KR20020061134A - Process for producing l-threonine - Google Patents

Abstract

PURPOSE: Provided is a manufacturing method for L-threonine by using a microorganism having its original threonine operon on its chromosomal DNA as well as at least one copy of threonine operon inserted into a specific site of the chromosomal DNA, thereby increasing the expression of threonine synthase and consequently improving the production of L-threonine dramatically. CONSTITUTION: The manufacturing method of L-threonine is characterized by inserting at least one copy of threonine operon into a specific site, lacZ site, of a chromosomal DNA in a microorganism to increase the expression of threonine synthase including thrA(aspartokinase I-hormoserine dehydrogenase), thrB(homoserine kinase), and thrC(threonine synthase) and consequently to improve L-threonine production. Wherein, the used microorganism is characteristically E. coli and the inserted operon shows resistance to threonine analogue, isoleucine analogue, and methionine analogue.

Description

Process for producing L-threonine

The present invention relates to the production of L-threonine by using a microorganism, and more particularly, in addition to the original threonine operon present in the chromosomal DNA of the microorganism used, at least one copy of the threonine operon is added to the chromosomal DNA. The present invention relates to a method for producing L-threonine, which is characterized by improving the production of L-threonine by increasing the expression level of threonine biosynthesis-related enzymes by inserting in a specific site.

L-Threonine is an essential amino acid, widely used in feed and food additives, and as a synthetic raw material for fluids and pharmaceuticals. L-threonine is produced by fermentation method using artificial mutants derived from wild strains of E. coli, Coryneform bacteria, Serratia bacteria, Prodencia strains. Such mutant strains are known as amino acid analogs and drug-resistant strains or artificial strains in which diaminopimeric acid, methionine, lysine and isoleucine nutrient composition are given to these resistant strains (Japanese Patent Application Laid-Open No. 2-219582, Appl.Microbiol.Biotechnol., 29.550-553 (1988), Korean Patent Publication No. 92-8365).

In general, as a method for increasing the expression level of a specific gene, there is a method for increasing the number of genes of one microorganism. For this purpose, a plasmid in which the number of copies per microorganism is maintained is usually used. (Sambrook et al, Molecular cloning, 2nd edition, 1989, 1.3-1.5). In other words, by inserting the desired gene in the plasmid and transforming the recombinant plasmid back to the microorganism can be expected to increase the gene by the number of copies of the plasmid per microorganism. Partial success has been reported by attempting to improve the productivity of threonine in this way (US Pat. No. 5,538,873). However, the technique using such plasmids causes a large burden on the host microorganism by overexpressing only a specific gene in most cases, and causes problems such as plasmid stability such that plasmids are lost during cultivation of recombinant strains.

Therefore, in order to solve this problem, methods using plasmids capable of adding antibiotics or controlling expression of cultures have been developed (Sambrook et al., Molecular cloning, 2nd edition, 1989, 1.5 ~ 1.6, 1.9 ~ 1.11). In the case of using a plasmid capable of expression control, it is a method of obtaining a target product by culturing in a condition where gene expression does not occur in the growing season to relieve the burden on the host microorganism, and then inducing expression temporarily after the microorganism is fully grown. However, most of these plasmids are only relevant if the end object is a protein. If the target product is a primary metabolite, since it is closely related to the growth of microorganisms, it is difficult to expect an increase in the primary metabolite by gene expression unless the effect of expression of the target gene is seen in the growing season. The same applies to threonine, a type of primary metabolite.

Therefore, in an effort to compensate for these shortcomings, there has been a case where a method of inserting a specific gene related to threonine biosynthesis into chromosomal DNA was used for threonine production (US Pat. No. 5,939,307). However, in this case, it was difficult to expect a dramatic increase in the expression of threonine operon genes because the method of replacing a promoter exchanged with an inducible promoter was replaced with the same gene present in the chromosome.

Therefore, the present inventors, unlike the conventional substitution method, leaving the original gene existing in the chromosome of the host microorganism as it is and additionally inserting a threonine operon into a specific site (lacZ gene region) of the chromosome DNA, which the original host microorganism has While utilizing the efficacy of the gene as it is found that the advantage of the gene of the gene of the threonine operon inserted into the chromosome can be further utilized to complete the present invention. In addition, the present invention has the feature that it is also possible to insert two or more genes if desired.

Accordingly, an object of the present invention is to solve the instability and growth inhibition of the plasmid, which is pointed out as a disadvantage of the recombinant strain using the plasmid, and simultaneously increase the number of threonine operon by expression of threonine operon genes by increasing the number of threonine operon. L-Threonine provides a manufacturing method that can achieve dramatic threonine productivity increase through increase.

Figure 1 illustrates the construction of a recombinant plasmid for chromosomal insertion of threonine operon.

In order to achieve the above object, the present invention, in the production of L-threonine by using a microorganism, in addition to the original threonine operon present in the chromosomal DNA of the microorganism used, at least one copy of the threonine operon It provides a method for producing L-threonine, characterized in that inserted in a specific region in the chromosomal DNA.

In the present invention, by increasing the number of threonine operon in the chromosomal DNA to two or more to increase the expression of threonine biosynthesis related enzymes (thrA: aspartokinase I-homoserine dehydrogenase, thrB: homoserine kinase, thrC: threonine synthase) It can improve the production of L-threonine.

Microorganisms that can be used in the present invention may be any strain capable of producing L-threonine, such as E. coli, Coryneform bacteria, Serratia bacteria, Prodencia strain, etc., but it is particularly preferable that the microorganism is E. coli Do.

The threonine operon further inserted into the microorganism of the present invention is particularly preferably obtained from microorganisms (artificial mutant strains) that are resistant to threonine analogues, lysine analogues, isoleucine analogues and methionine analogues.

The threonine operon to be inserted in the present invention may be inserted at any site other than the original threonine operon in the chromosomal DNA, but it is particularly preferable that the insertion site is the lacZ site.

Preferably, the present invention is a threonine operon cloned from the chromosome of Escherichia coli TF4076 (KFCC10718, Korean Patent Application No. 90-22965), which is a production strain of L-threonine, on the chromosome of the parent strain TF4076. It provides a method for producing L-threonine, characterized in that the insertion.

Looking at the present invention in more detail as follows.

1.Threonine Operon

The threonine operon was cloned from the chromosome of TF4076 (KFCC10718, Korean Patent Application No. 90-22965), which is a methionine-required, threonine analogue (AHV: α-amino-β-hydroxy valeric acid). Resistance to lysine analogues (AEC: S- (2-aminoethyl) -L-cysteine), resistance to isoleucine analogues (α-aminobutyric acid), analogues of methionine (ethionine) It has characteristics such as resistance to.

2. Insertion vector

Vectors for chromosomal insertion include plasmids of the pBRINT-Ts family, a vector developed for this purpose (Ref .: Sylvie Le Beatriz et al. (1998), pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal) integration into the lacZ gene of Escherichia coli.Gen. 223, 213-219. These vectors have the characteristic that when cloned at 37 ℃, the gene cloned into the plasmid is inserted into the lacZ gene region of chromosomal DNA, and when the temperature is raised to 44 ℃, the remaining plasmids in the cytoplasm are lost. Have

3. Recombinant Vector

First, the threonine operon obtained from the chromosome of TF4076 was cloned into Hind III and BamH I sites of pBRINT-TsGm, pBRINT-TsKm, and pBRINT-TsCm, respectively, to prepare pGmTN, pKmTN, and pCmTN, and transform the vector into TF4076. After incubation at 37 ° C. to induce insertion into the lacZ gene region of the chromosomal DNA of the cloned threonine operon, and further culture at 44 ° C. to induce loss of plasmids remaining in the cytoplasm of the host mother strain.

4. Screening method

Appearance selection methods of recombinant strains are resistant to gentamicin or kanamycin, or chloramphenicol and susceptibility to carbenicillin, depending on the type of vector used for cloning, and blue color in X-gal and IPTG added solid medium. Colonies showing non-white color were selected. This is based on the principle that the insertion of threonine operon into the lacZ gene in the chromosomal DNA results in the loss of lacZ activity and the ability to degrade X-gal, a colorant.

In this way, the selected recombinant strains were compared with the parent strain in the Erlenmeyer flask containing the titer medium. As a result, the mother strain produced 20 g / L at 48 hours, while the threonine operon was inserted into the chromosomal DNA. The best-performing strain, pGmTN14 (KFCC-11228), achieved a yield improvement of about 25% by producing 25 g / L of threonine (see Example 3). In addition, the 5-liter fermenter produced 95.0 g / L of threonine with about 26% improvement in yield compared to the parent strain (75.3 g / L) for this strain (see Example 4).

Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.

Example 1 Preparation of Chromosome DNA Insertion Vector Containing Threonine Operon

TF4076 was used as a threonine producing parent strain and DH5α was used as a host strain for DNA manipulation. The threonine operon used pAT94 (Korean Patent Application No. 92-24732), a recombinant plasmid vector prepared by cloning from chromosomal DNA of TF4076. As a vector for chromosomal DNA insertion, pBRINT-TsCm, pBRINT-TsKm, and pBRINT-TsGm, which are vectors of the pBRINT-Ts family obtained from the National University of Mexico, were used (Reference: Sylvie Le Beatriz et al. (1998), pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.Gen. 223, 213-219.). The concentration of marker antibiotics for the culture of Escherichia coli containing the pBRINT-Ts family of plasmids was 50 mg of carbenicillin, 30 mg of kanamycin, 30 mg of chlorampenicol, and gentamicin per liter of medium. gentamycin) was added to 5 mg. As culture medium, LB (5 g / L of yeast extract, 10 g / L of bactotriptone, 10 g / L of sodium chloride, pH 7.0) was used. The construction of the recombinant plasmid is shown in FIG. First, pAT94 was double-cut with restriction enzymes Hind III and BamH I, and electrophoresis was performed to recover 6.4 kb of threonine operon DNA fragments. pBRINT-Ts series plasmid vectors were also double-cleaved with Hind III and BamH I to recover only DNA fragments that were completely cut in the same manner. The cleaved plasmid vector and the recovered threonine operon DNA fragment were conjugated using T4 DNA ligase and transformed into Escherichia coli DH5α by electric field shock method. LB solid medium containing antibiotics (5 g / L of yeast extract, Bactot Single colonies grown at 10 g / L of lipton, 10 g / L of sodium chloride, 1.7% of bactoa, pH 7.0) were recovered. The recovered colonies were cultured and recovered to isolate plasmids, and the size of the contained plasmids was checked first, and the second was again double-cut with BamH I and Hind III to determine whether a 6.4 kb DNA fragment was produced. The recombinant plasmid pCmTN (14.5 kb), pGmTN (13.3 kb) and pKmTN (14.7 kb) were prepared.

Example 2: Screening for Chromosome Insertion Strains of Recombinant Plasmids

First, the recombinant plasmids pCmTN, pGmTN, and pKmTN isolated from DH5α were transformed into TF4076, a threonine producing strain, and an LB solid medium (5 g / L yeast extract, 10 g / L bactotryptone, chloride) was added. Naturium 10 g / L, bactoa was 1.7%, pH 7.0) and incubated at 30 ℃ 60 hours. Single colonies were inoculated in 0.5 ml of LB and incubated for 4 hours at 30 ° C., and then a part of the culture was transferred to 10 ml of LB, incubated for 6 hours at 30 ° C., and then incubated overnight at 37 ° C. The culture was diluted to 10-3 to 10-6 and plated in LB solid medium to which the appropriate antibiotic was added. At this time, 12 μl of 0.1 M IPTG and 60 μl of 2% X-gal were plated together. The concentration of the antibiotic marker for the selection of the plasmid-inserted strain on the chromosome was 15 mg of kanamycin, 15 mg of chloramphenicol and 5 mg of gentamicin per 1 liter of medium. The plated plates were incubated at 44 ° C. for 24 hours to select carbenicillin-sensitive colonies among white colonies.

Example 3: Comparison of Threonine Production Titers in Erlenmeyer Flasks of Recombinant Strains

Twenty single colonies of recombinant strains in which three recombinant plasmids were inserted into the chromosome were selected for each type, and threonine productivity was compared in an Erlenmeyer flask using a threonine titer shown in Table 1. Single colonies cultured overnight in LB solid medium in a 32 ° C. incubator were inoculated in a loop in 20 ml titer medium and incubated at 32 ° C. at 250 rpm for 48 hours. The results of the analysis are shown in Table 2. The parent strain TF4076 was 20 g / L, whereas the recombinant strains generally showed better results than the parent strain, especially the recombinant strains prepared using pGmTN showed the best results. For this strain, the yield was improved by about 25% compared to the parent strain by producing up to 25 g / L of threonine, and named as mutant pGmTN-14. It was deposited with the association and was assigned accession number KFCC-11228.

Threonine titer Composition Concentration (per liter) glucose 70 g Ammonium Sulfate 28 g KH ₂ PO ₄ 1.0 g MgSO ₄ .7H ₂ O 0.5 g FeSO ₄ .7H ₂ O 5 mg MnSO ₄ .8H ₂ O 5 mg Calcium carbonate 30 g L-methionine 0.15 g Yeast Extract 2 g pH (7.0)

Flask titer test results of recombinant strains 20 g / L or less 20-22 g / L 22-24 g / L 24 g / L or more pCmTN 4 6 5 5 pGmTN 2 2 6 10 pKmTN 6 5 6 3

Example 4: Comparison of threonine using a fermenter

Among the three recombinant strains, threonine productivity was compared in a 5-L fermenter using one strain of each selected strain and the parent strain TF4076. Initial medium compositions are shown in Table 3. As for the spawn culture, a medium in which 10 g / L of glucose and 0.1 g / L of L-methionine was added to the LB medium was used, and the initial inoculation volume of the fermenter was adjusted at 3-5% of the initial culture volume. Additional sugars were added six times and the concentration of glucose after addition was 5% and the additional time point was when the glucose was depleted. In addition, when adding glucose, potassium phosphate (KH 2 PO 4) was added together by 1% by weight. The initial culture volume is 1.5 L, the final volume is 3.0 L and the concentration of total glucose added after fermentation is 250 g / L. Stirring speed was 700 -1000 rpm and pH and temperature were set at 7.0 and 32 ℃ respectively. The pH of the culture was adjusted to 25-28% ammonia water. In addition, the air flow rate was adjusted to 0.5 vvm. The experimental results are shown in Table 4, and all three recombinant strains showed superior performances in the produced threonine concentration and yield compared to the parent strain TF4076. Among them, the pGmTN-14 strain (KFCC-11228) was the most excellent. Compared to the above, it showed a significant improvement of about 26% in concentration and yield. In addition, there is no delay in the fermentation time sugar consumption due to the growth inhibition phenomenon commonly seen in recombinant strains, but the fermentation time is 3 hours faster than the parent strain.

Initial medium composition of 5 liter fermenter Composition Concentration (per liter) glucose 50 g KH ₂ PO ₄ 4 g (NH ₄ ) ₂ SO ₄ 6 g Yeast Extract 3 g MgSO ₄ .7H ₂ O 2 g L-methionine 1 g FeSO ₄ .7H ₂ O 40 mg MnSO ₄ .8H ₂ O 10 mg CaCl ₂ .2H ₂ O 40 mg CoCl ₂ .6H ₂ O 4 mg H ₃ BO ₃ 5 mg Na ₂ MoO ₄ .2H ₂ O 2 mg ZnSO ₄ .7H ₂ O 2 mg pH 7.0

Fermentation results in 5 liter fermenters of recombinant strains Strain name Threonine (g / L) Fermentation time (hours) yield(%) TF4076 75.3 78 30.1 pGmTN-14 95.0 75 38.0 pCmTN-3 92.5 74 37.0 pKmTN-8 87.8 80 35.1

As described above, according to the present invention, by increasing the number of threonine operons in the chromosomal DNA to two or more, threonine biosynthesis related enzymes (thrA: aspartokinase I-homoserine dehydrogenase, thrB: homoserine kinase, thrC: threonine synthase) Increasing the amount of expression can significantly improve the production of L-threonine by more than 25% compared to the parent strain.

Claims (9)

In producing L-threonine using microorganisms, in addition to the original threonine operon present in the chromosomal DNA of the microorganism used, at least one copy of threonine operon is inserted into a specific portion of the chromosomal DNA. Method for producing L-threonine The method of claim 1, wherein the microorganism is Escherichia coli. The preparation of L-threonine according to claim 1, wherein the inserted threonine operon is obtained from a microorganism that is resistant to threonine analogue, lysine analogue, isoleucine analogue and methionine analogue. Way. The method for producing L-threonine according to claim 1, wherein the insertion site in the chromosomal DNA of the inserted threonine operon is a lacZ site. The method of claim 1, L- threonine production strain E. coli TF4076 (KFCC 10718) cloned from the chromosome of threonine operon (operon) cloned from the chromosome of the parent strain TF4076 characterized in that the insertion Method for preparing threonine. The method of claim 1, wherein the microorganism is prepared using the recombinant plasmid pGmTN shown in FIG. The method of claim 1, wherein the microorganism is prepared using the recombinant plasmid pCmTN shown in FIG. The method of claim 1, wherein the microorganism is produced using the recombinant plasmid pKmTN shown in FIG. Escherichia coli pGmTN14 to produce L-threonine (KFCC-11228).