# Patent

## Title
Method of producing L-threonine using Escherichia coli strain with phosphoenolpyruvate carboxylase promoter replaced with cysteine synthase promoter

## Abstract

An L-threonine-producing Escherichia coli in which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome is substituted with a promoter of a cysteine synthase (cysK) gene and a method of producing L-threonine by using the same are disclosed. The recombinant Escherichia coli may produce L-threonine in a high yield, and thus may be widely used in medical, pharmaceutical, and feed industries, particularly for an animal feed.

## Background
This application is a division of U.S. patent application Ser. No. 12/812,203, which is the United States national stage application of PCT/KR09/000,066, filed Jan. 7, 2009, which claims priority to Korean Patent Application No. 10-2008-0002310, filed Jan. 8, 2008, under provisions of 35 U.S.C. 119 and the International Convention for the protection of Industrial Property, the disclosures of each are incorporated by reference in their entirety.


TECHNICAL FIELD
One or more embodiments of the present invention relate to an L-threonine-producing Escherichia coli (E. coli) strain with enhanced L-threonine productivity in which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome is substituted with a promoter of a cysteine synthase (cysK) gene, and a method of producing L-threonine using the same.
BACKGROUND ART
L-threonine is an essential amino acid and is widely used as an animal feed additive or food additive, and is also used as a raw material for medical fluid or drug synthesis. While animal protein contains a sufficient amount of L-threonine, vegetable protein is deficient in L-threonine. Thus, L-threonine is likely to be deficient in animals mainly on vegetarian diets, and thus in particular it is widely used as an additive for animal feed.
L-threonine is produced mainly by a fermentation process using Escherichia coli (E. coli) or Corynebacterium, which is developed by artificial mutagenesis or genetic recombination. To produce L-threonine, a mutant strain derived from a wild-type strain of Escherichia coli (E. coli), Corynebacteria sp., Serratia sp., or Providencia sp is used. Examples of the mutant strain include an amino acid analogue- or drug-resistant mutant strain, and a diaminopimellic acid, a methionine, a lysine, or an isoleucine auxotrophic mutant strain that has also an amino acid analogue- or drug-resistance. Among methods of producing L-threonine using a mutant strain, a method of using a microorganism that belongs to Escherichia coli species, has diaminopimellic acid and methionine auxotroph phenotypes, and is mutated so that biosynthesis of L-threonine is not affected by feedback inhibition of threonine is disclosed in Japanese Patent No. 10037/81.
A fermentation process using a recombinant strain can also be used in production of L-threonine. For example, Japanese Patent Application Publication No. 05-227977 discloses a method of producing L-threonine using a recombinant E. coli into which a threonine operon consisting of genes encoding aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase is introduced in a plasmid form, and a method of producing L-threonine using threonine-producing Brevibacterium flavum into which a threonine operon derived from E. coli is introduced (TURBA E, et al, Agric. Biol. Chem. 53:2269 ̃2271, 1989).
In general, the expression of a specific gene in a microorganism may be enhanced by increasing the copy number of the gene in the microorganism. For this, a plasmid that gives a greater copy number to a microorganism is used [Sambrook et al., Molecular Cloning, 2th, 1989, 1.3-1.5]. That is, the number of the gene may be increased by as many as the copy number of the plasmid per a single microorganism by inserting a target gene into the plasmid whose copy number may be maintained at a high level and then transforming the microorganism with the obtained recombinant plasmid. Attempts have also been made to enhance the productivity of threonine using this method and a partial success was reported (U.S. Pat. No. 5,538,873). However, this technology using a plasmid induces excessive expression of only a specific gene in most cases, thereby imposing a heavy burden on a host microorganism. Furthermore, plasmids may be lost during culturing of a recombinant strain, thereby decreasing plasmid stability. To address these problems of the method of producing threonine by using a recombinant strain into which a plasmid is introduced, addition of an antibiotic into a culture and methods of using a plasmid whose expression is controllable have been developed [Sambrook et al. Molecular Cloning, 2th, 1989, 1.5-1.6, 1.9-1.11]. In the case of using the plasmid whose expression is controllable, to alleviate the burden on a host microorganism and obtain a large amount of cells, during the growth phase, a host microorganism is cultured under conditions where the expression of a target gene on the plasmid does not occur, and after the sufficient growth of the host microorganism, temporary expression of the gene is induced, thereby obtaining a target material. However, methods using plasmids whose expression is controllable can be used only when a final gene product is a protein or a secondary metabolite. In a case where a gene product is a primary metabolite that is produced at the same time when microorganisms begin to grow, expression of a target gene must be induced during the growth phase. Otherwise, it is difficult to anticipate the accumulation of the primary metabolite. Since threonine belongs to a primary metabolite, the latter case is also applied to threonine.
Thus, to enhance the productivity of threonine, which is a primary metabolite, inserting genes involved in threonine biosynthesis into chromosomal DNA of a microorganism is disclosed in U.S. Pat. No. 5,939,307, instead of using a method of introducing a plasmid with threonine biosynthesis-related genes into a microorganism. Methods of increasing the threonine biosynthesis-related genes and the expression thereof have been diversely developed, but there is still a need for developing a method of more economically producing L-threonine in a high yield.
To increase the production yield of L-threonine, research on a biosynthesis pathway from oxaloacetate to threonine has been intensively conducted. With regards to this, we intended to first induce the flow of carbon along a pathway from phosphoenolpyruvate to oxaloacetate by enhancing the activity of phosphoenolpyruvate carboxylase involved in a step right before the biosynthesis of L-threonine. For this, we studied and found that a microorganism strain capable of producing L-threonine in which a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome was substituted with a promoter of a gene encoding cysteine synthase (cysK) so as to increase the expression of a gene encoding ppc, which is a first enzyme in the biosynthesis of L-threonine after glycolysis, produced L-threonine in a high yield, thus completing the present invention.
DISCLOSURE OF INVENTION
Technical Problem
One or more embodiments of the present invention provide an Escherichia coli (E. coli) strain capable of producing L-threonine in high yield.
One or more embodiments of the present invention also provide a method of producing L-threonine using the E. coli strain, whereby L-threonine may be efficiently produced.
Technical Solution
The present invention provides an L-threonine-producing microorganism in which a promoter of a natural phosphoenolpyruvate carboxylase (ppc) gene on the chromosome is substituted with a promoter of a cysteine synthase (cysK) gene.
In the L-threonine-producing microorganism, the expression of ppc increases in such a way that a promoter of a gene encoding ppc, which converts phosphoenolpyruvate produced after glycolysis to oxaloacetate, the starting material of L-threonine biosynthesis, is substituted with the promoter of the cysK gene, and the productivity of L-threonine may increase, accordingly.
The microorganism may include a prokaryotic or eukaryotic cell capable of producing L-threonine in which a promoter of a ppc gene on the chromosome is substituted with a promoter of a cysK gene. For example, the microorganism may be a microorganism strain belonging to Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, or Brevibacterium genus. In particular, the microorganism may be a microorganism belonging to Enterobacteriaceae family, and more particularly, a microorganism belonging to Escherichia genus. Most particularly, the microorganism may be Escherichia coli CA030031 (KCCM 10910P).
The E. coli CA030031 is derived from E. coli KCCM 10541 which is derived from a L-threonine-producing E. coli, KFCC 10718 (Korean Patent Publication No. 92-8395). The E. coli KFCC 10718 has a resistance to an L-methionine analogue, a methionine auxotroph phenotype, a resistance to an L-threonine analogue, a leaky isoleucine auxotroph phenotype, a resistance to an L-lysine analogue, and a resistance to α-aminobutyric acid, and is capable of producing L-threonine. Thus, the microorganism may also include a mutant microorganism for producing L-threonine, in addition to a wild-type microorganism. For example, the mutant microorganism may be a microorganism that has a resistance to an L-methionine analogue, a methionine auxotroph phenotype, a resistance to an L-threonine analogue, a leaky isoleucine auxotroph phenotype, a resistance to an L-lysine analogue, and a resistance to α-aminobutyric acid, and belongs to E. coli capable of producing L-threonine.
In an embodiment, the microorganism may be E. coli that has a methionine auxotroph phenotype and resistances to a threonine analogue, a lysine analogue, an isoleucine analogue and a methionine analogue. For example, the L-methionine analogue may be at least one compound selected from the group consisting of D,L-methionine, norleucine, α-methylmethionine, and L-methionine-D,L-sulfoximine, the L-threonine analogue may include at least one compound selected from the group consisting of α-amino-β-hydroxy valeric acid and D,L-threonine hydroxamate, and the L-lysine analogue may be at least one compound selected from the group consisting S-(2-aminoethyl)-L-cysteine and δ-methyl-L-lysine. Examples of the mutant microorganism may include a microorganism in which a pckA gene involved in converting oxaloacetate (OAA) into phosphoenol pyruvate (PEP), which is an intermediate involved in the biosynthesis of L-threonine, is inactivated, a microorganism in which a tyrR gene repressing a lysC gene which is involved in conversion of oxaloacetate into aspartate is inactivated, or a microorganism in which a galR gene repressing the expression of a galP gene which is involved in the influx of glucose is inactivated.
In the microorganism of the present invention, the promoter of the ppc gene on the chromosome is substituted with the promoter of the cysK gene so as to increase the expression thereof. The promoter of the cysK gene used herein may be derived from a cysK gene with a high expression rate, and may have a nucleotide sequence of SEQ ID NO: 10.
The present invention also provides a method of producing L-threonine, the method including: culturing a transformed microorganism with enhanced L-threonine productivity in which a promoter of a natural ppc gene on the chromosome is substituted with a promoter of a cysK gene; and isolating L-threonine from the culture of the microorganism.
In the method of producing L-threonine, the transformed microorganism may be E. coli, for example, E. coli CA030031 (KCCM 10910P).
One or more embodiments of the present invention will now be described more fully with reference to the following examples. However, these examples are provided only for illustrative purposes and are not intended to limit the scope of the present invention.
Advantageous Effects
As described above, according to the present invention, in a microorganism in which a promoter of a ppc gene on the chromosome is substituted with a promoter of a cysK gene, the expression of the ppc gene, which is an enzyme converting phosphoenolpyruvate to oxaloacetate that is a precursor of L-threonine biosynthesis, increases, thereby significantly enhancing the productivity of L-threonine by 16% or higher. The microorganism may produce L-threonine in a high yield, and thus may be widely used in medical, pharmaceutical, and feed industries, particularly for an animal feed.



DESCRIPTIONS OF DRAWINGS
FIG. 1 is a diagram illustrating a process of constructing a recombinant vector pUC-PcysK; and
FIG. 2 is a diagram illustrating a process of constructing a recombinant vector pUCpcysKmloxP.



MADE FOR INVENTION
Example 1
Construction of Recombinant Vector pUCpcysKmloxP
Preparation of Pcysk Fragment
To obtain 0.3 kb DNA fragment containing a promoter of a cysK gene (SEQ ID NO: 10), the genomic DNA (gDNA) of W3110, which is E. coli wild type strain, was extracted using a QIAGEN Genomic-tip system, and a polymerase chain reaction (PCR) was performed using the gDNA as a template and a PCR HL premix kit (manufactured by BIONEER, Korea). To amplify the promoter of the cysK gene, the PCR was performed using primers of SEQ ID NOS: 1 and 2 as follows: 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 2.5 minutes.
The PCR products were digested with KpnI and EcoRV, electrophoresized on a 0.8% agarose gel, and then eluted to obtain 0.3 Kb DNA fragment (hereinafter, referred to as “PcysK fragment”).
(2) Construction of Recombinant Vector pUC-PcysK
FIG. 1 is a diagram illustrating a process of constructing recombinant vector pUC-PcysK containing a promoter of a cysK gene.
Plasmid pUC19 (New England Biolabs, USA) and the PcysK fragment obtained according to Example 1-(1) were each digested with restriction enzymes KpnI and Smal, and ligated with each other to construct vector pUC-PcysK.
(3) Construction of Recombinant Vector pUCpcysKmloxP
FIG. 2 is a diagram illustrating a process of constructing a recombinant vector pUCpcysKmloxP.
In general, in an experiment of gene deletion caused by one-step inactivation, whenever one gene is deleted, one sequence of a recombinase recognition site loxP remains on a chromosomal DNA. It has been reported that due to the sequences of loxP remaining on the chromosomal DNA, when the strains are additionally modified for further development, the efficiency may be significantly decreased (Nagy A., Genesis, 26:99, 2000). An improved method of gene deletion using loxP mutants, which are named lox71 and lox 66 has been proposed by Suzuki (Appl. Environ. Microbiol. 71:8472, 2005). Thus, to more efficiently substitute a promoter of a ppc gene on the chromosome with a promoter of a cysK gene by using the loxP mutants, we constructed vector pUCpcysKmloxP having both a mutant loxP-CmR-loxP cassette and the promoter of the cysK gene.
As shown in FIG. 2, the PCR was performed using plasmid pACYC184 (New England Biolab) as a template by using primers of SEQ ID NOS: 3 and 4 as follows: 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 1 minute to obtain 1.1 kb of PCR fragment. The vector pUC-PcysK constructed according to Example 1-(2) and 1.1 kb of DNA fragment obtained using pACYA184 as a template were each digested with restriction enzymes NdeI/KpnI, ligated with each other, and transformed into E. coli. Then, cell having DNA accurately ligated with the vector were selected using a general method, and plasmid pUCpcysKmloxP was purified from the culture of the cells.
Example 2
Preparation of Recombinant E. coli KCCM 10541-PcyK-ppc
To substitute a native promoter of a ppc gene (SEQ ID NO: 9) encoding phosphoenolpyruvate carboxylase on the chromosome with a promoter of a cysK gene, a known one-step inactivation method (Warner et al., PNAS, 6; 97(12):6640, 2000) was performed on E. coli KCCM 10541.
First, PCR was performed using the plasmid pUCpcysKmloxP constructed according to Example 1 as a template by using primers of SEQ ID NOS: 5 and 8 as follows: 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 1 minute to obtain DNA fragments and the obtained DNA fragments were purified using a QIAGEN kit (PCR Purification kit). Subsequently, PCR was further performed using primers of SEQ ID NOS: 6 and 7 and the purified DNA fragments as a template as follows: 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 1 minute. The resulting DNA fragments were purified, the purified DNA fragments were introduced by electroporation into E. coli KCCM 10541 into which vector pKD46 was introduced (Proc. Natl. Acad. Sci. U.S.A. 97(12), 6640-6645 (2000)), and a single colony was selected on a Luria-Bertani (LB) plate containing 15 μg/mL of chloramphenicol. The selected strain was a strain in which the DNA fragment was inserted into a promoter site of a ppc gene. Vector pJW168 (BMG Biothechnol. 2001; 1:7. Epub 2001 Sep. 26) was introduced into the selected strain to prepare recombinant E. coli KCCM 10541-PcyK-ppc in which the naive promoter of the ppc gene was substituted with the promoter of the cysK gene, by removing antibiotics-resistance gene.
Example 3
Comparison in L-Threonine Productivity Between Recombinant Microorganisms
The recombinant microorganism prepared according to Example 2 was cultured in a threonine titer medium, as shown in Table 1 below, in an Erlenmeyer flask, and it was confirmed whether the productivity of L-threonine was improved.










TABLE 1







Composition
Concentration (per liter)




















Glucose
70
g



KH2PO4
2
g



(NH4)2SO4
25
g



MgSO4•7H2O
1
g



FeSO4•7H2O
5
mg



MnSO4•4H2O
5
mg



DL-methionine
0.15
g



Yeast extract
2
g



Calcium carbonate
30
g










pH
6.8










1 platinum loop of E. coli KCCM 10541 and E. coli KCCM 10541-PcysK-ppc that were cultured in a LB solid medium in an incubator at 33° C. overnight were each inoculated in 25 ml of titer medium, as shown in Table 1, and cultured in the incubator at 33° C. at 200 rpm for 48 hours. The results are shown in Table 2 below.
As shown in Table 2, when the parent strain E. coli KCCM 10541 was cultured for 48 hours, it produced 29.8 g/L of L-threonine, while the E. coli KCCM 10541-PcysK-ppc of Example 2 produced 34.7 g/L of L-threonine, which has a 4.9 g/L higher productivity than that of the parent strain. Thus, it was confirmed that the recombinant strain in which the promoter of the ppc gene was substituted with the promoter of the cysK gene has enhanced L-threonine productivity. The transformed E. coli KCCM 10541-PcysK-ppc was named E. coli CA030031 and deposited in Korean Culture Center of Microorganisms (KCCM) on Dec. 20, 2007 (Accession No: KCCM 10910P).










TABLE 2







Strain
L-threonine (g/L)









KCCM10541 (parent strain)
29.8



KCCM10541-PcysK-ppc
34.7










While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

## Claims

1. A method of producing L-threonine, the method comprising:
culturing an Escherichia coli strain to produce L-threonine and
isolating L-threonine from the culture of the Escherichia coli strain,
wherein the Escherichia coli strain is modified by substituting a promoter of a phosphoenolpyruvate carboxylase (ppc) gene on the chromosome of said Escherichia coli strain with a promoter of a cysteine synthase (cysK) gene,
wherein the promoter of the cysK gene has the nucleotide sequence of SEQ ID No:10, and wherein the expression of the ppc gene in the modified Escherichia coli strain is increased relative to an Escherichia coli strain without said substitution.

2. The method of producing L-threonine according to claim 1, wherein the Escherichia coli strain is the Escherichia coli stain having Korean Culture Center of Microorganism Accession No: KCCM 10910P).