base sequence of which has an amber stop codon (TAG) or a gene encoding glutamic acid (GAG) or leucine (CTG) instead of glutamine at position 33. The "amber stop codon" means a stop codon with the base sequence TAG on the coding strand in a DNA molecule corresponding to UAG on the mRNA read by this DNA molecule. On the rpoS mRNA containing the amber stop codon, the translation reinitiates at position 54, resulting in a smaller rpoS protein consisting of 277 amino acids with a molecular weight of 30 kDa (T.Atlung, et al, MoI. Genet. Genomics, 266:873-881, 2002). It has been known that Escherichia coli containing the mutant rpoS proteinshows reduced activity, as compared to those containing wild type rpoS, and thus the enzyme activity of catalase (decomposing hydrogen peroxide) is reduced (P.R.Subbarayan, M.Sarkar, MoI. Genet. Genomics, 270:533-538, 2004).
[13] In order to overcome the plasmid instability that is frequently observed in recombinant strains, the present invention is characterized in that an artificial chromosome is used as a recombinant vector instead of using known multi-copy plasmids, since the artificial chromosome is very stable without antibiotics, and maintained in a microorganism as a single copy to avoid side effects due to over- expression of specific genes.
[14] The bacterial artificial chromosome of the present invention (hereinafter, abbreviated to "Bac") may be a vector capable of independent replication in a host cell, that is, a self replication vector. Alternatively, when the vector is introduced into the host cell, the vector may be integrated into the host cell genome and be replicated with the integrated chromosome. Examples of the artificial chromosome may include pBeloBac and pccBac, and preferably pccBac (Copy Control Bacterial Artificial Vector: hereinafter, abbreviated to "pccBac").
[15] In a specific embodiment of the present invention, a mutant rpoS gene containing the base sequence of SEQ ID NO. 5, which encodes the rpoS protein having leucine at position 33, is introduced into the artificial chromosome pccBac to complete a recombinant vector, which is designated as "pccBac-mrpoS" (Fig. 1). [16] In the vector, the DNA sequence should be operably linked to a suitable promoter.
The promoter may be any DNA sequence exhibiting transcriptional activity in a selected host cell, and may be derived from genes encoding proteins that are either homologous or heterologous to the host cell. In particular, examples of the promoter, which regulates the transcription of the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6 in a microbial host, may include the lac operon promoter ofEscherichia coli (E. colϊ) and the dagA promoter of agarose gene of Streptomyces coelicolor.
[17] In one embodiment, the present invention provides a method for fabricating the artificial chromosome recombinant vector, pccBac-mrpoS.
[18] The recombinant vector of the present invention may be fabricated by using various recombination techniques known in the art, for example, DNA recombination technique comprising the steps of (a) introducing the gene fragment comprising the base sequence of SEQ ID NO. 5 or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6 into a cloning vector; (b) performing PCR using the cloning vector as a template and primers of SEQ ID NOs. 3 and 4; and (c) ligating the PCR product to the artificial chromosome vector. Any recombination technique can be employed, as long as it can fabricate the recombinant vector by introducing DNA comprising the base sequence encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, into the artificial chromosome, but is not limited thereto. Any known method may be employed to fabricate the recombinant vector of the present invention.
[19] Further, the recombinant vector of the present invention can be fabricated by a mutagenic technique suitable for replacement of gene or allele. In one specific embodiment of the present invention, the recombinant vector pccBac-mrpoS of the present invention can be fabricated using the DNA recombination technique and site- directed mutagenesis, comprising the steps of (a) preparing a wild type rpoS gene fragment from Escherichia coli chromosome; (b) introducing the gene fragment into the cloning vector; (c) performing PCR using the cloning vector as a template to induce site directed mutation; and (d) ligating the mutated PCR product to the artificial chromosome vector. In step (c), PCR amplification for the induction of site directed mutation may be preferably performed with the primers of SEQ ID NOs. 3 and 4.
[20] In the methods for fabricating the recombinant vector, any cloning vector may be used, preferably pCR2.1-TOPO. In the case of using pCR2.1-TOPO as the cloning vector, the resulting vector was designated as "TOPO2.1-rpoS". The artificial chromosome may be preferably pccBac. In the case of using pccBac as the artificial chromosome vector, the resulting recombinant vector was designated as "pccBac- mrpoS".
[21] In the method for fabricating the recombinant vector pccBac-mrpoS, the wild type rpoS gene fragment derived from Escherichia coli chromosome in step (a) may be prepared by the steps of (D) extracting chromosomal DNA from Escherichia coli to prepare the DNA fragment comprising the rpoS open reading frame and promoter; and (D) performing PCR to amplify the DNA fragment, and PCR may be performed using the primers of SEQ ID NOs. 1 and 2. Further, in step (c), PCR amplification for the induction of site directed mutation may be performed using the primers of SEQ ID NOs. 3 and 4. The Escherichia coli may be a known strain MC4100, but is not limited thereto. Any Escherichia coli can be used, as long as it contains the DNA fragment comprising the rpoS open reading frame and promoter.
[22] In one embodiment, the present invention provides a transformed microorganism prepared by introducing the recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence having 95% or more homology with SEQ ID NO. 5 and encoding the amino acid sequence of SEQ ID NO. 6, into a microbial host.
[23] In another embodiment, the present invention provides a method for preparing the transformed microorganism, comprising the steps of (a) preparing the recombinant vector pccBac-mrpoS; and (b) introducing the vector into the host cell.
[24] Examples of the microbial host to be used in the present invention may include microorganisms belonging to gram negative bacteria, and preferably microorganisms belonging to the species Escherichia coli.
[25] In particular, L-threonine producing strains may be used as the transformed mutant strain harboring the pccBac vector, in which the mutant rpoS is introduced to the artificial chromosome. Examples thereof include microorganisms belonging to Escherichia coli, which is capable of producing L-threonine and has resistance to a L- methionine analogue, methionine auxotroph, resistance to L-threonine analogue, isoleucine-leaky auxotroph, and resistance to an L-lysine analog and α-aminobutyric acid; and mutant microorganisms, in which an endogeneous phosphoenol pyruvate carboxylase (PEPCase) gene (ppc) and genes in the threonine operon and additionally, at least one copy of ppc gene and thrA, thrB, thrc inthe threonine operon are inserted in the chromosomal DNA. Examples of the L-methionine analogue may include at least one selected from the group consisting of D, L-methionine, norleucine, α- methylmethionine, and L-methionine-D,L-sulphoximine. Further, examples of the L- threonine analogue may include at least one selected from the group consisting of α- amino-β-hydroxy valerate and D,L-threonine hydroxamate. Examples of the L-lysine analogue may include at least one selected from the group consisting of S- (2-aminoethyl)-L-cysteine and δ-methyl-L-lysine. Examples of other mutant microorganisms may include microorganisms, in which is inactivated the pckA gene that is involved in the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the biosynthesis of L-methionine; and microorganisms, in which is inactivated the galR gene that inhibits the expression of the galP gene involved in glucose transfer.
[26] Accordingly, in a specific embodiment, the present invention provides a microorganism transformed with the recombinant vector pccBac-mrpoS by using Escherichia coli KCCM 10541 as a host cell (accession number: KCCM10815P, hereinafter designated as "CA030016").
[27]
[28] Further, in one embodiment, the present invention provides a method for producing
L-threonine by culturing the transformed microorganism.
[29] In the production method of L-threonine of the present invention, the cultivation of the transformed microorganism may be conducted in suitable media and under culture conditions known in the art. According to strains used, the culturing procedures can be readily adjusted by those skilled in the art. Examples of the culturing procedures include batch type, continuous type and fed-batch type manners, but are not limited thereto. Various culturing procedures are disclosed in literature, for example, "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp 138-176, 1991).
[30] In one specific embodiment, the production method of L-threonine of the present invention may comprise the steps of (a) culturing the microorganism transformed with the artificial chromosome recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence that has 95% or more homology with SEQ ID NO. 5 and encodes a protein having the same activity as the rpoS protein having leucine at position 33; (b) enriching L-threonine in the broth or microorganisms; and (c) separating residual L-threonine and all or any parts of constituent of the fermentation broth and/or the biomass (>0 to 100). The transformed microorganism may be preferably accession number: KCCMl 0815P.
[31] The media used in the culture method should preferably meet the requirements of a specific strain. Typically culture media contain various carbon sources, nitrogen sources and minerals. Examples of the carbon sources useful in the present invention include carbohydrates such as glucose, fructose, sucrose, lactose, maltose, starch, and cellulose, lipids such as soybean oil, regular sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohol such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources useful in the present invention include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soy bean, and inorganic nitrogen sources such as urea (CO(NH ) ), ammonium sulfate ((NH ) SO ), ammonium chloride (NH Cl),
2 2 4 2 4 4 ammonium phosphate ((NH ) HPO ), ammonium carbonate ((NH ) CO ) and ammonium nitrate (NH NO ). These nitrogen sources may be used alone or in combination. To the media, phosphorus sources such as potassium dihydrogen phosphate (KH PO ), dipotassium hydrogen phosphate (K HPO ) or corresponding sodium-containing salts may be added. In addition, the media may contain metal salts such as magnesium sulfate or ferrous sulfate. Further, the media may be supplemented with amino acids, vitamins, and appropriate precursors. These media or precursors may be added to cultures by a batch type or continuous type method.
[32] During cultivation, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be properly added so as to adjust the pH of the cultures. Defoaming agents such as fatty acid polyglycol ester may be properly added so as to reduce the formation of foams in cultures. To maintain the cultures in aerobic states, oxygen or oxygen-containing gas (e.g., air) may be injected into the cultures. The cultures are maintained at 20 to 450C and preferably at 25 to 4O0C. The cultivation may be continued until a desired amount of L-methionine is obtained, and preferably for 10 to 160 hrs.
[33] The isolation of L-threonine from the culture broth can be performed by the conventional method known in the art. Examples thereof may include centrifugation, filtration, ion-exchange chromatography, and crystallization. For example, the cultures are subjected to low- speed centrifugation to remove the biomass, and the supernatant was separated by ion-exchange chromatography.
[34] As described in the following Examples, it was found that the L- threonine-producing mutant CA030016 of the present invention (accession number: KCCM10815P) that is expressed by introducing the mutant rpoS gene, the amino acid sequence of which has leucine at position 33, into the artificial chromosome is a strain capable of producing L-threonine in a high yield.
[35] Further, in one embodiment, the present invention provides a method for preparing an L-threonine containing feed supplements by mixing L-threonine that is prepared and isolated according to the method of the present invention and conventional feed supplements, and a feed supplement prepared by the same. Specifically, the method for preparing a feed supplement of the present invention comprises the steps of (a) culturing the microorganism transformed with the artificial chromosome recombinant vector, which contains the DNA sequence comprising the base sequence of SEQ ID NO. 5 encoding the rpoS protein, the amino acid sequence of which has leucine at position 33, or the base sequence that has 95% or more homology with SEQ ID NO. 5 and encodes a protein having the same activity as the rpoS protein having leucine at position 33; (b) enriching L-threonine in the broth or microorganisms; (c) separating residual L-threonine and all or any parts of constituent of the fermentation broth and/or the biomass (>0 to 100); and (d) mixing with conventional feed supplements. The transformed microorganism may be preferably accession number: KCCM10815P.
[36] The term "feed" as used herein refers to any natural or artificial dietary, one meal, or ingredients of one meal that is eaten, consumed, and digested by animals, and examples thereof may include concentrated feeds, bulky feeds and/or specialized feeds.
[37] The term "conventional feed supplement" as used herein belongs to the category of feed additives under the Control of Livestock and Fish Feed Act (Korea), and refers to substances that are added to the feed for the purpose of nutrient supplement and weight loss prevention, improvement of dietary fiber digestion, milk quality, and pregnancy rate, and prevention of reproductive disorder and heat stress. The livestock of the present invention refers to livestock being suitable for domestication and increasing farm income, defined by Article 2-1 of the Livestock Industry Act (Korea) and each Section, Article 2 of the administrative regulation (Korea), and examples thereof include cow, horse, mule, donkey, goat, sheep, lamb, deer, swine, rabbit, dog, and poultry such as chicken, turkey, duck, ostrich, goose, and covey, but are not limited thereto. Further, the feed supplement according to the present invention can be processed in a liquid or solid form.
[38]
Mode for the Invention
[39] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
[40]
[41] Example 1. Preparation of recombinant L-threonine producing strain by artificial chromosome vector carrying wild type rpoS gene
[42] Example 1-1. Preparation of wild type rpoS I EcoR I gene fragment
[43] Chromosomal DNA was extracted from Escherichia coli MC4100 strain using a
Genomic-tip system (Qiagen), and polymerase chain reaction (hereinafter, abbreviated to "PCR" was performed using the chromosomal DNA as a template to amplify a DNA fragment of 2357 bp containing the rpoS open reading frame (hereinafter, abbreviated to "ORF" and promoter. In this connection, the used primer set was as represented by SEQ ID NOs. 1 and 2, and the used reaction solution was a PCR HL premix kit (BIONEER Co., hereinafter the same). PCR conditions included 30 cycles of de- naturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min 30 sec.
[44] The PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of the desired size was excised out from the gel.
[45] The rpoS gene fragment obtained from PCR was ligated into a pCR2.1-TOPO vector using a TA cloning kit (Invitrogen Co., hereinafter the same). Then, Escherichia coli Top 10 (Invitrogen Co., hereinafter the same) was transformed with the vector, and smeared on an LB (Lurina-Bertani) solid medium (Bacto tryptone: 10 g/L, Bacto Yeast extract: 5 g/L, sodium chloride: 10 g/L) containing ampicillin (100 mg/L), followed by incubation at 37°C overnight.The rpoS base sequence was confirmed by DNA sequencing analysis (data not shown). The TOPO2.1-rpoS plasmid was treated with a restriction enzyme EcoRI, and then excised from the 0.8% agarose gel to give a DNA fragment of about 2.4 kb, which was designated as "wild type rpoSIEcoRl gene fragment".
[46]
[47] Example 1-2. Construction of recombinant vector
[48] The wild type rpoSIEcoRl gene fragment prepared in Example 1-1 was ligated to the linear artificial chromosome, pccBac vector (EPICENTRE, hereinafter the same) treated with EcoRI using a Rapid DNA ligation kit (ROCHE, hereinafter the same) for 30 min, and then Escherichia coli Top 10 (Invitrogen) was transformed with the vector, and smeared on a LB solid medium containing chrolamphenicol (15 mg/L), followed by incubation at 37°C overnight.
[49] Using a platinum loop, the colonies grown on the solid medium were inoculated in
3 ml of a liquid LB medium containing chloramphenicol (same compositions as LB solid medium of Example 1-1), followed by incubation overnight. Then, plasmid DNA was recovered using a plasmid miniprep kit (QIAGEN, hereinafter the same) to confirm the size of the recombinant vector (data not shown), which was designated as "recombinant vector pccBac-wrpoS".
[50]
[51] Example 2. Preparation of recombinant L-threonine producing strain by artificial chromosome vector carrying amber rpoS gene
[52] Example 2-1. Preparation of amber rpoS I EcoR I gene fragment
[53] Chromosomal DNA was extracted from the threonine producing strain, FTR2533 using a Genomic-tip system (Qiagen), and PCR was performed using the chromosomal DNA as a template to amplify DNA fragment of 2357 bp containing the rpoS ORF and promoter. In this connection, the used primer set was as represented by SEQ ID NOs. 1 and 2, and the used reaction solution was a PCR HL premix kit. PCR conditions included 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min 30 sec.
[54] The PCR product was subjected to electrophoresis on a 0.8% agarose gel, and then a band of the desired size was excised out from the gel.
[55] The rpoS gene fragment obtained from PCR was ligated into a pCR2.1-TOPO vector using a TA cloning kit. Then, Escherichia coli Top 10 was transformed with the vector, and smeared on an LB solid medium containing ampicillin (100 mg/L), followed by incubation at 37°C overnight. The base sequence of amber rpoS was confirmed by DNA sequencing analysis (data not shown).
[56] The TOPO2.1-rpoS plasmid was treated with a restriction enzyme EcoRl, and then excised from the 0.8% agarose gel to give a DNA fragment of about 2.4 kb, which was designated as "amber rpoSIEcoRl gene fragment".
[57]
[58] Example 2-2. Construction of recombinant vector
[59] The amber rpoSIEcoRl gene fragment prepared in Example 2-1 was ligated to the linear artificial chromosome, pccBac vector treated with EcoRl using a Rapid DNA ligation kit for 30 min, and then Escherichia coli Top 10 was transformed with the vector, and smeared on a LB solid medium containing chloramphenicol, followed by incubation at 37°C overnight.
[60] Using a platinum loop, the colonies grown on the solid medium were inoculated in
3 ml of a liquid LB medium containing chloramphenicol, followed by incubation overnight. Then, plasmid DNA was recovered using a plasmid miniprep kit to confirm the size of the recombinant vector (data not shown), which was designated as "recombinant vector pccBac-arpoS".
[61]
[62] Example 3. Preparation of recombinant L-threonine producing strain by artificial chromosome vector carrying mutant rpoS gene having leucine at position 33
[63] Example 3-1. Preparation of mutant rpoS I EcoR I gene fragment
[64] In order to prepare the mutant rpoS genehaving an amino acid sequence with leucine at position 33 (designated as "stop331eu"), PCR was performed using the
TOPO2.1-rpoS vector prepared in Example 1-1 as a template and SEQ ID NOs. 3 and
4 as a primer for site directed mutation (QuikChange Site-Directed Mutagenesis Kit, STRATAGENE). PCR conditions included 18 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 1 min and extension at 68°C for 6 min 30 sec. Then, Escherichia coli Top 10 was transformed with 12 uL of each PCR product, and smeared on a solid medium containing ampicillin, followed by incubation at 37°C overnight.
[65] Using a platinum loop, the colonies grown on the solid medium were inoculated in
3 ml of a liquid LB medium containing ampicillin, followed by incubation overnight. Then, plasmid DNA was recovered using a plasmid miniprep kit, and the rpoS base sequence was confirmed by DNA sequencing analysis (data not shown). The TOPO2.1-rpoS plasmid having an amino acid sequence with leucine at position 33 was treated with a restriction enzyme EcoRI, and then excised from the 0.8% agarose gel to give a DNA fragment of about 2.4 kb, which was designated as "mutant rpoSIEcoRl gene fragment".
[66]
[67] Example 3-2. Construction of recombinant vector pccBac-mrpoS
[68] The mutant rpoSIEcoRl gene fragment, stop331eu prepared in Example 3-1 was ligated to the linear artificial chromosome, pccBac vector treated with EcoRI using a Rapid DNA ligation kit for 30 min, and then Escherichia coli Top 10 was transformed with the vector, and smeared on a solid medium containing chloramphenicol, followed by incubation at 370C overnight.
[69] Using a platinum loop, the colonies grown on the solid medium were inoculated in
3 ml of a liquid LB medium containing chloramphenicol, followed by incubation overnight. Then, plasmid DNA was recovered using a plasmid miniprep kit to confirm the size of the recombinant vector (data not shown), which was designated as "recombinant vector pccBac-mrpoS" (Fig. 1).
[70]
[71] Example 4. Comparison of L-threonine productivity of strains transformed with artificial chromosome vector carrying rpoS gene
[72] Example 4-1. Preparation of transformed strains
[73] Three types of vectors, pccBac-wrpoS, pccBac-arpoS, and pccBac-mrpoS that were prepared in Examples 2-1, 3-1, and 4-1 were introduced into the L-threonine producing strain, KCCM 10541 by electroporation, respectively. Then, each transformed KCCM 10541 was smeared on an LB solid medium containing chloramphenicol to confirm the L-threonine production (Table X).
[74]
[75] Example 4-2. Comparison of L-threonine productivity of transformed strains
[76] The transformed strains prepared in Example 4-1 were cultured in Erlenmeyer flasks containing threonine titer medium of Table 1, and the L-threonine productivity was compared.
[77] Table 1
Figure imgf000013_0001
Figure imgf000014_0001
[78] From KCCM 10541, wild type KCCM10541/pccBac-wrpoS, amber type KCCM10541/pccBac-arpoS, and mutant KCCM10541/pccBac-mrpoS cultured on LB solid media in a 330C incubator overnight, the colonies were inoculated in 25 ml of titer media of Table 1, and then cultured at 330C and 200 rpm for 48 hrs. The results are shown in Table 2.
[79] As shown in Table 2, it was found that in the case of culturing for 48 hrs, the parent strain KCCM10541 produced 22.5 g/L of L-threonine, the amber strain KCCM10541/pccBac-arpoS harboring the amber gene produced 21.0 g/L, the wild type strain KCCM10541/pccBac-wrpoS harboring the wild type gene produced 22.1g/L, and the mutant strain KCCM10541/pccBac-mrpoS harboring the mutant rpoS genewithamino acid sequence havingleucine at position 33 produced 24.9 g/L. Based on the result, in the KCCM10541/pccBac-mrpoS strain transformed with the artificial chromosome vector harboring the mutant rpoS genewithamino acid sequence ofleucine at position 33, the productivity was increased by 2.8 g/L and 2.4g/L, as compared to the strain harboring the wild type rpoS gene and the KCCM 10541 strain used as a control, respectively (Table 2), which indicates that the mutant rpoS gene with amino acid sequence having leucine at position 33 was introduced to increase the L- threonine productivity. The transformed strain was designated as "CA030016", and deposited at the Korean Culture Center of Microorganisms (KCCM) under an accession number of KCCM10815P.
[80] Table 2
Figure imgf000014_0002
[81] It will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope and spirit of the invention. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within meets and bounds of the claims, or equivalents of such meets and bounds are therefore intended to be embraced by the claims.
Industrial Applicability
[82] The microorganism of the present invention can produce L-threonine in a high yield, thereby being used in medicinal, pharmaceutical, and feed industries, in particular, as an animal feed.
[83]
Figure imgf000015_0001
ai f.n d
Figure imgf000015_0002
I ιfϊTN"i iL-rc/vrκ;.N ^" . 1 1 r [ϊiiϊϋejtLV-
u ;,ι;s-(f, i ii jf-, Di IiH1' J J-J "J — )
ill o^1 "y __
Figure imgf000015_0003
[84]

Claims

Claims
[1] A threonine-producing microorganism transformed with a vector comprising a gene encoding an rpoS protein, the amino acid sequence of which has leucine at position 33.
[2] The threonine-producing microorganism according to claim 1, wherein the amino acid sequence having leucine at position 33 is represented by SEQ ID NO. 6.
[3] The threonine-producing microorganism according to claim 1 or 2, wherein the microorganism is E.coli CA030016 (KCCM- 10815P).
[4] A method for producing L-threonine, comprising the steps of culturing the microorganism of claim 1; enriching L-threonine in broth or microorganisms; and separating residual L-threonine and all or any parts of constituent of the fermentation broth and/or the biomass.
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