KR20150035917A - Microorganisms for production of l-threonine and process for producing l-threonine using the same - Google Patents

Abstract

The present invention relates to microorganisms for production of L-threonine and a method for producing L-threonine using the same and, more specifically, to microorganisms having an enhanced ability for production of L-threonine and the method for producing L-threonine using the same at high yield rate.

Description

L-threonine producing microorganism and the production method of L-threonine using the same {MICROORGANISMS FOR PRODUCTION OF L-THREONINE AND PROCESS FOR PRODUCING L-THREONINE USING THE SAME}

The present invention relates to an RNA polymerase sigma-32 factor variant and a method for producing L-threonine using the same.

L-threonine is a kind of essential amino acid and is widely used as a feed and food additive, and as an animal growth promoter, and is also useful as a synthetic raw material for pharmaceuticals and as an infusion solution for medicine.

L- threonine is mainly produced by fermentation using Escherichia coli or coryneform bacteria (Corynebacterium) developed by artificial mutation methods or recombinant methods. Artificial mutants derived from wild-type strains of Escherichia coli, Coryneform bacteria, Serratia and Providencia are widely used for the production of L-threonine.

With the development of gene recombination technology, site-specific gene substitution, gene amplification and deletion were introduced into L-threonine producers developed by random mutation method to develop more improved L-threonine producers. The technologies for this have been reported. Genes involved in the biosynthesis of threonine and methods for increasing their expression have been developed in various ways, but there is still a need for a method capable of producing L-threonine in a higher yield.

The global transcription machinery is responsible for controlling transcriptomes in all cellular systems (prokaryotic and eukaryotic). Global transcription machinery engineering provides a recombinant cell comprising a global transcriptional regulator transformed by mutating a nucleic acid encoding a global regulator, or a promoter that regulates its expression. Cells with an improved phenotype can be produced by the method.

The sigma factor is a kind of global transcriptional regulator that plays an important role in regulating global transcription by focusing on the promoter preference of RNA polymerase holoenzyme. Sigma factor (σ factor) is a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to the promoter of a gene. Different sigma factors are activated in response to different environmental conditions, and all RNA polymerase molecules contain one sigma factor. E. coli has at least eight sigma factors, and it is known that the number of sigma factors depends on the bacterial species.

The present inventors have conducted an improvement study on the rpoH gene encoding sigma-32, which is known to control the heat shock response, with the aim of further enhancing the resistance to stress at high temperatures of E. coli having L-threonine-producing ability. As a result, the present invention was developed by developing an RNA polymerase sigma-32 factor variant that regulates the transcriptional mechanism so that the threonine biosynthesis does not significantly decrease even at high temperatures, and introduced it into a threonine producing strain to produce a threonine producing strain. Completed.

An object of the present invention is to provide an RNA polymerase sigma-32 factor variant.

Another object of the present invention is to provide a nucleotide sequence encoding the RNA polymerase sigma-32 factor variant.

Another object of the present invention is to provide a vector containing the nucleotide sequence.

Another object of the present invention is to provide a microorganism of the genus Escherichia having the ability to produce L-threonine expressing the above variant.

Another object of the present invention is to provide a method for producing L-threonine using the recombinant Escherichia microorganism.

In order to achieve the above object, the present invention provides an RNA polymerase sigma-32 factor variant having the amino acid sequence of SEQ ID NO: 17.

The present invention also provides a nucleotide sequence encoding the RNA polymerase sigma-32 factor variant.

The present invention also provides a recombinant microorganism of the genus Escherichia having the ability to produce L-threonine expressing the above variant.

The present invention also provides a method for producing L-threonine using the recombinant Escherichia microorganism.

The strain transformed with a vector containing a nucleotide sequence encoding an RNA polymerase sigma-32 factor variant resistant to stress caused by high temperature according to the present invention is Not only is temperature resistance imparted, but also the yield of L-threonine is improved, so that significantly higher threonine can be produced than conventional strains even during high temperature culture, so it can be usefully used to produce threonine in high yield.

Hereinafter, the present invention will be described in detail.

The present invention provides an RNA polymerase sigma-32 factor variant having the amino acid sequence set forth in SEQ ID NO: 17.

The terms used in the present invention "sigma ^{32 (sigma-32, σ 32} ) factor" is known as the primary sigma factor that controls the heat shock response in log phase (log phase) as sigma factor design adjustment broadband transfer, by rpoH gene Is coded.

Variants of the present invention are also included in the present invention having homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more with respect to the amino acid sequence of the variant. Homology to the amino acid sequence is determined, for example, by the literature algorithm BLAST [Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Based on this algorithm BLAST, a program called BLASTN or BLASTX has been developed [see: www.ncbi.nlm.nih.gov ]. Variants of the present invention include mutants having deletions, insertions, amino acid substitutions, etc. with respect to the amino acid sequence of the mutants, and also mutants having codon substitutions.

In a preferred embodiment of the invention extracts from the mutant E. coli wild own random mutant DNA pools consisting of the introduced rpoH (random mutation) of W3110 (rpoH mutated DNA pool) by rpoH ^{2 -} was named ^G6. As a result of nucleotide sequence analysis, it was confirmed that mutations in the amino acid sequence occurred compared with the amino acid sequence (SEQ ID NO: 16) of the wild-type RNA polymerase sigma-32 factor (Example 3).

The present invention also provides a nucleotide sequence encoding the variant.

In a preferred embodiment of the present invention, the RNA polymerase sigma-32 factor variant may preferably have a nucleotide sequence of SEQ ID NO: 15.

In a preferred embodiment of the present invention, the nucleotide sequence encoding the variant may have a degenerate genetic code. Genetic code degeneracy is a phenomenon that has no meaning at the end of the letters in the genetic code. If the first two bases are the same, the code in place is the same, even if the code is different.

Accordingly, the nucleotide sequence of the present invention is 80% or more homology, preferably 90% or more homology, more preferably 95% or more homology, and most preferably 99% or more of the nucleotide sequence described in SEQ ID NO: 15. You can have the same sex.

The present invention also provides a vector comprising the nucleotide sequence.

The vector used in the present invention is not particularly limited as long as it is replicable among hosts, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector, and as a plasmid vector, pBR system, pUC system, pBluescript II system , pGEM system, pTZ system, pCL system, pET system, etc. can be used. The vector usable in the present invention is not particularly limited, and a known expression vector may be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, and the like may be used. Most preferably, pACYC177, pACYC184, pCL, pCC1BAC vectors may be used.

The present invention also provides a recombinant microorganism of the genus Escherichia having the ability to produce L-threonine expressing the above variant.

In the present invention, the expression of the variant may be achieved by transforming with a recombinant vector operably containing a gene encoding the variant, or by inserting a polynucleotide encoding the variant into a chromosome, but is not particularly limited thereto. .

In the present invention, the term "transformation" means introducing a vector containing a polynucleotide encoding a protein of interest into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Introduced polynucleotides can be placed within the chromosome of the host cell or outside the chromosome, as long as the introduced polynucleotide can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide structure containing all elements necessary for self-expression. The expression cassette usually includes a promoter operably linked to an open reading frame (hereinafter abbreviated as "ORF") of the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. . The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.

In the present invention, preferably, the transformation is characterized in that a vector containing a nucleotide sequence encoding an RNA polymerase sigma-32 factor variant is transduced or the nucleotide sequence is inserted into a chromosome. Not limited.

The microorganism of the present invention includes any prokaryotic microorganism as long as it can produce L-threonine. For example, Escherichia, Erwinia, Serratia, Providencia, Corynebacterium and Brevibacterium Microbial strains belonging to the genus may be included. Preferably, it is a microorganism belonging to the genus Escherichia, more preferably Escherichia coli .

In a preferred embodiment of the present invention, the present invention may utilize a threonine-producing strain as a parent strain. The threonine-producing strain may be E. coli having methionine demand, resistance to threonine analogs, resistance to lysine analogs, resistance to isoleucine analogs, and resistance to methionine analogs. In addition, the threonine-producing strain may be a recombinant E. coli engineered with a threonine biosynthesis gene, for example, phosphoenol pyruvate carboxylase (ppc) gene and aspartokinease L -Recombinant E. coli with 1 copy of operon each containing homoserine dehydrogenase (thrA), homoserine kinase (thrB), and threonine synthase (thrC). Alternatively, the operon gene (tdcBC) and phosphoenol pyruvate carboxy kinase (PEP carboxykinase, pckA) involved in the L-threonine degradation process may be all inactivated recombinant E. coli.

Preferably, E. coli FTR2533 KCCM10541P (Korean Patent No. 10-0576342), E. coli ABA5G/ pAcscBAR'-M, pC-Ptrc-scrAB (Korean Registration No. 10-1145943) and E. coli KCCM11167P (Korean Patent Laid-Open Patent) No. 10-2012-0083795) can be used.

In a preferred embodiment of the present invention, the microorganism transformed according to the present invention may preferably be E. coli FTR2700.

In a preferred embodiment of the present invention, the microorganism of the present invention may be E. coli having one or more preferred phenotypes for enhancing L-threonine productivity. Preferably, at least one preferred phenotype is a temperature tolerant type.

The present invention also provides a method for producing L-threonine using the transformed strain.

The culturing process of the present invention may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Examples of the culture method include, but are not limited to, batch, continuous, and fed-batch culture.

The medium and other culture conditions used for culturing the microorganisms of the present invention may be any medium used for culturing the microorganisms of the genus Escherichia. However, the requirements of the microorganisms of the present invention must be appropriately satisfied.

As a specific aspect of the present invention, the microorganism of the present invention is cultivated in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. under aerobic conditions while controlling temperature, pH, and the like.

At this time, the carbon source includes carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, and sorbitol, alcohols and organic acids such as sugar alcohol, glycerol, pyruvic acid, lactic acid and citric acid, and amino acids such as glutamic acid, methionine and lysine, Natural organic nutrients such as starch hydrolyzate, molasses, black strap molasses, rice winter, cassava, sugarcane and corn steep liquor can be used, preferably glucose and sterilized pretreated molasses (i.e. molasses converted to reducing sugar) ), etc., and other appropriate carbon sources can be used in various ways without limitation. Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and a corresponding sodium-containing salt as personnel. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used, and other amino acids, vitamins, and appropriate precursors may be included. These media or precursors can be added to the culture in a batch or continuous manner.

The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture in an appropriate manner during the culture. In addition, during cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without the injection of gas to maintain the anaerobic and microaerobic state. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 37°C. The cultivation period can be continued until the production amount of the desired useful substance is obtained, and is preferably 10 to 100 hours.

The L-threonine produced in the cultivation step of the present invention may further include a step of purifying or recovering, and the purification or recovery method may include a method of culturing the microorganism of the present invention, for example, batch, continuous or The desired L-threonine can be purified or recovered from the culture medium using a suitable method known in the art according to a fed-batch culture method or the like.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

< Example >

Example 1: recombinant vector pCC1BAC - rpoH Making of

(One) rpoH Preparation of gene fragment

rpoH Gene (SEQ ID NO: 14, amino acid SEQ ID NO: 16) to obtain a DNA fragment of about 1.0 kb, using Qiagen's Genomic-tip system to extract the chromosomal DNA (gDNA) of the E. coli wild line W3110, and the gDNA A polymerase chain reaction (hereinafter abbreviated as "PCR") was performed using a PCR HL premix kit (manufactured by BIONEER, hereinafter the same) as a template.

PCR for amplifying the rpoH gene consisted of denatured at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute using the primers of SEQ ID NOs: 1 and 2. The cycle was repeated 27 times.

The PCR product was digested with EcoR I to obtain a 1.0 Kb-sized DNA fragment (hereinafter, referred to as “rpoH fragment”) by electrophoresis on a 0.8% agarose gel, followed by elution.

(2) Recombinant vector pCC1BAC - rpoH Making of

Copy control pCC1BAC EcoR I cloning-ready vector (Copycontrol pCC1BAC EcoR I cloning-ready vector; EPICENTRE (USA)) and the rpoH fragment obtained in Example 1- (1) were treated with restriction enzyme EcoR I, respectively, and ligation ( ligation) to prepare a pCC1BAC-rpoH plasmid.

Example 2: recombinant vector pCC1BAC - rpoH of Variant Library creation

(One) error - prone PCR Using rpoH Variant Ready

Introduced random mutations In order to obtain a DNA pool consisting of rpoH variant fragments, the user's manual of the diversify PCR random mutagenesis kit (catalog No. K1830-1) of Clonetech Co., Ltd. as a template using the gDNA of W3110 extracted in Example 1-(1) PCR was performed under the conditions of mutagenesis reactions 4 shown in Table III. PCR was performed 25 times in a cycle consisting of denatured at 94°C for 30 seconds and elongation at 68°C for 1 minute using the primers of SEQ ID NOs: 1 and 2.

The PCR result was digested with EcoR I to obtain a 1.0 Kb-sized DNA fragment (hereinafter referred to as " rpoH ^m fragment") by electrophoresis on a 0.8% agarose gel, followed by elution. .

(2) Recombinant vector pCC1BAC - rpoH of Variant Creation of the library

The copy number control pCC1BAC EcoR I cloning-ready vector was ligated with the rpoH ^m fragment obtained in Example 2-(1) to prepare a pCC1BAC-rpoH ^m vector.

This was transformed into TransforMax EPI300 Electrocompetent E. coli (EPICENTRE (USA)), and LB plate + 15 µg/ml chloramphenicol + 40 µg/ml X-Gal + 0.4mM IPTG It was confirmed that blue colonies did not appear by selection at, and the obtained colonies were collected and plasmid prep was performed to prepare a mutant library of pCC1BAC-rpoH.

(3) pCC1BAC - rpoH of Variant Library Threonine Introduction of production strain

In E. coli KCCM 10541, a threonine-producing strain prepared in a competent state, the mutant library of pCC1BAC-rpoH obtained in Example 1-(2) and pCC1BAC-rpoH obtained in Example 2-(2), respectively Transformed and introduced, the obtained strains were named as KCCM 10541/pCC1BAC-rpoH and KCCM 10541/pCC1BAC-rpoH mutant libraries, respectively.

E. coli KCCM10541, the parent strain used in this example, is resistant to methionine auxotroph, isoleucine lyki-type requirements, and L-threonine analogs (e.g., α-amino-β-hydroxy valeric acid, AHV). , Resistance to L-lysine analogs (e.g. S-(2-aminoethyl)-L-cysteine, AEC), resistance to isoleucine analogs (e.g., α-aminobutyric acid), analogs of methionine (e.g. It is a strain with improved L-threonine-producing ability by inactivating the tyrR gene and galR gene present inside the chromosome of Escherichia coli KCCM 10236, which has characteristics such as resistance to thionine). Korean Patent Registration No. 10-0576342).

Example 3: resistant to temperature RNA Polymerase Sigma-32 Factor Variant Selection

In this example, an experiment was conducted to select an RNA polymerase sigma-32 factor variant that is resistant to temperature.

The E. coli KCCM 10541/pCC1BAC-rpoH and E. coli KCCM 10541/pCC1BAC-rpoH mutant library prepared in Example 2-(3) were cultured overnight in LB solid medium in an incubator at 37°C and 33°C, respectively. Each platinum was inoculated into the 25 mL titer medium shown in Table 1, and then incubated for 48 hours in a shaking incubator at 37°C and 33°C and 200 rpm.

Composition Concentration (per liter) glucose 70 g KH ₂ PO ₄ 2 g (NH ₄ ) ₂ SO ₄ 27.5 g MgSO ₄ 7H ₂ O 1 g FeSO ₄ 7H ₂ O 5 mg MnSO ₄ 4H ₂ O 5 mg DL-methionine 0.15 g Yeast extract 2 g Calcium carbonate 30 g pH 6.8

by culturing them to each of the colonies it was introduced rpoH mutant libraries from 37 ℃ screening the variants that increase the concentration of threonine compared KCCM10541 / pCC1BAC-rpoH, and repeat these evaluation by culturing at 33 ℃ the rpoH mutant library The evaluation was carried out. Through this process, clones to which both temperature tolerance and yield improvement were simultaneously imparted were selected. Extracting vector from the clone pCC1BAC-rpoH ^{2 -} was named ^G6.

The pCC1BAC-rpoH ^{2 -} In order to confirm the variation of the ^G6, can control pCC1BAC EcoR I cloning Cloning - the PCR product sequences were PCR to ^G6 and the resulting ^analysis-pIB provided as a primer for confirmation to the ready vector kit FP (SEQ ID NO: 3) and pIB RP (SEQ ID NO: 4) pCC1BAC-rpoH ² using. Sequence analysis, the mutant of the rpoH rpoH ^{2 - G6} (SEQ ID NO: 15) confirmed that it has the amino acid sequence of SEQ ID NO: 17.

Example 4: Of recombinant strains L- Threonine Productivity compare

Escherichia coli KCCM 10541 / pCC1BAC-rpoH ² to ^G6 transformed into E. coli KCCM 10541 ^{- -} vector pCC1BAC ^rpoH-2 obtained in Example 3 was prepared in the ^G6.

Parent strain E. coli KCCM 10541, Escherichia coli KCCM 10541 / pCC1BAC-rpoH and Escherichia coli KCCM 10541 / pCC1BAC-rpoH ^{2 -} cultured in an Erlenmeyer flask using threonine titer medium of Table 1, respectively to the ^G6 L- threonine Productivity was confirmed. The results are shown in Table 2 below.

Strain L-Threonine (g/L) 33℃ 37℃ KCCM 10541 (parent strain) 30.6 25.0 KCCM 10541/pCC1BAC-rpoH 30.5 26.1 KCCM 10541/pCC1BAC-rpoH ^{2 - G6} 31.1 31.8

As shown in Table 2, the parent strain, E. coli KCCM10541 and the control strain, KCCM10541/pCC1BAC-rpoH, produced 30.6 g/L and 30.5 g/L of L-threonine when cultured for 48 hours. The obtained Escherichia coli KCCM 10541/pCC1BAC-rpoH ^{2 - G6} strain produced 31.1 g/L of L-threonine, showing about 1% improved L-threonine production ability compared to the parent strain.

At 37°C, the yield of the parent strain (KCCM 10541) and the control strain (KCCM10541/pCC1BAC-rpoH) decreased, whereas the KCCM 10541/pCC1BAC-rpoH ^{2 - G6} strain did not show a decrease in concentration according to temperature. By producing 31.8 g/l of L-threonine, it was confirmed that about 8% improved threonine-producing ability was imparted to the control group during high temperature culture.

Example 5: selected rpoH Variant (rpoH ^2-G6 )of Threonine Comparison of effects by production strain

(One) ABA5G / pAcscBAR' -M, pC - Ptrc - scrAB In strain rpoH Variant ( rpoH ^{2 - G6} ) Check the effect

Example 4 The effect of the confirmation vector pCC1BAC-rpoH ^{2 -} by introducing a write to ^G6 Leo non-producing strain of ABA5G / pAcscBAR'-M, pC- Ptrc-scrAB ( Republic of Korea Patent No. 10-1145943 No.) ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pCC1BAC- rpoH ^{2 - G6} were prepared, and titer evaluation was performed by preparing a titer medium as shown in Table 3 below. The results are shown in Table 4 below.

The parent strains ABA5G/ pAcscBAR'-M and pC-Ptrc-scrAB used in this example are the threonine-producing strain ABA5G strain (a strain produced by induction of NTG mutation of the parent strain, E. coli W3110, and methionine requirement , Isoleucine leaky, α-amino-β-hydroxy bareric acid resistance, 2-aminoethyl-1-cysteine resistance, 1-azetidine-2-carboxylic acid resistance) in pAcscBAR'-M gene group And pC-Ptrc-scrAB gene group.

Composition Concentration (per liter) Wondang 70 g KH ₂ PO ₄ 2 g (NH ₄ ) ₂ SO ₄ 27.5 g MgSO ₄ 7H ₂ O 1 g FeSO ₄ 7H ₂ O 5 mg MnSO ₄ 4H ₂ O 5 mg DL-methionine 0.15 g Yeast extract 2 g Calcium carbonate 30 g pH 6.8

Strain L-Threonine (g/L) 33℃ 37℃ ABA5G/ pAcscBAR'-M, pC-Ptrc-scrAB 22.8 19.8 ABA5G/ pAcscBAR'-M, pC-Ptrc-scrAB, pCC1BAC- rpoH ^{2 - G6} 22.8 22.0

As shown in Table 4 above, even when introduced into a threonine-producing strain other than E. coli KCCM10541, introduction of the vector pCC1BAC-rpoH ^2-G6 similarly to that confirmed in Table 2 above results in a threonine yield at 33°C. It was maintained, and it was confirmed that the threonine yield was maintained at a level similar to that of 33℃ even at 37℃.

(2) E. coli KCCM11167P In strain rpoH Variant ( rpoH ^{2 - G6} ) Check the effect

E. coli KCCM11167P by introducing the ^{vector pCC1BAC-rpoH 2 - G6} into another threonine producing strain, E. coli KCCM11167P (Korean Patent Application No. 2011-0005136). /pCC1BAC-rpoH ^{2 -G6} was prepared, and the titer medium of Table 1 was prepared to evaluate the threonine production ability. The results are shown in Table 5 below.

E. coli KCCM11167P, the parent strain used in this example, is a strain that inactivates tdcB in KCCM10541 strain (Korean Patent Registration No. 10-0576342) and enhances NAD kinase activity by enhancing nadK by 2 copies.

Strain L-Threonine (g/L) 33℃ 37℃ KCCM11167P 30.1 26.2 KCCM11167P/pCC1BAC- rpoH ^{2 - G6} 30.2 29.8

^{Looking at the results of the titer evaluation in Table 5, when pCC1BAC-rpoH 2-G6} is introduced into a threonine-producing strain, as when introduced into other threonine-producing strains as in 5-(1), 33 It was confirmed that the threonine yield was maintained at °C, and the threonine yield was maintained similar to the yield at 33 °C at 37 °C.

Example 6: Selected rpoH Variant (rpoH ^2-G6 )of Further insertion into the chromosome

(One) rpoH ^{2 - G6} Cassette for insertion ( integration cassette ) Preparation of the short story

The rpoH ² screening in Example ^{3-a G6} variants were produced linear insertion cassette (linear integration cassette) for insertion in order to add to the chromosome. The cassette for linear insertion was manufactured according to the following method.

PCR was performed using E. coli W3110 gDNA as a template using the primers of SEQ ID NOs: 5 and 6. PCR was repeated 27 times of a cycle consisting of denatured at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 30 seconds. The DNA fragment thus obtained was designated as a homologous region 1.

^{PCR was performed to amplify the mutant loxP-Cm r} -loxP cassette using the primers of SEQ ID NOs: 7 and 8 as a template, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and 1 at 72°C. The cycle consisting of elongation of minutes was repeated 27 times. The pMloxCmt used as a template here is a vector produced by a person skilled in the art by applying the content that Suzuki et al. reported about an improved gene deletion method using mutant loxP named lox71 and lox66 (Suzuki N. et al. al ., Appl. Environ. Microbiol. 71:8472, 2005).

The homologous region 1 and the mutant loxP-Cm ^r -loxP cassette portion thus obtained were subjected to overlap extension PCR using SEQ ID NOs: 5 and 8 to obtain a homologous region 1-mutant loxP-Cm ^r -loxP. At this time, PCR was repeated 5 times a cycle consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute and 30 seconds in the absence of a primer, and then adding primers and performing 23 additional cycles. I did.

By using primers of SEQ ID NOS: 9 and 10 pCC1BAC-rpoH ^{2 -} to the ^G6 as the template, PCR was performed. A cycle consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute was repeated 27 times.

PCR was performed using E. coli W3110 gDNA as a template using primers of SEQ ID NOs: 11 and 12. A cycle consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and stretching at 72°C for 30 seconds was repeated 27 times. The DNA fragment thus obtained was designated as a homologous region 2.

RpoH ² above ^- the portion of the ^G6 and homologous region 2 by using the SEQ ID NO: 9 and 12 to perform the overlap extension PCR rpoH ^{2 -} to give a ^G6 -homologus region 2. At this time, PCR was repeated 5 times a cycle consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute and 30 seconds in the absence of a primer, and then adding primers and performing 23 additional cycles. I did.

To ^G6 to the -homologus region 2 as the template by using the SEQ ID NO: 5 and 12 perform the ^{overlap extension PCR rpoH 2 - - homologus} region 1-mutant loxP-Cm r -loxP, rpoH 2 obtained above to prepare a cassette for insertion ^G6 . At this time, PCR was repeated 5 times a cycle consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 3 minutes in the absence of a primer, and then the primer was added and 23 cycles were additionally performed. . Through this process rpoH ^2, such as SEQ ID NO: 13 ^- to prepare a cassette for insertion ^G6.

(2) On chromosomes rpoH ^{2 - G6} Mutant Preparation of additionally inserted recombinant strains

Selected rpoH group on chromosome ^{2 - G6} The embodiment in E. coli KCCM10541 to insert a variant example 6 (1) ² produced in the rpoH ^{- G6} insert separating the cassette DNA fragment to give the active step of the known fire for (Warner et al, PNAS, 6 ; 97 (. 12): the portion of the chromosome 6640, the existing inherent rpoH back in the same manner as in 2000) rpoH ^{2 - G6} Additional variants were inserted. After that, the antibiotic resistance marker gene was removed to obtain rpoH ^{2 - G6} A strain into which the variant was additionally inserted was prepared, and it was confirmed that no PCR error was introduced through nucleotide sequence analysis. The fabricated rpoH ^{2 -} was added to the ^G6 insertion strain named FTR2700, the transformed Escherichia coli FTR2700 On February 5, 2013, it was deposited with the Korean Culture Center of Microorganisms (hereinafter abbreviated as "KCCM") (accession number KCCM11368P).

(3) L- Threonine Productivity check

The recombinant microorganism prepared in 6-(2) was cultured in an Erlenmeyer flask using the threonine titer medium of Table 1 to confirm L-threonine productivity.

E. coli KCCM 10541 and E. coli KCCM11368P cultured overnight in LB solid medium in an incubator at 33° C. and 37° C. were inoculated into the 25 mL titer medium of Table 1, respectively, and then inoculated at 33° C. and 37° C. 200 rpm. Incubated for 48 hours in a shaking incubator of.

As shown in Table 6 below, when the parent strain, E. coli KCCM10541 strain, was cultured for 48 hours, 30.3 g/L of L-threonine was produced, and KCCM 11368P produced in Example 6-(2) of the present invention The E. coli strain produced 30.0 g/L of L-threonine, showing similar L-threonine productivity to the parent strain. At 37℃, the yield of the parent strain (KCCM 10541) decreased, while KCCM 11368P The strain did not show a decrease in concentration according to the temperature and showed a pattern of increasing the concentration.

Accordingly, it was confirmed that the threonine-producing ability at a level similar or higher than that of 33°C culture was given even at 37°C in the same manner as the transformed strain by introducing it in the form of a vector.

Strain L-Threonine (g/L) 33℃ 37℃ KCCM 10541 (parent strain) 30.3 26.0 KCCM 11368P 30.0 32.0

From the above description, those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it is to be understood that the examples and experimental examples described herein are illustrative in all respects and do not limit the scope of the present invention. The scope of the present invention should be construed as including the meaning and scope of the claims to be described later rather than the above-described detailed description, and all changes or modified forms derived from the concept of equivalents thereof to be included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11368P 20130205

<110> CJ Cheiljedang Corporation <120> Microorganisms for production of L-threonine and process for producing L-threonine using the same <130> PA15-0125 <160> 17 <170> KopatentIn 2.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cagtatccgg aattcgcttg cattgaactt gtgga 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtcataccgg aattccttaa tagcggaaat tacgc 35 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cagtatccgg aattcgcttg cattgaactt gtgga 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtcataccgg aattccttaa tagcggaaat tacgc 35 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atctagaaag cgcagcgcaa actgttc 27 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttcgtataat gtatgctata cgaacggtaa ccccggactc tcatccaggg 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gcagagaacc ctggatgaga gtccggggtt accgttcgta tagcatacat 50 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtgattttat ccacaagttc aatgcaagcg gtacctaccg ttcgtataat 50 <210> 9 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tagcatacat tatacgaacg gtaggtaccg cttgcattga acttgtggat 50 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 catccagggt tctctgctta atagcggaaa ttacgcttca atggcagcac gc 52 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaaattgcg tgctgccatt gaagcgtaat ttccgctatt aagcagagaa 50 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aaagctttgt ttcgggtcac aggcatcg 28 <210> 13 <211> 3136 <212> DNA <213> Artificial Sequence <220> <223> rpoH2-G6 integration cassette <400> 13 aagcgcagcg caaactgttc ttcaacctgc gtaaaaccaa gcagcgtctg ggctggttta 60 accaggatga agtcgaaatg gtggcccgtg aactgggcgt aaccagcaaa gacgtacgtg 120 agatggaatc acgtatggcg gcacaggaca tgacctttga cctgtcttcc gacgacgatt 180 ccgacagcca gccgatggct ccggtgctct atctgcagga taaatcatct aactttgccg 240 acggcattga agatgataac tgggaagagc aggcggcaaa ccgtctgacc gacgcgatgc 300 agggtctgga cgaacgcagc caggacatca tccgtgcgcg ctggctggac gaagacaaca 360 agtccacgtt gcaggaactg gctgaccgtt acggcgtttc cgctgagcgt gtacgccagc 420 tggaaaagaa cgcgatgaaa aaattgcgtg ctgccattga agcgtaattt ccgctattaa 480 gcagagaacc ctggatgaga gtccggggtt aggtgacact atagaacgcg gccgccagct 540 gaagctttac cgttcgtata gcatacatta tacgaagtta tctgccctga accgacgacc 600 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt 660 agcaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca 720 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacagac 780 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt 840 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact 900 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg 960 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg 1020 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa 1080 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat 1140 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa 1200 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg 1260 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg 1320 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc 1380 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa 1440 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt 1500 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg 1560 tatttattcg gcgcaaagtg cgtcgggtga tgcataactt cgtatagcat acattatacg 1620 aacggtaccc atcagatcca ctagcttgca ttgaacttgt ggataaaatc acggtccgat 1680 aaaacaatga atgataacct cgttgctctt aagctctggc acagttgttg ctaccactga 1740 agcgccagaa gatatcgatt gagaggattt gaatgactga caaaatgcaa agtttagctt 1800 tagccccagt tggcaacctg gattcctaca tccgggcagc taacgcgtgg ccgatgttgt 1860 cggctgacga ggagcgggcg ctggctgaaa agctgcatta ccatggcgat ctggaagcag 1920 ctaaaacgct gatccagtct cacctgcggt ttgttgttca tattgctcgt aattatgcgg 1980 gctatggcct gccacaggcg gatttgattc aggaaggtaa catcggcctg atgaaagcag 2040 tgcgccgttt caacccggaa gcgggtgtgc gcctggtctc cttcgccgtt cactggatca 2100 aagcagagat ccacgaatac gttctgcgta actggcgtat cgtcaaagtt gcgaccacca 2160 aagcgcagcg caaactgttc ttcaacctgc gtaaaaccaa gcagcgtctg ggctggttta 2220 accaggatga agtcgaaatg gtggcccgtg aactgggcgt aaccagcaaa gaagtacgtg 2280 agatggaatc acgtatggcg gcacaggaca tgacctttga cctgtcttcc gacgacgatt 2340 ccgacagcca gccgatggct ccggtgctct atctgcagga taaatcatct aactttgccg 2400 acggcattga agatgatatc tgggaagagc aggcggcaaa ccgtctgacc gacgcgatgc 2460 agggtctgga cgaacgcagc caggacatca tccgtgcgcg ctggctggac gaagacaaca 2520 agtccacgtt gcaggaactg gctgaccgtt acggcgtttc cgctgagcgt gtccgccagc 2580 tggaaaagaa cgcgatgaaa aaattgcgtg ctgccattga agcgtaattt ccgctattaa 2640 gcagagaacc ctggatgaga gtccggggtt tttgtttttt gggcctctgt aataatcaat 2700 ttcccctccg gcaaaacgcc aatccccacg cagattgtta ataaactgtc aaaatagcta 2760 ttccaatatc ataaaaatcg ggtatgtttt agcagagtat gctgctaaag cacgggtagt 2820 catgcataaa acgaaataaa gtgctgaaaa acaacatcac aacacacgta ataaccagaa 2880 gaatggggat tctcaggatg aacataaagg gtaaagcgtt actggcagga tgtatcgcgc 2940 tggcattcag caatatggct ctggcagaag atattaaagt cgcggtcgtg ggcgcaatgt 3000 ccggtccggt tgcgcagtac ggtgaccagg agtttaccgg cgcagagcag gcggttgcgg 3060 atatcaacgc taaaggcggc attaaaggca acaaactgca aatcgtaaaa tatgacgatg 3120 cctgtgaccc gaaaca 3136 <210> 14 <211> 984 <212> DNA <213> Escherichia coli <400> 14 gcttgcattg aacttgtgga taaaatcacg gtctgataaa acagtgaatg ataacctcgt 60 tgctcttaag ctctggcaca gttgttgcta ccactgaagc gccagaagat atcgattgag 120 aggatttgaa tgactgacaa aatgcaaagt ttagctttag ccccagttgg caacctggat 180 tcctacatcc gggcagctaa cgcgtggccg atgttgtcgg ctgacgagga gcgggcgctg 240 gctgaaaagc tgcattacca tggcgatctg gaagcagcta aaacgctgat cctgtctcac 300 ctgcggtttg ttgttcatat tgctcgtaat tatgcgggct atggcctgcc acaggcggat 360 ttgattcagg aaggtaacat cggcctgatg aaagcagtgc gccgtttcaa cccggaagtg 420 ggtgtgcgcc tggtctcctt cgccgttcac tggatcaaag cagagatcca cgaatacgtt 480 ctgcgtaact ggcgtatcgt caaagttgcg accaccaaag cgcagcgcaa actgttcttc 540 aacctgcgta aaaccaagca gcgtctgggc tggtttaacc aggatgaagt cgaaatggtg 600 gcccgtgaac tgggcgtaac cagcaaagac gtacgtgaga tggaatcacg tatggcggca 660 caggacatga cctttgacct gtcttccgac gacgattccg acagccagcc gatggctccg 720 gtgctctatc tgcaggataa atcatctaac tttgccgacg gcattgaaga tgataactgg 780 gaagagcagg cggcaaaccg tctgaccgac gcgatgcagg gtctggacga acgcagccag 840 gacatcatcc gtgcgcgctg gctggacgaa gacaacaagt ccacgttgca ggaactggct 900 gaccgttacg gcgtttccgc tgagcgtgta cgccagctgg aaaagaacgc gatgaaaaaa 960 ttgcgtgctg ccattgaagc gtaa 984 <210> 15 <211> 984 <212> DNA <213> Escherichia coli <400> 15 gcttgcattg aacttgtgga taaaatcacg gtccgataaa acaatgaatg ataacctcgt 60 tgctcttaag ctctggcaca gttgttgcta ccactgaagc gccagaagat atcgattgag 120 aggatttgaa tgactgacaa aatgcaaagt ttagctttag ccccagttgg caacctggat 180 tcctacatcc gggcagctaa cgcgtggccg atgttgtcgg ctgacgagga gcgggcgctg 240 gctgaaaagc tgcattacca tggcgatctg gaagcagcta aaacgctgat ccagtctcac 300 ctgcggtttg ttgttcatat tgctcgtaat tatgcgggct atggcctgcc acaggcggat 360 ttgattcagg aaggtaacat cggcctgatg aaagcagtgc gccgtttcaa cccggaagcg 420 ggtgtgcgcc tggtctcctt cgccgttcac tggatcaaag cagagatcca cgaatacgtt 480 ctgcgtaact ggcgtatcgt caaagttgcg accaccaaag cgcagcgcaa actgttcttc 540 aacctgcgta aaaccaagca gcgtctgggc tggtttaacc aggatgaagt cgaaatggtg 600 gcccgtgaac tgggcgtaac cagcaaagaa gtacgtgaga tggaatcacg tatggcggca 660 caggacatga cctttgacct gtcttccgac gacgattccg acagccagcc gatggctccg 720 gtgctctatc tgcaggataa atcatctaac tttgccgacg gcattgaaga tgatatctgg 780 gaagagcagg cggcaaaccg tctgaccgac gcgatgcagg gtctggacga acgcagccag 840 gacatcatcc gtgcgcgctg gctggacgaa gacaacaagt ccacgttgca ggaactggct 900 gaccgttacg gcgtttccgc tgagcgtgtc cgccagctgg aaaagaacgc gatgaaaaaa 960 ttgcgtgctg ccattgaagc gtaa 984 <210> 16 <211> 284 <212> PRT <213> Escherichia coli <400> 16 Met Thr Asp Lys Met Gln Ser Leu Ala Leu Ala Pro Val Gly Asn Leu 1 5 10 15 Asp Ser Tyr Ile Arg Ala Ala Asn Ala Trp Pro Met Leu Ser Ala Asp 20 25 30 Glu Glu Arg Ala Leu Ala Glu Lys Leu His Tyr His Gly Asp Leu Glu 35 40 45 Ala Ala Lys Thr Leu Ile Leu Ser His Leu Arg Phe Val Val His Ile 50 55 60 Ala Arg Asn Tyr Ala Gly Tyr Gly Leu Pro Gln Ala Asp Leu Ile Gln 65 70 75 80 Glu Gly Asn Ile Gly Leu Met Lys Ala Val Arg Arg Phe Asn Pro Glu 85 90 95 Val Gly Val Arg Leu Val Ser Phe Ala Val His Trp Ile Lys Ala Glu 100 105 110 Ile His Glu Tyr Val Leu Arg Asn Trp Arg Ile Val Lys Val Ala Thr 115 120 125 Thr Lys Ala Gln Arg Lys Leu Phe Phe Asn Leu Arg Lys Thr Lys Gln 130 135 140 Arg Leu Gly Trp Phe Asn Gln Asp Glu Val Glu Met Val Ala Arg Glu 145 150 155 160 Leu Gly Val Thr Ser Lys Asp Val Arg Glu Met Glu Ser Arg Met Ala 165 170 175 Ala Gln Asp Met Thr Phe Asp Leu Ser Ser Asp Asp Asp Ser Asp Ser 180 185 190 Gln Pro Met Ala Pro Val Leu Tyr Leu Gln Asp Lys Ser Ser Asn Phe 195 200 205 Ala Asp Gly Ile Glu Asp Asp Asn Trp Glu Glu Gln Ala Ala Asn Arg 210 215 220 Leu Thr Asp Ala Met Gln Gly Leu Asp Glu Arg Ser Gln Asp Ile Ile 225 230 235 240 Arg Ala Arg Trp Leu Asp Glu Asp Asn Lys Ser Thr Leu Gln Glu Leu 245 250 255 Ala Asp Arg Tyr Gly Val Ser Ala Glu Arg Val Arg Gln Leu Glu Lys 260 265 270 Asn Ala Met Lys Lys Leu Arg Ala Ala Ile Glu Ala 275 280 <210> 17 <211> 284 <212> PRT <213> Escherichia coli <400> 17 Met Thr Asp Lys Met Gln Ser Leu Ala Leu Ala Pro Val Gly Asn Leu 1 5 10 15 Asp Ser Tyr Ile Arg Ala Ala Asn Ala Trp Pro Met Leu Ser Ala Asp 20 25 30 Glu Glu Arg Ala Leu Ala Glu Lys Leu His Tyr His Gly Asp Leu Glu 35 40 45 Ala Ala Lys Thr Leu Ile Gln Ser His Leu Arg Phe Val Val His Ile 50 55 60 Ala Arg Asn Tyr Ala Gly Tyr Gly Leu Pro Gln Ala Asp Leu Ile Gln 65 70 75 80 Glu Gly Asn Ile Gly Leu Met Lys Ala Val Arg Arg Phe Asn Pro Glu 85 90 95 Ala Gly Val Arg Leu Val Ser Phe Ala Val His Trp Ile Lys Ala Glu 100 105 110 Ile His Glu Tyr Val Leu Arg Asn Trp Arg Ile Val Lys Val Ala Thr 115 120 125 Thr Lys Ala Gln Arg Lys Leu Phe Phe Asn Leu Arg Lys Thr Lys Gln 130 135 140 Arg Leu Gly Trp Phe Asn Gln Asp Glu Val Glu Met Val Ala Arg Glu 145 150 155 160 Leu Gly Val Thr Ser Lys Glu Val Arg Glu Met Glu Ser Arg Met Ala 165 170 175 Ala Gln Asp Met Thr Phe Asp Leu Ser Ser Asp Asp Asp Ser Asp Ser 180 185 190 Gln Pro Met Ala Pro Val Leu Tyr Leu Gln Asp Lys Ser Ser Asn Phe 195 200 205 Ala Asp Gly Ile Glu Asp Asp Ile Trp Glu Glu Gln Ala Ala Asn Arg 210 215 220 Leu Thr Asp Ala Met Gln Gly Leu Asp Glu Arg Ser Gln Asp Ile Ile 225 230 235 240 Arg Ala Arg Trp Leu Asp Glu Asp Asn Lys Ser Thr Leu Gln Glu Leu 245 250 255 Ala Asp Arg Tyr Gly Val Ser Ala Glu Arg Val Arg Gln Leu Glu Lys 260 265 270 Asn Ala Met Lys Lys Leu Arg Ala Ala Ile Glu Ala 275 280

Claims (8)

An RNA polymerase sigma-32 factor mutant comprising the amino acid sequence of SEQ ID NO: 17. A nucleotide sequence encoding the mutant of claim 1 represented by SEQ ID NO: 15. A vector comprising the nucleotide sequence of claim 2. A recombinant Escherichia genus microorganism having L-threonine producing ability expressing the mutant of claim 1. 5. The method of claim 4,
The expression may be expressed by introducing a recombinant vector containing a nucleotide sequence encoding an RNA polymerase sigma-32 factor mutant containing the amino acid sequence of SEQ ID NO: 17, Wherein the recombinant Escherichia genus is L-threonine-producing microorganism. 6. The method of claim 5,
The recombinant Escherichia genus microorganism having L-threonine producing ability, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 15. 6. The method of claim 5,
The Escherichia genus microorganism is Escherichia that the coli) which is characterized, L- threonine recombinant microorganism of the genus Escherichia having the production capacity. A method for producing L-threonine comprising culturing a recombinant Escherichia genus microorganism having L-threonine producing ability according to any one of claims 4 to 7 and separating L-threonine from the culture liquid, Nin production method.