# Patent

## Title
Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism

## Abstract

Disclosed is a strain of Escherichia sp., capable of producing O-acetyl homoserine in high yield, with the introduction and enhancement therein of the activity of: homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase; and at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase. Also, a method of producing O-acetyl homoserine using the strain is provided.

## Background
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200187—407_SEQUENCE_LISTING.txt. The text file is 26 KB, was created on Mar. 7, 2011, and is being submitted electronically via EFS-Web.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a strain of Escherichia sp. capable of producing O-acetyl homoserine in high yield. More particularly, the present invention relates to a strain of Escherichia sp. capable of producing O-acetyl homoserine in high yield, in which the activity of homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase, in combination with at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase, are introduced and enhanced. Also, the present invention is concerned with a method of producing O-acetyl homoserine using the strain.
2. Description of the Related Art
Methionine, an essential amino acid for the body, finds a variety of applications in the food and medical industries, such as the use thereof as an additive in animal feed and foods and as a material for parenteral nutrient solutions and medicines. Methionine acts as a precursor for choline (lecithin) and creatine and is used as a material useful for the synthesis of cysteine and taurine. Together with cysteine, methionine is one of two sulfur-containing proteinogenic amino acids. S-Adenosyl methionine, derived from L-methionine, serves as a methyl donor in vivo and is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is also found to prevent lipid accumulation in the liver and arteries and to alleviate depression, inflammation, liver diseases and muscle pain (Jeon B R et al., J Hepatol., 2001 March; 34(3): 395-401).
As summarized below, methionine and/or S-adenosyl-L-methionine has been thus far known to have the in vivo functions of:
1) suppressing lipid accumulation in arteries and in the liver, where lipid metabolism is mediated, and improving blood circulation in the brain, the heart and the kidneys (J Hepatol. Jeon B R et al., 2001 March; 34(3): 395-401).
2) promoting the digestion, detoxication and excretion of toxic substances and the excretion of heavy metals such as Pb.
3) acting as an antidepressant when methionine is administered in a daily dose of from 800 to 1,600 mg (Am J Clin Nutr. Mischoulon D. et al., 2002 November; 76(5): 1158S-61S)
4) improving liver functions against liver diseases (FASEB J. Mato J M., 2002 January; 16(1): 15-26), particularly, against alcohol-induced liver injury (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235)
5) showing an anti-inflammatory effect on osteoarthritis and promoting the healing of joints (ACP J Club. Sander O., 2003 January-February; 138(1): 21, J Fam Pract., Soeken K L et al., 2002 May; 51(5): 425-30).
6) acting as an essential nutrient to hair formation and preventing brittle hair and depilation (Audiol Neurootol., Lockwood D S et al., 2000 September-October; 5(5): 263-266).
Methionine for use in animal feed, foods and medicines can be synthesized chemically or biologically.
In the chemical synthesis route, on the whole, methionine is produced through the hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. However, the synthesized methionine is disadvantageously present in a mixture of L- and D-forms which needs a difficult additional process to separate them from each other. In order to solve this problem, the present inventors developed a biological method for selectively synthesizing L-methionine, a chemical which a patent (WO 2008/103432) has already been applied for. The method, termed in brief “a two-step process”, comprises the fermentative production of an L-methionine precursor and the enzymatic conversion of the L-methionine precursor to L-methionine. The methionine precursor preferably includes O-acetylhomoserine and O-succinyl homoserine. The two-step process is evaluated on terms of having overcome the problems from which the conventional methods suffer, such as sulfide toxicity, feedback regulation in methionine synthesis by methionine and SAMe, and degradation of intermediates by cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase and O-acetylhomoserine sulfhydrylase. Also, compared to the conventional chemical synthesis method of producing DL-methionine, the two-step process has the advantage of being selective for L-methionine only, with the concomitant production of organic acids, such as succinic acid and acetic acid as useful by-products.
Found as an intermediate in the biosynthesis pathway of methionine, O-acetyl-homoserine is used as a precursor for the production of methionine (WO 2008/013432). O-acetyl-homoserine is synthesized from L-homoserine and acetyl-CoA with the aid of O-acetyl transferase as shown in the following formula:

L-Homoserine+Acetyl-CoA→O-Acetyl-Homoserine.

In the U.S. patent application Ser. No. 12/062,835 of the present assignee are disclosed a microorganism strain into which a thrA gene responsible for aspartate kinase and homoserine dehydrogenase activity and a Deinococcus-derived metX gene coding for homoserine acetyl transferase are introduced to improve the biosynthesis of L-homoserine and O-acetyl-homoserine, respectively, and a method for producing O-acetyl homoserine at high yield using the same.
In this context, the present inventors conceived that the enhancement of the other three enzymes responsible for the catalytic reactions in the homoserine biosynthesis pathway, that is, phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC) and aspartate semi-aldehyde dehydrogenase (asd), would increase a higher production yield of O-acetyl homoserine than would the method of U.S. Ser. No. 12/062,835.
Like the concomitant enhancement of a series of the enzymes involved in the conversion from phosphoenolpyruvate to O-acetylhomoserine according to the present invention, attempts have been made to increase L-amino acid productivity by simultaneously expressing the enzymes which play important roles in the biosynthesis pathways of aspartate-derived L-amino acids, such as L-lysine, L-threonine and L-methionine.
EP00900872 is directed to the effective production of L-lysine, featuring an increase in the activities of a series of enzymes involved in the lysine biosynthesis, including dihydropicolinate synthase (dapA), aspartokinase (lysC), dihydropicolinate reductase (dapB), diaminopimelate dehydrogenase (ddh), tetrahydropicolinate succinylase (dapD), succinyl diaminopimelate diacylase (lysE), aspartate semi-aldehyde dehydrogenase (asd), phosphoenolpyruvate carboxylase (ppc), in E. coli. Japanese Patent Nos. JP2006-520460 and JP2000-244921 describes the effective production of L-theronine in E. coli by increasing the activities of aspartate semi-aldehyde dehydrogenase (asd), phosphoenolpyruvate carboxylase (ppc), aspartokinase (thrA), homoserine dehydrogenase (thrA), homoserine kinase (thrB) and threonine synthase (thrC). Also, WO 2007/012078 discloses a recombinant strain of Corynebacterium capable of producing increased levels of L-methionine in which genes coding for aspartokinase (lysC), homoserine dehydrogenase (hom), homoserine acetyl transferase (metX), O-acetylhomoserine sulfhydrylase (metY), cystathionine gamma synthase (metB), cobalamin-dependent transmethylase (metH); cobalamin-independent methionine synthase (metE), methyltetrahydrofolate reductase (metF), and glucose 6-phosphate dehydrogenase (zwf) are increased in expression level while genes coding for methionine repressor protein (mcbR), homoserine kinase (hsk), S-adenosylmethionine synthetase (metK), and threonine dehydratase (livA) are decreased in expression level.
All of the patents are related to the effective production of aspartate-derived L-amino acids, that is, L-lysine, L-threonine and L-methionine, respectively, featuring the employment of gene combinations depending on the respective products.
In the present invention, a series of enzymes responsible for the catalytic steps from phosphoenolpyruvate to O-acetylhomoserine in the O-acetylhomoserine biosynthesis pathway are designed to be increased in expression level to produce O-acetylhomoserine in higher yield, which has been mentioned nowhere in previous documentation. Further, the enzyme combination employed in the present invention is different from that employed for the production of the aspartate-derived L-amino acid, such as L-lysine, L-threonine or L-methionine, as the final products are different.
Leading to the present invention, intensive and thorough research into the production of O-acetyl homoserine in maximal yield, conducted by the present inventors, resulted in the finding that the concomitant enhancement of the genes encoding aspartate kinase and homoserine dehydrogenase (thrA), and homoserine acetyl transferase (metX) plus a gene encoding at least one enzyme selected from among phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), and aspartate semi-aldehyde dehydrogenase (asd) in the form of a genomic DNA and/or a plasmid in a microorganism strain could bring about a significant increase in the production of O-acetyl homoserine.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a microorganism strain capable of producing O-acetyl homoserine in high yield, which is designed to fortify a series of genes responsible for the enzymes involved in the homoserine biosynthesis pathway from phosphoenolpyruvate to O-acetyl homoserine.
It is another object of the present invention to provide a method of producing O-acetyl homoserine in high yield, using the microorganism strain.
In accordance with an aspect of the present invention, there is provided a strain of Escherichia sp., capable of producing O-acetyl homoserine in high yield, with the introduction and enhancement therein of the activity of: (a) homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase; and (b) at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase.
In accordance with another aspect of the present invention, there is provided a method of producing O-acetyl homoserine in a culture medium, comprising fermenting the strain in the culture medium.
In accordance with a further aspect of the present invention, there is provided a method of producing L-methionine and acetate, comprising: (a) fermentating the strain to produce O-acetyl homoserine; (b) separating the O-acetyl homoserine; and (c) converting the O-acetyl homoserine, together with methyl mercaptan, into L-methionine and acetate in the presence of an enzyme selected from a group consisting of cystathionine ganuna synthase, O-acetyl homoserine sulfhydrylase, and O-succinyl homoserine sulfhydrylase.
According to the present invention, therefore, O-acetyl homoserine can be produced in high yield by fermenting a strain of Escherichia sp. which anchors all of the six genes aspartate kinase and homoserine dehydrogenase (thrA), homoserine acetyl transferase (metX), phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), and aspartate semi-aldehyde dehydrogenase (asd), responsible for the biosynthesis pathway from phosphoenolpyruvate to O-acetyl homoserine, in the form of chromosomal DNA or plasmid DNA. In addition, the O-acetyl-L-homoserine produced by the strain of the present invention can be converted, as disclosed in WO2008/013432, entitled “Microorganism producing L-methionine precursor and method of producing L-methionine and organic acid from the L-methionine precursor”, issued to the present inventors, into L-methionine in high yield.



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a schematic view showing an O-acetyl homoserine biosynthesis pathway of the strain according to the present invention;
FIG. 2 is a schematic view showing the genetic map and construction of a pSG-2ppc vector for chromosomal integration;
FIG. 3 is a schematic view showing the genetic map and construction of a pSG-2aspC vector for chromosomal integration;
FIG. 4 is a schematic view showing the genetic map and construction of a pSG-2asd vector for chromosomal integration; and
FIG. 5 is a schematic view showing the genetic map and construction of an expression vector pCJ-thrA(M)-metX-CL;



DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with an aspect thereof, the present invention pertains to a strain of Escherichia sp. capable of producing O-acetyl homoserine in high yield, with the introduction and enhancement therein of the activity of (a) homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase activities; and (b) at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase.
As used herein, the term “L-methionine precursor” is intended to refer to a metabolite found on the methionine biosynthesis pathway or a derivative thereof, and particularly to O-acetyl homoserine.
As used herein, the term “O-acetyl homoserine-producing strain” is intended to refer to a eukaryotic or prokaryotic microorganism which can produce O-acetyl homoserine intracellularly or extracellularly and particularly to a genetically modified microorganism which can accumulate O-acetyl homoserine therein. Examples of the strain useful in the present invention include Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacteria sp., Pseudomonas sp., Leptospira sp., Salmonellar sp., Breuibacteria sp., Hypomononas sp., Chromobacterium sp., Norcardia sp., fungi and yeasts, with preference for Escherichia sp., Corynebacteria sp. and Leptospira sp. and yeast. More preferred is Escherichia sp. Far more preferred is Escherichia coli. Further far more preferred is a strain of E. coli which can produce L-lysine, L-threonine, L-isoleucine or L-methionine. Most preferred is one derived from the strain of E. coli of Accession No. KCCM 10921P (deposited at Korean Culture Center of Microorganism, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea, on Jan. 23, 2008) or KCCM 10925P deposited by the present assignee (deposited at Korean Culture Center of Microorganism, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea, on Feb. 12, 2008; U.S. Ser. No. 12/062,835), or from FTR2533 (Accession No. KCCM 10541, deposited at Korean Culture Center of Microorganism, Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea on Dec. 9, 2003).
As used herein, the term “introduction and enhancement of activity” is intended to mean an increase in the intracellular activity of an enzyme encoded for by the corresponding gene, which can be generally achieved by the overexpression of the gene. There are many approaches to the overexpression of a target gene. For example, the overexpression may be implemented by the modification of a base in the promoter region and/or 5′-UTR for the target gene, by introducing the extra copy of the target gene on the chromosome, or by the introduction of the target gene in combination with an autologous or a heterologous promoter onto a vector, followed by the transformation of the vector into a microorganism strain. Further, a mutation in the ORF (open reading frame) of the target gene may result in the overexpression thereof. In numerical terms, when overexpression occurs, the corresponding protein increases in activity or concentration by 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, 1000% or up to 2000%, compared to when it is expressed in a natural state. Approaches to the introduction and enhancement of the activity of a gene include transformation with a plasmid carrying the corresponding gene, an increase in the number of gene copies, employment of a strong promoter for the gene, or a mutation in a preexisting promoter for the gene.
In a preferred embodiment thereof, the present invention provides a microorganism strain capable of producing O-acetyl homoserine in higher yield, with the introduction and enhancement of the activity of a series of six enzymes consisting of aspartate kinase and homoserine dehydrogenase (thrA), homoserine acetyl transferase (metX), phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), and aspartate semi-aldehyde dehydrogenase (asd), and a method of producing O-acetyl homoserine using the same. Preferably, aspartate kinase and homoserine dehydrogenase (thrA) or homoserine acetyl transferase (metX) are introduced into and enhanced in cells by transformation with an expression vector carrying the corresponding genes while two or more copies of a gene coding for at least one selected from among phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC) and aspartate semi-aldehyde dehydrogenase (asd) may be located in the genome of the microorganism strain. Most preferably, all of these three genes are located in two or more copies in the genome of E. coli. 
In greater detail, the microorganism strain is designed to increase the level of the metX gene coding for homoserine O-acetyltransferase responsible for the first step of the methionine bionsynthesis pathway, leading to an improvement in the synthesis of the L-methionine precursor O-acetyl homoserine. Herein, metX refers generally to a gene encoding a protein having the activity of homoserine O-acetyltransferase. For use in the present invention, new, exogenous homoserine O-acetyltransferase may originate from a variety of microorganisms. Examples of the microorganisms from which a gene coding for homoserine O-acetyltransferase can be obtained include Corynebacterium sp., Leptospira Deinococcus sp., Pseudomonas sp., or Mycobacterium sp., but are not limited thereto. Preferably, the homoserine O-acetyltransferase may be encoded by a gene originating from a strain selected from a group consisting of Corynebacterium glutamicum, Leptospira meyeri, Deinococcus radiodurans, Pseudomonas aeruginosa and Mycobacterium smegmatis. More preferably, the homoserine O-acetyltransferase has an amino acid sequence of UniProt Database Accession No. Q9RVZ8 (SEQ ID NO. 18), NP 249081 (SEQ ID NO. 19), or YP 886028 (SEQ ID NO. 20). The metX gene originating from Leptospira meyeri is known to show resistance to feedback inhibition (J Bacteriol. 1998 January; 180(2):250-5. Belfaiza J et al.). The other homoserine O-acetyltransferases were also found to be refractory to feedback inhibition in previous studies of the present inventors.
For example, the introduction and enhancement of homoserine O-acetyltransferase may be implemented by the introduction of metX or by the modification of a base in the 5′-UTR and/or promoter region for the target gene. Preferably, the target gene in combination with an autologous or a heterologous promoter is inserted into a vector, followed by the transformation of the vector into a microorganism strain. The introduction and enhancement of metX results in an increase in the synthesis of the methionine precursor.
In addition, the microorganism strain is designed to increase the activity of aspartokinase or homoserine dehydrogenase so as to improve the synthesis of the O-acetyl homoserine precursor homoserine. Herein, thrA refers generally to a gene encoding a peptide having the activity of aspartokinase and homoserine dehydrogenase. Preferably, the aspartokinase and homoserine dehydrogenase is encoded by a gene of Uniprot database Accession No: AP—000666. The thrA gene may be preferably introduced via a plasmid and remain as a plasmid DNA. That is, an expression vector carrying the thrA gene may be transformed into the strain. More preferably, both metX and thrA are introduced into the strain and remain as plasmid DNAs in the strain. That is, an expression vector carrying both metX and thrA is transformed into the strain.
In an embodiment of the present invention, the O-acetyl-L-homoserine-producing microorganism strain may be prepared as follows.
First, the microorganism strain is designed to accumulate O-acetyl-L-homoserine by increasing the number of copies of genes respectively encoding phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC) and aspartate semi-aldehyde dehydrogenase (asd). For this, these genes are cloned into respective pSG vectors useful for the integration of a gene into a chromosome, followed by transformation with the pSG vectors to increase the number of the respective genes to two or more copies. As a result, the expression of the genes is improved. Next, the genes coding for aspartate kinase and homoserine dehydrogenase (thrA) and homoserine acetyl transferase (metX) are introduced as plasmid DNAs into the microorganism strain. In this regards, a thrA-metX operon composed of a thrA gene (aspartate kinase and homoserine dehydrogenase), a metX gene (homoserine acetyl transferase) derived from Deinococcus, and a CJ1 promoter is constructed and cloned into pCL1920, a low copy plasmid, followed by the transformation of the recombinant plasmid into the strain which has 2 copies of each of the genes (phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), and aspartate semi-aldehyde dehydrogenase (asd)). Therefore, the microorganism strain is improved in every step of the biosynthesis pathway from phsphoenolpyruvate to O-acetyl homoserine.
A series of the enzymes are responsible for the catalytic steps of the biosynthesis pathway from phosphoenolpyruvate to O-acetyl homoserine as shown in the following reaction formulas. Accordingly, the overexpression of the serial genes leads to the intracellular accumulation of O-acetyl homoserine.

Phosphoenolpyruvate+H2O+CO2<->Oxaloacetate+Phosphate

Oxaloacetate+Glutamate<->Aspartate+a-ketoglutarate

Aspartate+ATP<->Aspartyl-4-phosphate+ADP

Aspartyl-4-phosphate+NADPH<->Aspartate-semialdehyde+Phosphate+NADP+

Aspartate-semialdehyde+NADPH<->Homoserine

Homoserine+Acetyl-CoA<->O-Acetyl-homoserine+CoA

Genes coding respectively for phosphoenolpyruvate carboxylase, aspartate aminotransferase, aspartate semi-aldehyde dehydrogenase, aspartate kinase and homoserine dehydrogenase, and homoserine acetyl transferase are generally expressed as ppc, aspC, asd, thrA, and metX. These genes may be obtained from the genome sequences of Escherichia coli and Deinococcus radiodurans R1 disclosed previously (Mol Syst Biol. 2006; 2:2006.0007. Epub 2006 Feb. 21, Science. 1999 Nov. 19; 286(5444):1571-7). Also, the gene sequences may be obtained from public databases such as those constructed by the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ). For example, GenBank ID. No. 89110074 is given to ppc, GenBank ID. No. 85674274 to aspC, GenBank ID. No. 89110578 to asd, GenBank ID. No. 89106886 to thrA, and GenBank ID. No. 1799718 to metX. The microorganism strain thus prepared is improved in a series of catalytic steps extending from aspartate to O-acetyl homoserine on the biosynthesis pathway, thereby producing O-acetyl-L-homoserine in high yield. This O-acetyl homoserine-producing strain, CJM-XPA2 (pCJ-thrA(M)-metX-CL), named “Escherichia coli CA05-0567” was deposited at KCCM (Korean Culture of Microorganism, Eulim build, Hongje-1-Dong, Seodaemun-ku, Seoul, 361-221, Korea) on Aug. 11, 2009, with the accession No. KCCM11025P.
An L-methionine-producing strain may be prepared on the basis of an L-lysine-, an L-threonine-, or an L-isoleucine-producing strain, and preferably on the basis of an L-threonine-producing strain. In this case, these strains have already been adapted to synthesize homoserine and can be further engineered to produce the methionine precursor in a large amount by increasing the expression of metX.
As used herein, the term “L-threonine-producing strain” is intended to refer to a prokaryotic or eukaryotic microorganism which can produce L-threonine intracellularly. Examples of the strain useful in the present invention include Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacteria sp., Pseudomonas sp. or Brevibacteria sp., with preference for Escherichia sp. More preferred is Escherichia coli. 
In a preferred embodiment of the present invention, the L-threonine-producing strain FRT2533 disclosed in WO 2005/075625 may be used. FTR2533 is derived from Escherichia coli TFR7624 which originates from the Escherichia coli Accession No. KCCM10236 which is in turn based on Escherichia coli TF4076. Escherichia coli Accession No. KCCM10236 expresses in high levels the ppc gene encoding an enzyme responsible for the formation of oxaloacetate from PEP, together with the genes encoding enzymes essential for the biosynthesis of threonine from aspartate, including thrA (aspartokinase, 1-homoserine dehydrogenase), thrB (homoserine kinase), and thrC (threonine synthase), thus showing increased productivity of L-threonine. Escherichia coli TFR7624 (KCCM10538) carries an inactivated tyrR gene which represses the expression of tyrB gene necessary for L-threonine biosynthesis. Escherichia coli FTR2533 (KCCM10541) is an L-threonine-producing E. coli strain carrying an inactivated galR gene.
In a preferred embodiment of the present invention, CJM2-X/pthrA(M)-CL (Accession No. KCCM 10925P), disclosed in U.S. Ser. No. 12/062,835, may be used. This strain is derived from E. coli FTR2533 by deleting metB, thrB, metJ and metA genes and inserting a Deinococcus radiodurans-derived metX gene at the metA locus, followed by transformation with an expression vector carrying a thrA gene.
Also, in a preferred embodiment of the present invention, CJM-X/pthrA(M)-CL (Accession No. KCCM 10921P), disclosed in U.S. Ser. No. 12/062,835, may be used. This strain is derived from E. coli CJM002 (Accession No. KCCM10568) by deleting metB, thrB, metJ and metA genes and inserting a Deinococcus radiodurans-derived metX gene at the metA locus, followed by transformation with an expression vector carrying a thrA gene.
In concrete examples of the present invention, two copies of each of ppc, aspC and asd genes are integrated into the chromosome of E. coli. To this end, recombinant plasmids for the integration of respective genes are constructed, as shown in FIG. 2 for pSG-2ppc, FIG. 3 for pSG-2aspC, and FIG. 4 for pSG-2asd. Also, a recombinant expression vector pCJ-thrA(M)-metX-CL is constructed to express both thrA and metX simultaneously (FIG. 5). The recombinant vectors pSG-2ppc, pSG-2aspC and pSG-2asd are sequentially transformed into the CJM-X/pthrA(M)-CL strain enhanced with thrA and metX genes, disclosed in U.S. Ser. No. 12/062,835 (Accession No. KCCM 10921P). The transformed strain has two copies of each of the genes ppc, aspC, and asd integrated into the chromosome thereof and is named CJM-XPA2. Following transformed with the pCJ-thrA(M)-metX-CL vector, this mutant strain is cultured in flasks to quantitatively analyze the production of O-acetyl homoserine. Compared to the control CJM-X/pthrA(M)-CL (accession no. KCCM 10921P), the production yield of O-acetyl homoserine was found to increase by 3.6% from 29.1% to 32.7% in the strain having two copies of each of ppc, aspC, asd genes (responsible for the conversion of phosphoenolpyruvate to aspartate) integrated into the chromosome thereof, and by as high as 16.9% from 29.1% to 46% in the strain anchoring all of the genes ppc, aspC, asd, thrA and metX (responsible for the biosynthesis pathway from phosphoenolpyruvate to O-acetyl homoserine) in the form of chromosomal DNA or plasmid DNA. In consideration of the fact that the production yield of O-acetyl homoserine was 32.7% upon the enhancement of only the genes ppc, aspC and asd (responsible for the conversion of phosphoenolpyruvate to aspartate) and 37.5% upon the enhancement of only thrA and metX, when all of the genes responsible for the entire biosynthesis pathway extending from phosphoenolpyruvate to O-acetyl homoserine are enhanced together, the production yield of O-acetyl homoserine is further increased to 46% (Example 2, Table 2). Therefore, the strain prepared according to the present invention produces O-acetyl homoserine in greater yield than does the wild-type counterpart.
In accordance with another aspect thereof, the present invention is directed to a method of producing O-acetyl-homoserine, comprising the fermentation of the O-acetyl-homoserine producing E. coli strain in a culture medium to accumulate O-acetyl-homoserine in the medium.
In accordance with a further aspect thereof, the present invention is directed to a method of producing L-methionine and acetate, comprising (a) producing O-acetyl-homoserine through the fermentation of the O-acetyl homoserine-producing the strain of Escherichia sp. of the present invention; (b) separating the O-acetyl homoserine; and (c) converting the separated O-acetylhomoserine, together with methylmercaptan, into L-methionine and acetate in the presence of a transferase selected from among cystathionine gamma synthase, O-acetylhomoserine sulfhydrylase and O-succinylhomoserine sulfhydrylase.
When used in connection with the strain of the present invention, the method of producing L-methionine, which is based on the use of the converting enzyme, cystathionine gamma synthase, O-acetylhomoserine sulfhydrylase or O-succinylhomoserine sulfhydrylase as disclosed in WO 2008/013432, issued to the present inventors, can bring about a higher yield in L-methionine production.
The O-acetyl-L-homoserine-producing strain prepared above can be cultured in a medium and conditions known in the art. As is well understood by those familiar with the art, the culture method may be adjusted according to the strain used. The fermentation may be carried out in a batch, a continuous culture, or a fed-batch type, but is not limited thereto. A variety of fermentation methods are described in the following reference: “Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176.
The culture medium has to meet the culture conditions for a specific strain. A variety of microorganism culture mediums are described in the following reference: “Manual of Methods for General Bacteriology” by the American Society for Bacteriology, Washington D.C., USA, 1981. Generally, a culture medium includes various carbon sources, nitrogen sources and trace elements. Examples of the carbon source include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; fats such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of the nitrogen source include organic nitrogen sources, such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and bean flour, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, which may be used alone or in combination. Additionally, the medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate and/or the corresponding sodium-containing salts thereof. Also, metal may be contained in the form of salts, like magnesium sulfate or iron sulfate, in the medium. In addition, amino acids, vitamins and proper precursors can be added as well. The mediums or the precursors can be added to the culture by batch-type or continuous type.
The pH of the culture can be adjusted with a suitable compound, for example, ammonium hydroxide, potassium hydroxide, ammonia, phosphate acid, and sulfuric acid. In order to inhibit the generation of bubbles in the culture, a defoaming agent such as fatty acid polyglycol ester may be used. To create aerobic conditions, the culture medium may be aerated with oxygen or oxygen-containing gas (e.g., air). The culture medium is maintained at 20 ̃45° C. and preferably at 25 ̃40° C. The strain is cultured to a desired level of the L-methionine precursor preferably for 10 ̃160 hrs.
A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.
EXAMPLE 1
Preparation of O-Acetyl Homoserine-Producing Strain
<1-1> Construction of pSG Vector for Chromosomal Integration of ppc
For use in the integration of ppc into the chromosome of E. coli, a pSG-2ppc vector was constructed.
The base sequence of the ppc gene was obtained from the GenBank database of the NIH (NCBI-gi: 89110074). On the basis of this base sequence, two sets of primers for the amplification of the ppc gene were synthesized: one set starting from 200 bp upstream of the start codon of the ppc ORF and containing the restriction enzyme sites EcoRI and Sad (SEQ ID NOS. 1 and 2); and the other set starting from 200 bp upstream of the start codon of the ppc ORF and containing the restriction enzyme sites Sad and KpnI (SEQ ID NOS. 3 and 4).
While the chromosomal DNA of Escherichia coli W3110 served as a template, PCR was performed using a set of primers of SEQ ID NOS. 1 and 2 or SEQ ID NOS. 3 and 4 in the presence of high-fidelity DNA polymerase PfuUltraTM (Stratagene), with 30 cycles of denaturation at 96° C. for 30 sec; annealing at 50° C. for 30 sec; and extension at 72° C. for 4 min.
The PCR products thus obtained were two kinds of about 3.1 kb ppc genes containing EcoRI and Sad sites, and Sad and KpnI sites therein, respectively.
After digestion with the restriction enzymes EcoRI and SacI, and SacI and KpnI, respectively, the two amplified ppc genes were ligated to each other and inserted into a pSG76-C vector treated with restriction enzymes EcoRI and KpnI (J Bacteriol. 1997 July; 179(13):4426-8), to construct a recombinant plasmid pSG-2ppc carrying two copies of the ppc gene. FIG. 2 shows the genetic map and construction of the vector pSG-2ppc for chromosomal integration of 2 copies of ppc.
<1-2> Construction of pSG Vector for Chromosomal Integration of aspC
For use in the integration of aspC into the chromosome of E. coli, a pSG-2aspC vector was constructed.
The base sequence of the aspC gene was obtained from the GenBank database of the NIH (NCBI-gi: 85674274). On the basis of this base sequence, a set of primers for the amplification of the aspC gene was designed to start from 200 bp upstream of the start codon of the aspC ORF and contain the restriction enzyme site BamHI (SEQ ID NOS. 5 and 6).
While the chromosomal DNA of Escherichia coli W3110 served as a template, PCR was performed using a set of primers of SEQ ID NOS. 5 and 6 in the presence of high-fidelity DNA polymerase PfuUltraTM (Stratagene), with 30 cycles of denaturation at 96° C. for 30 sec; annealing at 50° C. for 30 sec; and extension at 72° C. for 2 min.
The PCR product thus obtained was an about 1.5 kb aspC gene containing a BamHI site therein.
After digestion with the restriction enzyme BamHI, the amplified apsC gene was ligated to a pSG76-C vector treated with the same restriction enzyme to construct a recombinant plasmid pSG-2aspC carrying two copies of the aspC gene. FIG. 3 shows the genetic map and construction of the vector pSG-2aspC for chromosomal integration of 2 copies of aspC.
<1-3> Construction of pSG Vector for Chromosomal Integration of asd
For use in the integration of asd into the chromosome of E. coli, a pSG-2asd vector was constructed.
The base sequence of the asd gene was obtained from the GenBank database of the NIH (NCBI-gi: 89110578). On the basis of this base sequence, two sets of primers for the amplification of the asd gene were synthesized: one set starting from 200 bp upstream of the start codon of the asd ORF and containing the restriction enzyme sites EcoRI and XbaI (SEQ ID NOS. 7 and 8); and the other set starting from 200 bp upstream of the start codon of the asd ORF and containing the restriction enzyme sites XbaI and EcoRI (SEQ ID NOS. 9 and 10).
While the chromosomal DNA of Escherichia coli W3110 served as a template, PCR was performed using a set of primers of SEQ ID NOS. 7 and 8 or SEQ ID NOS. 9 and 10 in the presence of high-fidelity DNA polymerase PfuUltraTM (Stratagene), with 30 cycles of denaturation at 96° C. for 30 sec; annealing at 50° C. for 30 sec; and extension at 72° C. for 2 min.
The PCR products thus obtained were two kinds of about 1.5 kb asd genes containing EcoRI and XbaI sites, and XbaI and EcoRI sites therein, respectively.
After digestion with the restriction enzymes EcoRI and XbaI, the amplified asd genes were ligated to each other and inserted into a pSG76-C vector treated with restriction enzyme EcoRI to construct a recombinant plasmid pSG-2asd carrying two copies of the asd gene. FIG. 4 shows the genetic map and construction of the vector pSG-2asd for chromosomal integration of 2 copies of asd.
<1-4> Construction of Recombinant pCJ-thrA(M)-metX-CL for Expression of ThrA and MetX
For the biosynthesis of O-acetyl homoserine, thrA and metX were enhanced by introduction of a recombinant expression vector carrying the genes.
A nucleotide sequence of metX gene was obtained from NIH GenBank (NCBI gi: 1799718). On the basis of this nucleotide sequence, a set of primers was designed to cover a metX ORF ranging from ATG to TAA and have the restriction enzyme site HindIII at both ends thereof (SEQ ID NOS. 11 and 12).
Using the primers of SEQ ID NOS. 11 and 12, PCR was performed in the presence of high-fidelity DNA polymerase with 30 cycles of denaturation at 96° C. for 30 sec; annealing at 50° C. for 30 sec; and extension at 72° C. for 2 min during which the chromosomal DNA of Deinococcus radioduran R1 served as a template.
The PCR product thus obtained was an about 1 kb metX gene containing the restriction enzyme site HindIII.
After digestion with the restriction enzyme HindIII, the amplified metX gene was ligated to the thrA expression vector pCJ-thrA(M)-CL plasmid, disclosed in U.S. Ser. No. 12/062,835, which was previously treated with the same restriction enzyme, so as to construct a recombinant expression vector carrying both thrA and metX, termed pCJ-thrA(M)-metX-CL (FIG. 5).
<1-5> Preparation of O-Acetyl-Homoserine-Producing Strain
The plasmid pSG-2ppc carrying two copies of ppc gene, constructed in Example <1-1>, was transformed into the strain disclosed in U.S. Ser. No. 12/062,835, CJM-X/pthrA(M)-CL (Accession No. KCCM 10921P), followed by incubation on LB-Cm plates (Yeast extract 10 g/L, NaCl 5 g/L, Tryptone 10 g/L, chloramphenicol 25 μg/L) to select 10 chloramphenicol-resistant colonies for each transformant. The selected transformant anchored the pSG-2ppc vector at the chromosomal ppc site thereof. Then, the strain with two copies of ppc gene inserted thereinto was transformed with pAScep, an expression vector carrying the restriction enzyme I-SceI, so as to cleave the I-SceI site present in the pSG vector, followed by selection on LB-Ap (Yeast extract 10 g/L, NaCl 5 g/L, Tryptone 10 g/L, Ampicillin 100 μg/L). As a result, selected was a strain in which 2 copies of ppc gene were anchored at the chromosome thereof, with the pSG76-C vector removed therefrom. The same procedure as in the pSG-2ppc plasmid was repeated for pSG76C-2aspC and pSG76C-2asd vectors, constructed in Examples <1-2> and <1-3>, respectively, in order. Finally, a strain was derived from CJM-X/pthrA(M)-CL (Accession No. 10921P) with two copies of each of ppc, asd, and aspC inserted into the chromosome thereof, and was named CJM-XPA2.
Further, the CJM-XPA2 strain was transformed with the pCJ-thrA(M)-metX-CL vector constructed in Example <1-4> and then cultured on LB-Sp (Yeast extract 10 g/L, NaCl 5 g/L, Tryptone 10 g/L, Spectinomycin 25 μg/L) to select 10 colonies resistant to spectinomycin. The CJM-XPA2 (pCJ-thrA(M)-metX-CL), named “Escherichia coli CA05-0567” was deposited at KCCM (Korean Culture of Microorganism, Eulim build, Hongje-1-Dong, Seodaemun-ku, Seoul, 361-221, Korea) on Aug. 11, 2009, with the accession No. KCCM11025P. They were compared to each other for O-acetyl homoserine productivity.
EXAMPLE 2
Fermentation for O-Acetyl Homoserine Production
In order to examine the strains prepared in Example 1 for ability to produce the methionine precursor O-acetyl homoserine, they were cultured in Erlenmeyer flasks.
For this culture, the O-acetyl-homoserine titer medium shown in Table 1 was employed.







TABLE 1







Composition of Medium for O-Acetyl-Homoserine Production










Composition
Concentration(per liter)















Glucose
60
g



Ammonium Sulfate
17
g



KH2PO4
1.0
g



MgSO4•7H2O
0.5
g



FeSO4•7H2O
5
mg



MnSO4•8H2O
5
mg



ZnSO4
5
mg



CaCO3
30
g



Yeast Extract
2
g



Methionine
0.15
g



Threonine
0.15
g










Single colonies which were generated on LB plates during incubation overnight at 32° C. were taken with platinum loops and inoculated respectively into 25 mL of the O-acetyl homoserine titer medium, followed by culturing at 32° C. for 42 ̃64 hrs with shaking at 250 rpm. Each culture was quantitatively analyzed for O-acetyl homoserine using HPLC. The analysis data are summarized in Table 2, below.
Compared to the control CJM-X/pthrA(M)-CL (accession no. KCCM 10921P), as shown in Table 2, the production yield of O-acetyl homoserine was found to increase by 3.6% from 29.1% to 32.7% in the strain having two copies of each of ppc, aspC, asd genes, responsible for the conversion of phosphoenolpyruvate to aspartate, integrated into the chromosome thereof, and by as high as 16.9% from 29.1% to 46% in the strain anchoring all of the genes ppc, aspC, asd, thrA and metX, responsible for the biosynthesis pathway from phosphoenolpyruvate to O-acetyl homoserine, in the form of chromosomal DNA or plasmid DNA.
Taken together, the data obtained in the flask tests indicate that, in consideration of the fact that the production yield of O-acetyl homoserine is 32.7% upon the enhancement of only the genes ppc, aspC and asd, responsible for the conversion of phosphoenolpyruvate to aspartate, and 37.5% upon the enhancement of only thrA and metX, when all of the genes responsible for the entire biosynthesis pathway extending from phosphoenolpyruvate to O-acetyl homoserine are enhanced together, the production yield of O-acetyl homoserine is further increased to 46%. Therefore, the strain prepared according to the present invention produced O-acetyl homoserine in greater yield than does the wild-type counterpart.







TABLE 2







Flask Tests for O-Acetyl-Homoserine Production












OAH
Yield


Strain
Plasmid
production (g/L)
(%)





CJM-X/pthrA(M)-CL
—
17.5
29.1


(Accession No.
pCJ-thrA(M)-metX-
22.5
37.5


KCCM 10921P)
CL


CJM-XPA2
—
19.6
32.7



pCJ-thrA(M)-metX-
27.6
46.0



CL









INDUSTRIAL APPLICABILITY
As described hitherto, the present invention provides a strain of Escherichia sp. which produces O-acetyl homoserine in high yield in a culture medium when fermented in the medium. In addition, the O-acetyl homoserine can be converted, along with methyl mercaptan, by the two-step process into L-methionine, with the concomitant production of acetic acid.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

## Claims

1. A strain of Escherichia coli, capable of producing O-acetyl homoserine in high yield, which overexpresses homoserine acetyl transferase, aspartokinase, homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, and aspartate semi-aldehyde dehydrogenase, wherein aspartokinase and homoserine dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, aspartate semi-aldehyde dehydrogenase, and homoserine acetyl transferase are encoded by respective genes thrA, ppc, aspC and asd from Escherichia coli and metX from Deinococcus radiodurans.

2. The strain as defined in claim 1, wherein the overexpression of the activity is achieved by transformation with a plasmid carrying a corresponding gene, by increasing a number of copies of a corresponding gene, or by an employment of a strong promoter for a corresponding gene.

3. The strain as defined in claim 2, wherein the overexpression of homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase is achieved by introducing a plasmid carrying metX and thrA genes.

4. The strain as defined in claim 1, wherein the homoserine acetyl transferase has an amino acid sequence of SEQ ID NO:18, 19, or 20.

5. The strain as defined in claim 1, wherein the homoserine acetyl transferase has an amino acid sequence from Deinococcus radiodurans Q9RVZ8.

6. The strain as defined in claim 1, being from a strain capable of producing L-threonine, L-isoleucine or L-lysine.

7. The strain as defined in claim 1, being from E. coli CJM-X/pthrA (M)-CL, Accession No. KCCM 10921P.

8. The strain as defined in claim 1, being from E. coli CJM2-X/pthrA(M)-CL, Accession No. KCCM 10925P.

9. The strain as defined in claim 1, being from E. coli FTR2533, Accession No. KCCM 10541.

10. The strain as defined in claim 1, being deposited with Accession No. KCCM 11025P.