WO2003097839A1

WO2003097839A1 - Nucleotide sequence of threonine operon irrepressible by isoleucine and method for producing l-threonine using transformed host cell containing the same

Info

Publication number: WO2003097839A1
Application number: PCT/KR2003/000955
Authority: WO
Inventors: Hee-Jong Lee; Seong-Jun Kim; Jin-Suck Sung; Seok-Won Song; Young-Hoon Park
Original assignee: Cj Corporation
Priority date: 2002-05-15
Filing date: 2003-05-15
Publication date: 2003-11-27
Also published as: CN1653180A; KR100459758B1; US7288393B2; CN1297665C; US20050221449A1; AU2003230344A1; KR20030088781A

Abstract

A nucleotide sequence of the threonine operon of E.coli with a deletion of all or part of a nucleotide fragmentof -56 to -18, a recombinant vector containing the above nucleotide sequence, and a transformed host cell containing the recombinant vector are provided. A method for producing L-threonine comprising culturingthe transformed host cell is also provided.

Description

NUCLEOTIDE SEQUENCE OF THREONINE OPERON

IRREPRESSIBLE BY ISOLEUCINE AND METHOD FOR PRODUCING

L-THREONINE USING TRANSFORMED HOST CELL CONTAINING

THE SAME

Technical Field

The present invention relates to a nucleotide sequence of the threonine operon of Escherichia coli with a deletion of all or part of a nucleotide fragment of -56 to -18, a recombinant vector containing the above nucleotide sequence, and a transformed host cell containing the recombinant vector. The present invention also provides a method for producing L-threonine comprising culturing the transformed host cell.

Background Art L-threonine is an essential amino acid and is widely used as a feed or food additive. In addition, L-threonine is used as a medical solution or a raw material for a drug synthesis.

L-threonine is produced by a fermentation process using a mutant strain derived from a wild-type strain of Escherichia coli (E.coli), Corynebacteria sp., Serratia sp., or Providencia sp. Examples of the mutant strain include an amino acid analogue- or drug-resistant mutant strain, or a variety of auxotroph to diaminopimeric acid, methionine, lysine, or isoleucine [Japanese Patent Laid-Open Publication No. Hei. 2-219582; Appl. Microbiol. Biotechnol., 29. 550-553 (1988); Korean Patent No. 1992-8365].

A fermentation process using a recombinant strain can also be used to produce L-threonine. Japanese Patent Laid-Open Publication No. Hei. 5-10076 discloses a method for producing threonine in large scale using a recombinant strain of Serratia sp. containing a DNA fragment with genetic information of aspartokinase, homoserine kinase, homoserine dehydrogenase, and threonine synthase. In addition, a method for mass-producing L-threonine using a gene derived from a strain of Providencia sp. resistant to antagonist of methionine metabolism is disclosed in Japanese Patent Laid-Open Publication No. Hei. 1-289493.

As a method for producing threonine by regulation of expression of threonine operon, replacing a promoter of the threonine operon with tac promoter (WO 98/04715) and replacing an expression regulatory region of the threonine operon with cl repressor and PR promoter of E. coli λ phage (EP 059.3792B1 ) are disclosed.

The threonine biosynthesis operon of E.coli is composed of genes, thrA, thrB, and thrC. The thrA gene encodes for aspartokinase and homoserine dehydrogenase, the thrB gene encodes for homoserine kinase, and the thrC gene encodes for threonine synthase. A leader sequence and attenuator of these genes precedes the threonine operon [Proc. Natl. Acad. Sci. America (1979), 76: 1706-1710]. One example of the nucleotide sequence of the threonine operon comprising the leader sequence, the attenuator, and the structural thr genes is as set forth in SEQ ID NO: 1. The ATG codon of nucleotide positions 337-339 of SEQ ID NO: 1 is a start codon for the threonine operon. In particular, the expression of the threonine operon is regulated by the intracellular levels of both threonine and isoleucine. This is similar to the regulatory mechanism of the tryptophan operon [J. Bacteriology (1975), 161-166].

Disclosure of the Invention

For the threonine-producing strain that is not repressed by threonine or isoleucine, the present inventors have achieved the goals of the present invention by providing the mutant threonine operon which contains a defective attenuator region.

Therefore, the present invention is to provide a nucleotide sequence of the threonine operon that is not repressible by isoleucine.

The present invention is also to provide a recombinant vector containing the above nucleotide sequence of the threonine operon.

The present invention is also to provide a transformed host cell containing the recombinant vector.

The present invention is further to provide a method for producing L-threonine comprising culturing the transformed host cell.

According to an aspect of the present invention, there is provided a nucleotide sequence of the threonine operon of E. coli with a deletion of all or part of a 39 bp nucleotide fragment of -56 to -18 that is attenuator of the structural genes of the threonine operon. In this case, the nucleotide sequences in an upstream region from the A of the start codon, ATG (+1 ) are numbered as negative numbers, and the nucleotide sequences in a downstream region as positive numbers. Unless specified otherwise, the nucleotide numbering is as defined in the above. According to another aspect of the present invention, there is provided a recombinant vector containing the above nucleotide sequence of the threonine operon of E.coli. The recombinant vector can be manufactured by a conventional method comprising digesting the threonine operon of E.coli and a suitable vector with restriction enzymes such as Apal and Pstl, followed by ligation. The suitable vector may be phage, plasmid, and cosmid, for examples. Examples of the phage and cosmid vectors include pWE15, M13, λ EMBL3, λ EMBL4, λ FIXII, λ- DASHII, λ ZAPII, λ. gt10, λ. gt11 , Charon4A, and Charon21A. Examples of the plasmid vector include pBR series, pUC series, pBluescriptll series, pGEM series, pTZ series, and pET series. In addition, various shuttle vectors that can replicate in a variety of host cells such as E.coli and Corynebacteria may be used. Preferably, pECCG122 (KFCC 10696), which has been deposited the Korean Federation of Culture Collection (KFCC) on June 20, 1990, is used as the cloning vector.

According to another aspect of the present invention, there is provided a novel microorganism containing the above recombinant vector. The microorganism may be manufactured by transforming a host cell with the recombinant vector using a conventional method [Sambrook, J. et al., 2th (1989), Cold Spring Harbor Laboratory Press].

A host cell may be gram-negative bacteria. Cells belong to the genus Escherichia sp. are most preferred. In particular, according to the preferred embodiment of the present invention, E.coli KCCM 10236 (Korean Patent Application No. 2001-6976) is used as a host cell. The E.coli KCCM 10236 is an auxotroph for methionine and is resistant to threonine analogue (AHV: α -amino-β -hydroxyvaleric acid), lysine analogue (AEC: S-(2-aminoethyl)-L-cysteine), isoleucine analogue (α -aminobutyric acid), and methionine analogue (ethionine). In addition, the E.coli KCCM 10236 has an extra copy of phosphoenol pyruvate carboxylase gene in a chromosomal DNA and at least one of the thrA, thrB, and thrC genes is present in an amplified state, thereby its productivity of L-threonine is increased. In the present invention, the effects of the above recombinant vector were confirmed by using Escherichia coli KFCC 10236 as a host cell. The Escherichia coli KFCC 10236 has been deposited in the Korean Federation of Culture Collection (KFCC) on December 29, 2000.

According to yet another aspect of the present invention, there is provided a method for producing L-threonine comprising culturing the transformed host cell and recovering L-threonine from the culture. Preferably, E.coli KCCM 10236/pTHR(+) is used as the transformed host cell.

The method for producing L-threonine of the present inveniton may be carried out under a conventional condition. Although the intracellular levels of isoleucine and threonine are high, the production of L-threonine is not inhibited. Thereby the productivity of L-threonine is increased.

Hereinafter, the present invention will be described more specifically by examples. However, the following examples are provided only for illustrations and thus the present invention is not limited to or by them.

Examples

Example 1 showed correlation between isoleucine concentration in a culture medium and L-threonine biosynthesis using a conventional L-threonine-producing strain. The nucleotide sequence of the threonine operon (hereinafter, simply referred to as "the thr operon") was cloned from the strain of Example 1 (Example 2). Then, mutation was induced on the pTHR (-) vector containing the cloned thr operon (Example 3) and strains of which the expression of the thr operon cannot be repressed by isoleucine and threonine were selected (Example 4). Finally, in Example 5, the thr operon of the selected strains was sequenced. As a result, a part of the attenuator in the thr operon was deleted.

Example 1 : Correlation between isoleucine concentration in culture medium and L-threonine biosynthesis

E.coli KCCM 10236 strains were fed-batch cultured in a fermenter with capacity of 30 liter for 77 hours while maintaining a culture temperature of 32 ^°C , a culture pH of 6.5, and aeration rate of 0.5 to 1.0 vvm. The composition and concentration of amino acids in the final culture medium were determined by an automatic amino acid analyzer. The results are presented in Table 1.

Table 1 Composition and concentration of amino acids in E.coli KCCM 10236 culture medium

In order to investigate the productivity of L-threonine depending on the added amount of L-isoleucine, 0 to 0.4 g/l of L-isoleucine was added to fermentation media and the resultant media were incubated in a flask for 48 hours. The results are shown in Table 2. Table 2 Effect of addition of L-isoleucine on productivity of L-threonine of E.coli

KCCM 10236

As shown in Table 2, in the case of a conventional threonine production strain, E.coli KCCM 10236, as increasing the amount of L-isoleucine , the productivity of L-threonine was rapidly reduced.

Example 2: Cloning of nucleotide sequence of thr operon in E.coli

KCCM 10236

The thr operon was amplified and cloned from E.coli KCCM 10236.

Primers for amplification of the thr operon were thr-F and thr-R as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The primer sequences were designed using the GenBank database (NCBI).

5447 bp of the thr operon containing promoter region was amplified using Expand™ High Fidelity PCR System (Boehringer Mannheim). The PCR reaction mixture contained 200 μ M of each dNTP, 300 nM of each primer, 0.1 μg of E.coli KCCM 10236 genomic DNA, 1x Expand HF buffer with 15 mM MgCI₂, and 2.6U of Expand™ High Fidelity PCR System enzyme mix. The PCR reaction was performed at 95 °C for 5 minutes, 95 ^°C for 30 seconds, 56 ^°C for 30 seconds, and 68 ^°C for 4 minutes and 30 seconds for 30 cycles to amplify the thr operon. The PCR reaction was maintained at 68 ^°C for 10 minutes and then terminated at 4^°C. The amplified thr operon was inserted into a cloning vector, pGEM-T (Promega, America). Then, E.coli DH5a was transformed with the cloning vector containing the thr operon to obtain a pGEM-Thr vector.

The pGEM-Thr and pECCG 122 (KFCC 10696) vectors were digested with restriction enzymes, Apal and Pst\, and ligated with the thr operon and pECCG122 vector using a conventional method. E.coli DH5a was transformed with the ligation product to obtain a pTHR(-) vector. According to sequencing analysis, the cloned thr operon contained the nucleotide sequence of a conventional thr operon as set forth in SEQ ID NO: 1.

Example 3: Mutation of THR(-)

In order to induce mutation on the thr operon, 2 μg of the pTHR(-) DNA containing the thr operon was dissolved in 200μi of buffer (0.1 M KH₂PO₄-1 mM EDTA, pH 6.0) containing 200-500 g/ml of N-methyl-N'-nitro-N-nitrosoguanidine and incubated at 37 ^°C for 10-30 minutes. The mutation-induced vector was recovered using a conventional alcohol precipitation and E.coli W3110 (ATCC27325) was transformed with the recovered vector. Then, the transformed E.coli W3110 culture was plated on a LB medium containing 50 mg/l of kanamycine to obtain colonies grown on the LB medium. The colonies were replica plated onto a selective minimal medium plate containing isoleucine hydroxamate as an isoleucine analogue and α -amino-β -hydroxyvaleric acid (AHV) as a threonine analogue and onto a control minimal medium plate without containing these analogues, Strains grown onto the selective minimal medium plate were selected. After the replica plating was repeated three times, 27 candidate strains were selected. Then, the selected candidate strains were again inoculated onto selective minimal media plate containing isoleucine hydroxamate as an isoleucine analogue and α -amino-β -hydroxyvaleric acid (AHV) as a threonine analogue to thereby reconfirm the growth of the candidate strains on the selective minimal media plate.

Example 4: Determination of nucleotide seguence of thr operon irrepressible by isoleucine and resistance of thr operon to isoleucine Mutation-induced pTHR(*) vectors were recovered using a conventional method from the 27 candidate strains obtained in Example 3 and then a threonine-producing strain, E.coli KCCM 10236 was transformed with the pTHRf) vectors. The recombinant E.coli KCCM 10236 strains were plated onto LB media containing 50 mg/l of kanamycin to obtain colonies grown on the LB media. The obtained recombinant strains containing the pTHR(^*) vectors, total 135 strains were cultured on solid media of Table 3. Then, one loopful of each culture was inoculated in a flask containing the fermentation medium of Table 4 and cultured for 48 hours. The accumulated amount of L-threonine was analyzed. According to the analysis results, two types of the pTHR(^*)-containing strains produced threonine at increased levels of 14% and 22%, respectively, when compared to the parent strain, E.coli KCCM 10236. In particular, the pTHR(*) vector obtained from the latter strain was designated as pTHR(+) vector. After the pTHR(+) vector was inserted into E.co//^' w31 10 (ATCC27325), the obtained strain was designated as E.coli w3110/pTHR(+) and then deposited in the Korean Federation of Culture Collection (KFCC) on April 16, 2002 (accession number: KCCM-10371 ). In order to determine the resistance of the recombinant strain,

E.coli KCCM 10236(pTHR) to L-isoleucine, the productivity of threonine was examined in varying concentration of L-isoleucine-containing media. The results are shown in Table 5.

Table 3 Solid medium for threonine production

Table 4 Liquid medium for threonine production

Table 5

Effect of concentration of L-isoleucine on productivity of L-threonine in E.coli KCCM 10236 containing pTHR(+)

As shown in Tables 2 and 5, in the case of the E.coli KCCM

10236/pTHR(+), reduction rate of the productivity of L-threonine depend on increase of the concentration of L-isoleucine was considerably decreased, when compared to the parent strain, E.coli KCCM 10236. Therefore, It can be seen that the pTHR(+) vector contains the mutated thr operon for L-threonine biosynthesis that is not repressible by L-isoleucine.

Example 5: Identification of mutation site

In order to identify mutation site in the thr operon as obtained in Example 4, the nucleotide sequence of the thr operon within the pTHR(+) vector was analyzed. For this, PCR was carried out using 0.1 mg of pTHR(+) as a template, 2 mM of thr-F (SEQ ID NO: 3) as a primer, and 1 μl of BigDye™ Terminator Cycle Sequencing v2.0 Ready Reaction (PE Biosystems). PCR conditions were as follows: denaturation at 95 ^°C for 30 seconds, annealing at 56 ^°C for 30 seconds, and amplification at 72 ^°C for 30 seconds. After 30 cycles, the PCR reaction was terminated at 4 ^°C . The amplified nucleotide sequence was determined using ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). According to the analysis result, it was found that a 39 bp nucleotide sequence of -56 to -18 that maps before the structural thr operon was deleted. The nucleotide sequence of the obtained thr operon is as set forth in SEQ ID NO:2. The productivity of threonine in the obtained thr operon was investigated. For this, three types of strains, E.coli KCCM 10236, E.coli KCCM 10236/pTHR(+), and E.coli KCCM 10236/pECCG were cultured in flasks containing media for threonine production. The productivities of threonine in the strains were evaluated. In detail, the three strains were cultured overnight in 32 °C incubator containing the media for threonine production of Table 3. One loopful of each of the obtained single colonies was inoculated on 25 ml of the medium of Table 4 and cultured for 48 hours at 32 ^°C and 250 rpm. The concentration of the produced L-threonine was measured with High Performance Liquid Chromatography (HPLC). The results are presented in Table 6. Table 6

Comparison of threonine production between a parent strain and a selected recombinant strain in a flask

As shown in Table 6, the productivity of threonine in the recombinant strain, E.coli KCCM 10236/pTHR(+) was enhanced by 22%, when compared to the parent strain, E.coli KCCM 10236. In the presence of isoleucine at high concentration (200 mg/l), the productivity of threonine in the parent strain, E.coli KCCM 10236 was reduced by 46%. On the other hand, the productivity of threonine in the E.coli KCCM 10236/pTHR(+) was reduced only by 18.9%. Therefore, it can be seen that the strains containing the thr operon of the present invention have an increased resistance to isoleucine repression. Industrial Applicability

As apparent from the above description, the present invention provides a nucleotide sequence of the thr operon of E. coli with a deletion of all or part of a 39bp nucleotide fragment of -56 to -18 that is attenuator of the structural genes of the thr operon. In addition, a recombinant vector containing the nucleotide sequence of the thr operon and a transformed host cell containing the recombinant vector are provided.

Therefore, the transformed host cell of the present invention can produce L-threonine in large scale even in the presence of isoleucine.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule ttbts)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

Korean Culture Center of Microorganisms

Address of depositaiy institution (including postal code and country)

361-221 Yurim B/D, Hongje-1-dong, Seodaemun-gu/ Seoul 120-091 Republic of Korea

Date of deposit Accession Number

3 July 1990 KFCC-10696

C. ADDITIONAL INDICATIONS fleave blank if not applicable) This information is continued on an additional sheet ]

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications hsted below will be submitted to the International Bureau later (specify the general nature of the indications eg, "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

I j This sheet was received with the international application I I This sheet was received by the International Bureau on

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Form PCT/RO/134 (Julyl998)

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule \3bts)

A. The indications made below relate to the deposited microorganism or other biological mateπal referred to in the description on page 3 , line 33

Name of depositaiy institution

Korean Culture Center of Microorganisms

Address of depositaiy institution (including postal code and country)

361-221 Yurim B/O, Hongje-1-dong, Seodaemun-gu_/ Seoul 120-091 Republic of Korea

Date of deposit Accession Number

29 December 2000 KCCM-10236

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Stales)

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The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g , "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

I I This sheet was received with the international application I I This sheet was received by the International Bureau on

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Form PCT/RO/134 (Julyl998)

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 136u)

A. refeιτed to in the description

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet j

Name of depositary institution

Korean Culture Center of Microorganisms

Address of depositary institution (including postal code and country)

361-221, Yurim B/D, Hongje-1-dong, Seodaemun-gu, Seoul 120-091 Republic of Korea

Date of deposit Accession Number

11 April 2002 KCCM-10371

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet ~j

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Slates)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications eg, "Accession Number of Deposit")

For receiving Office use only For International Bureau use only j j This sheet was received with the international applicauon I I This sheet was received by the International Bureau on

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Form PCT/RO/134 (July 1998)

Claims

What is claimed is:

1. A nucleotide sequence of the threonine operon of E.coli with a deletion of all or part of an attenuator.

2. The nucleotide sequence according to claim 1 , which is the threonine operon with a deletion of 39 bp (-56 to -18) as set forth in SEQ ID NO: 2.

3. A recombinant vector containing the nucleotide sequence according to claim 1 or claim 2.

4. The recombinant vector according to claim 3, wherein a cloning vector for the recombinant vector is pECCG122 obtained from KFCC 10696.

5. The recombinant vector according to claim 3, which is pTHR(+).

6. A transformed host cell containing the recombinant vector according to claim 3.

7. The transformed host cell according to claim 6, wherein the host cell is Escherichia sp. or Corynebacteria sp.

8. The transformed host cell according to claim 6, wherein the host cell is E.coli KCCM 10236.

9. A method for producing L-threonine comprising: culturing the transformed host cell according to claim 6; and recovering L-threonine from the culture.