阅读说明：本技术 生产o-乙酰基高丝氨酸的微生物和用其生产o-乙酰基高丝氨酸的方法 (Microorganism producing O-acetylhomoserine and method for producing O-acetylhomoserine using the same ) 是由 金贤雅 徐主熙 申容旭 金素影 金相谦 罗光镐 裵智妍 孙晟光 柳慧连 崔珍根 于 2015-06-22 设计创作，主要内容包括：公开了生产O-乙酰基高丝氨酸的埃希氏菌属微生物,和利用该微生物高产率生产O-乙酰基高丝氨酸的方法。(Disclosed are an Escherichia microorganism producing O-acetylhomoserine, and a method for producing O-acetylhomoserine with high yield using the microorganism.)

1. A method for producing L-methionine, comprising:

(a) culturing a microorganism belonging to the genus Escherichia (Escherichia sp) to produce O-acetylhomoserine, wherein endogenous activity of citrate synthase of the microorganism is attenuated or inactivated; and

(b) contacting O-acetylhomoserine produced in step (a) with O-acetylhomoserine sulfhydrylase or a microorganism having O-acetylhomoserine sulfhydrylase.

2. The method of claim 1, wherein the microorganism having attenuated endogenous activity of citrate synthase has an amino acid sequence encoded by SEQ ID NO: 1 or SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the microorganism of (a) further has an activity of cystathionine gamma synthase, homoserine kinase, or both attenuated or inactivated compared to its endogenous activity.

4. The method according to claim 1, wherein the microorganism of (a) further has an introduced or enhanced homoserine O-acetyltransferase activity or an endogenous homoserine O-succinyltransferase further modified to have an amino acid sequence identical to SEQ ID NO: 16 and further has mutations at the G111E position and the L112T or L112H position to have homoserine O-acetyltransferase activity.

5. The method of claim 1, wherein the microorganism of (a) further has an introduced or enhanced activity of at least one protein selected from the group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semialdehyde dehydrogenase.

6. The process according to claim 1, wherein the microorganism of (a) is Escherichia coli (Escherichia coli).

7. The method according to claim 1, wherein the O-acetylhomoserine sulfhydrylase is derived from Leptospira sp, Chromobacterium sp, or Phomonas sp.

8. The method of claim 1, further comprising adding methyl mercaptan as a substrate in step (b).

9. The method according to claim 1, further comprising recovering O-acetylhomoserine produced in step (a).

10. The process of claim 1, further comprising recovering the L-methionine produced in step (b).

Technical Field

The present invention relates to an Escherichia sp microorganism producing O-acetylhomoserine, and a method for producing O-acetylhomoserine with high yield using the same.

Background

O-acetylhomoserine is a precursor of methionine, which is one of essential amino acids of the body. Methionine has been widely used as a component of medical infusions and as a raw material for medical products as well as animal feed and food additives.

Methionine can be synthesized biologically or chemically. Recently, a two-step process has been disclosed in which an L-methionine precursor produced by fermentation is converted into L-methionine by an enzymatic reaction (International publication No. WO 2008/013432). In the two-step process described above, O-succinylhomoserine and O-acetylhomoserine can be used as methionine precursor, and it is important to produce O-acetylhomoserine at high yield to produce methionine efficiently on a large scale.

Disclosure of Invention

Technical problem

The present inventors have found that reduction in the expression or activity of citrate synthase protein can significantly increase the production capacity of O-acetylhomoserine when they attempt to improve O-acetylhomoserine production, thereby completing the present invention.

Technical scheme

It is an object of the present invention to provide an O-acetylhomoserine producing microorganism with improved O-acetylhomoserine production ability.

It is another object of the present invention to provide a method for producing O-acetylhomoserine using the microorganism.

Advantageous effects

O-acetylhomoserine can be produced in a higher yield and in a more environment-friendly manner than by chemical synthesis using the microorganism having O-acetylhomoserine production ability according to the present invention. In addition, O-acetylhomoserine thus produced can be used as a precursor for methionine and acetic acid synthesis by O-acetylhomoserine sulfhydrylase to achieve bioconversion of L-methionine, and the L-methionine thus converted can be widely used for the production of human food or food additives and animal feed or animal feed additives.

Drawings

FIG. 1 is a design of an expression cassette for constructing a microorganism with reduced citrate synthase activity.

FIG. 2 is a restriction map of pBAD 24-citrate synthase antisense RNA (asRNA) vector.

Detailed Description

In one aspect, the present invention provides an Escherichia microorganism producing O-acetylhomoserine, in which the activity of an endogenous citrate synthase protein is attenuated or inactivated.

As used herein, the term "O-acetylhomoserine", which is a specific intermediate material in the methionine biosynthetic pathway of microorganisms, means an acetyl derivative of L-homoserine. O-acetylhomoserine can be produced by an enzyme activity of transferring acetyl group from acetyl-CoA to homoserine using homoserine and acetyl-CoA as substrates.

As used herein, the term "microorganism producing O-acetylhomoserine" includes microorganisms: a eukaryotic or prokaryotic microorganism producing O-acetylhomoserine in a living organism, which has O-acetylhomoserine producing ability, and a parent microorganism thereof does not have O-acetylhomoserine producing ability; or a microorganism which: endogenously has O-acetylhomoserine producing ability.

O-acetylhomoserine production ability can be provided or promoted by modification of species. Microorganisms having an O-acetylhomoserine producing ability may include microorganisms belonging to the genera Escherichia, Erwinia (Erwinia sp.), Serratia (Serratia sp.), Providencia (Providecia sp.), Corynebacterium (Corynebacterium sp.), Pseudomonas (Pseudomonas sp.), Leptospira (Leptospira sp.), Salmonella (Salmonella sp.), Brevibacterium (Brevibacterium sp.), Hypomonas sp., Chromobacterium sp., and Nocardia (Norcardia sp.), or fungi or yeasts; specifically, microorganisms belonging to the genus Escherichia, Corynebacterium, Leptospira, and yeast; and more specifically, a microorganism belonging to the genus Escherichia, specifically, for example, Escherichia Coli (Escherichia Coli). The microorganism having O-acetylhomoserine producing ability may be a microorganism producing the following: l-lysine, L-threonine, L-isoleucine, or L-methionine or a derivative thereof, but not limited thereto.

As used herein, the term "citrate synthase (e.c. 2.3.3.1)" means an enzyme in the first step of the TCA cycle that mediates the reaction between oxaloacetate (oxaloacetate) and acetyl-coa. Specifically, citrate synthase mediates a condensation reaction between an acetate residue having two carbon atoms and oxaloacetate having four carbon atoms in acetyl-coa, thereby generating citrate having six carbon atoms (citrate). In E.coli, citrate synthase is named GltA, and citrate synthase and GltA are used interchangeably in the present invention.

Acetyl coenzyme A + oxaloacetate + H₂O → citrate + coenzyme A-SH

Specifically, the citrate synthase may be a citrate synthase derived from Escherichia, more specifically GltA derived from Escherichia coli. The citrate synthase may be a protein that: comprises the amino acid sequence of SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof; or with SEQ ID NO: 4, or a sequence having a homology of 70% or more, specifically 80% or more, or more specifically 90% or more. In addition, as a sequence having homology, if the amino acid sequence is a sequence having a sequence identical to SEQ ID NO: 4 the same or corresponding citrate synthase activity, amino acid sequences having a knockout, modification, substitution, or addition in a partial sequence are obviously also intended to be included in the scope of the present invention. In addition, polynucleotide sequences encoding the same amino acid sequences and variants thereof are intended to be included within the scope of the present invention based on the degeneracy of the genetic code.

The term "endogenous" activity, as used herein, means either the native state of a protein in a microorganism or the active state of the corresponding protein provided in the microorganism prior to modification.

By "attenuation or inactivation of protein activity as compared to its endogenous activity" is meant that the activity of the protein is reduced or eliminated when compared to the activity it has in its native state. Attenuation is a concept representing the following: due to the modification of the protein-encoding gene, the activity of the protein is reduced as compared with the activity of the protein originally possessed by the microorganism; the overall protein expression level is lower than that of a natural type strain of the microorganism; or a combination thereof, but is not limited thereto. Inactivation includes the following conditions: the gene encoding the protein is not expressed at all compared to the native type strain; and the gene is expressed, but not active.

Attenuation or inactivation of protein activity can be achieved by various methods known in the art. Examples of the method may include a method in which a gene encoding a protein on a chromosome is replaced with a modifying gene so that the enzyme activity (which includes a case where the activity of the protein is removed) can be decreased; a method of introducing a modification into an expression regulatory sequence of a gene encoding a protein on a chromosome; a method of replacing an expression regulatory sequence of a gene encoding a protein with a sequence having weak activity or no activity; a method of knocking out a part or the whole of a gene encoding a protein on a chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense RNA) that inhibits translation from mRNA to protein by complementarily binding to a transcript of a gene on a chromosome; a method for rendering ribosome attachment impossible-secondary structure is formed by artificially adding Shine-Dalgarno (SD) sequence and its complementary sequence to the front end of SD sequence of gene encoding protein; reverse Transcription Engineering (RTE) method-addition of a promoter to reverse transcribe at the 3' end of the Open Reading Frame (ORF) of the corresponding sequence; etc., and combinations thereof are also included, but not limited thereto.

Specifically, a method of knocking out a part or the whole of a gene encoding a protein can be performed by: the polynucleotide encoding the endogenous protein of interest in the chromosome is replaced with the polynucleotide or (when part of the polynucleotide sequence is knocked out) the marker via insertion of the chromosome into the vector of the microorganism. For example, a method of gene knockout by homologous recombination can be employed, but is not limited thereto. In addition, as used herein, the term "portion", although variable depending on the kind of polynucleotide, may specifically mean 1 nucleotide to 300 nucleotides, more specifically 1 nucleotide to 100 nucleotides, still more specifically 1 nucleotide to 50 nucleotides, but is not limited thereto.

In addition, the method of modifying the expression regulatory sequence can be performed by: inducing an expression control sequence alteration of the polynucleotide sequence by a knockout, insertion, conservative substitution, non-conservative substitution, or a combination thereof to further attenuate the activity of the expression control sequence; or replacing the polynucleotide sequence with a less active polynucleotide sequence. The polynucleotide sequence may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding domain, and sequences that regulate termination of transcription and translation.

In addition, the method of modifying the gene sequence on the chromosome can be performed by: inducing the sequence change-by knock out, insertion, conservative substitution, non-conservative substitution, or a combination thereof, to further attenuate the activity of the expression regulatory sequence; or replacing the sequence with a modified gene sequence having a weaker activity or a modified gene sequence having no activity, but is not limited thereto.

Specifically, with respect to the reduction of the citrate synthase protein activity, a part of amino acid(s) in the amino acid sequence of the citrate synthase protein may be substituted with other amino acid(s). More specifically, a citrate synthase having the following amino acid sequence may be included: the amino acid at position 145 or the amino acid at position 167 in the amino acid sequence of the citrate synthase protein is substituted with tyrosine (Y) or lysine (K) to other amino acid(s). Still more specifically, the citrate synthase can be a citrate synthase having a gene sequence encoding a modified polypeptide: wherein the amino acid at position 145 in the amino acid sequence of the citrate synthase protein is substituted with tyrosine (Y) to alanine (A), and the amino acid at position 167 is substituted with lysine (K) to alanine (A). Specifically, after setting the amino acid next to methionine encoded by the start codon to the 1 st amino acid, the number of amino acid residues is determined in the order of sequence. The polypeptides may have SEQ ID NOs: 1 or 2. In addition, if the activity of citrate synthase is weaker than that of the wild type, the citrate synthase may include a citrate synthase having a sequence identical to that of SEQ id no: 1 or 2, or an amino acid sequence having 80% or more, specifically 90% or more, more specifically 95% or more, still more specifically 97% or more homology thereto. As a sequence having homology, if the amino acid sequence is a sequence identical to SEQ ID NO: 1 or 2, and amino acid sequences having knockout, modification, substitution, or addition in partial sequences are also obviously included in the scope of the present invention.

The term "homology", as used herein, means the percentage of identity between two polynucleotide or polypeptide portions. Homology between the sequences of one portion and another can be determined by techniques known in the art. For example, homology can be determined by: sequence information is directly arranged and readily obtained between two different polynucleotide molecules or two different polypeptides using computer program alignment. Computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), and the like. In addition, the homology between polynucleotides can be determined by: hybridizing the polynucleotides under conditions that form a stable double strand between the homologous regions, digesting with a single strand specific nuclease, and determining digested fragments.

As used herein, the term "homology" means the relationship between proteins having "common evolutionary origin", including homologous proteins derived from all grammatical forms or spelling variants of a superfamily of proteins, and homologous proteins derived from different species. These proteins (and their encoding genes) have sequence homology reflected by a high level of sequence similarity. However, the term "homology", as commonly used and as used herein, means sequence similarity modified by an adjective such as "very high", rather than meaning a common evolutionary origin.

In exemplary embodiments of the invention, the microorganism may be a cystathionine gamma synthase (EC 2.5.1.48), a homoserine kinase (EC 2.7.1.39), or both, which have weaker activity than their endogenous activity or are inactivated.

As used herein, the term "cystathionine gamma synthase" means an enzyme that can synthesize cystathionine by the following chemical reaction using O-succinylhomoserine and L-cysteine as substrates. In the present invention, cystathionine gamma synthase derived from Escherichia coli (E.coli) was named "MetB".

O-succinyl-L-homoserine + L-cysteine → L-cystathionine + succinate

Specifically, cystathionine gamma synthase derived from escherichia coli, although not particularly limited thereto, may be a cystathionine gamma synthase including SEQ ID NO: 9 or an amino acid sequence corresponding to SEQ ID NO: 9, a protein having an amino acid sequence with 70% or more, specifically 80% or more, more specifically 90% or more homology. In addition, as a sequence having homology, if the amino acid sequence is a sequence identical to SEQ ID NO: 9 has the same or corresponding homoserine kinase activity, and amino acid sequences having deletions, modifications, substitutions, or additions in the partial sequences are also obviously included in the scope of the present invention. In addition, polynucleotide sequences encoding the same amino acid sequences and variants thereof are also intended to be included within the scope of the present invention based on the degeneracy of the genetic code.

The method for attenuating and inactivating cystathionine gamma synthase activity can be carried out according to the above-mentioned method.

As used herein, the term "homoserine kinase" means an enzyme that causes homoserine phosphorylation, which undergoes the following chemical reaction. In the present invention, homoserine kinase derived from Escherichia coli is named "ThrB".

ATP + L-homoserine → ADP + O-phospho-L-homoserine

Specifically, homoserine kinase derived from escherichia, although not particularly limited thereto, may be a homoserine kinase including SEQ ID NO: 11 or an amino acid sequence substantially identical to SEQ ID NO: 11, or a protein having an amino acid sequence with 70% or more homology, specifically 80% or more homology, or more specifically 90% or more homology. In addition, as a sequence having homology, if the amino acid sequence is a sequence identical to SEQ ID NO: 11 has the same or corresponding homoserine kinase activity, amino acid sequences having a deletion, modification, substitution, or addition in a partial sequence should be obviously included in the scope of the present invention. In addition, polynucleotide sequences encoding the same amino acid sequences and variants thereof are also intended to be included within the scope of the present invention based on the degeneracy of the genetic code.

The method for attenuating and inactivating the homoserine kinase activity can be performed as described above.

In a particular aspect of the invention, the microorganism may be a microorganism: wherein the activity of homoserine O-acetyltransferase is further introduced or enhanced, or endogenous homoserine O-succinyltransferase is further modified to have the activity of homoserine O-acetyltransferase.

As used herein, the term "homoserine O-acetyltransferase (EC 2.3.1.31)" means an enzyme having the activity of transferring acetyl group from acetyl-coa to homoserine.

Specifically, homoserine O-acetyltransferase activity can be introduced into the microorganism according to the present invention. The homoserine O-acetyltransferase can be derived from various microbial species, for example, a microorganism selected from the group consisting of: corynebacterium, Leptospira, Deinococcus (Deinococcus sp.), Deinococcus, Pseudomonas, and Mycobacterium (Mycobacterium sp.). Specifically, the homoserine O-acetyltransferase may be a homoserine O-acetyltransferase including an amino acid sequence represented by: SEQ ID NO: 13 (Leptospira mairei (Leptospira meyeri)), SEQ ID NO: 14 (Corynebacterium glutamicum), or SEQ ID NO: 15 (Deinococcus radiodurans), but is not limited thereto. In addition, the homoserine O-acetyltransferase can be a protein comprising the above amino acid sequence or an amino acid sequence having 70% or more, specifically 80% or more, or more specifically 90% or more homology with the above amino acid sequence. In addition, polynucleotide sequences encoding the same amino acid sequences and variants thereof are also intended to be included within the scope of the present invention based on the degeneracy of the genetic code.

Examples of homoserine O-acetyltransferase useful in the present invention are disclosed in Korean patent application laid-open No. 10-2011-0023703 and European patent application laid-open No. EP 2290051, the entire descriptions of which are incorporated herein by reference.

Further, a protein in which endogenous homoserine O-succinyl transferase (EC 2.3.1.46) is modified to have homoserine O-acetyl transferase activity means a polypeptide in which: wherein the substrate specificity of the polypeptide having homoserine O-succinyl transferase activity is changed from succinyl-CoA to acetyl-CoA. In addition, the modified protein, although not particularly limited thereto, may be a peptide having homoserine O-acetyltransferase activity which differs from its wild type by substitution of a part of the amino acid sequence of the polypeptide having homoserine O-succinyltransferase activity.

Examples of the homoserine O-succinyl transferase can be a polypeptide derived from Enterobacteriaceae (Enterobacteria sp.), Salmonella, Pseudomonas, Bacillus (Bacillus sp.), or Escherichia, specifically, a polypeptide having homoserine O-succinyl transferase activity derived from Escherichia, for example, a polypeptide having homoserine O-succinyl transferase activity derived from Escherichia coli. More specifically, homoserine O-succinyl transferase from escherichia coli can have the amino acid sequence of SEQ ID NO: 16, but is not limited thereto. The homoserine O-succinyl transferase from E.coli is designated "MetA".

The modified homoserine O-succinyl transferase can be a variant polypeptide, wherein SEQ ID NO: 16 or a polypeptide substantially similar to SEQ ID NO: 16 by substitution of glutamic acid for the 111 th amino acid and threonine or histidine for the 112 th amino acid in the polypeptide having 95% or more homology to the polynucleotide sequence. In particular, the variant polypeptide can be a polypeptide having the sequence of seq id NOS: 17 to 19. In addition, the variant polypeptide may be a protein comprising an amino acid sequence having 70% or more, specifically 80% or more, or more specifically 90% or more homology to the above-mentioned amino acid sequence. In addition, polynucleotide sequences encoding the same amino acid sequences and variants thereof are also intended to be included within the scope of the present invention based on the degeneracy of the genetic code. Information on the modified homoserine O-succinyl transferase can be obtained from Korean patent application laid-open No. 10-2012-0070531 or International publication No. WO2012/087039, the entire descriptions of which are incorporated herein by reference.

As used herein, the term "introducing or enhancing activity" means providing the activity of a particular protein to a microorganism that does not have that protein; or enhancing the intracellular activity of the protein in a microorganism having the protein, and the like, and means increasing the intracellular activity of the protein as compared with the endogenous activity of the protein.

As used herein, the term "introducing or enhancing protein activity" means not only achieving an effect higher than the original function due to the increase of the activity of the protein itself, but also increasing the activity of the protein due to the increase of the activity of an endogenous gene, amplification of an endogenous gene by internal or external factors, increase in copy number, introduction of a gene from the outside, increase in enzyme activity due to substitution, modification or mutation, and the like, but is not limited thereto.

In the above, the increase in the copy number of the gene, although not particularly limited thereto, may be performed in a state of being operably linked to a vector, or by inserting into a chromosome in a host cell. Specifically, the method may be performed by: introducing into a host cell a vector operably linked to a polynucleotide encoding a protein of the invention and capable of replication and function independently of the host; or introducing into the host cell a vector operably linked to the polynucleotide to insert the polynucleotide into the chromosome of the host cell, thereby increasing the copy number of the gene in the chromosome of the host cell.

The vector is a DNA construct: a polynucleotide sequence comprising a polynucleotide encoding a protein of interest operably linked to appropriate control sequences such that the protein of interest can be expressed in a suitable host, wherein the control sequences include a promoter to initiate transcription, a random operator sequence to control transcription, a sequence encoding an appropriate mRNA ribosome binding domain, and sequences that control transcription and translation. The vector may replicate or function independently of the host genome, or may be integrated into the host genome itself, following transformation into an appropriate host cell.

The vector used in the present invention may not be particularly limited as long as the vector is replicable in host cells, and any vector known in the art may be used. Examples of vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as phage vectors or cosmid vectors, pWE15, M13, λ MBL3, λ MBL4, λ ixi, λ ASHII, λ t10, λ t11, Charon4A, Charon21A, and the like; and as plasmid vectors, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

In addition, the polynucleotide encoding the endogenous protein of interest may be replaced with a modified polynucleotide using a vector inserted into the chromosome of the microorganism. Insertion of the polynucleotide into the chromosome can be performed using methods known in the art, for example, by homologous recombination. Since the vector of the present invention can be inserted into a chromosome by homologous recombination, a selection marker for confirming the insertion into a chromosome may be additionally included. Selection of transformed cells using a selection marker, i.e., confirming whether a polynucleotide of interest has been inserted, and markers providing alternative phenotypes such as drug resistance, nutritional requirements, cytotoxic agent resistance, and surface protein expression may be used, but are not limited thereto. In the case of treatment with a selection agent, only cells expressing the selection marker will survive or express other phenotypic characteristics, and thus transformed cells can be easily selected.

As used herein, the term "transformation" means the process of introducing a vector comprising a polynucleotide encoding a protein of interest into a host cell, thereby effecting expression of the polynucleotide encoding the protein in the host cell. With respect to the transformed polynucleotide, it does not matter whether it is inserted into and located in the chromosome of the host cell or is located extrachromosomally, as long as it can be expressed in the host cell. In addition, polynucleotides include DNA and RNA encoding proteins of interest. The polynucleotide may be inserted in any manner so long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, a genetic construct that includes all the essential elements required for self-expression. The expression cassette may conveniently include a promoter, transcription termination signal, ribosome binding domain, and translation termination signal operably linked to a polynucleotide, and may be in the form of an expression vector capable of autonomous replication. In addition, the polynucleotide may be introduced into the host cell as such, and operably linked to sequences necessary for its expression in the host cell. In addition, as used herein, the term "operably linked" means a functional linkage between a promoter sequence (which initiates and mediates transcription of a polynucleotide encoding a protein of interest) and a gene sequence.

Then, modification of the expression control sequence to increase polynucleotide expression may be performed, although not specifically limited thereto, by: inducing a polynucleotide sequence change via a knockout, insertion, conservative substitution, non-conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence; or replacing the polynucleotide sequence with a more active polynucleotide sequence. Expression control sequences, although not particularly limited thereto, may include a promoter, an operator sequence, a sequence encoding a ribosome binding domain, and sequences that control termination of transcription and translation, and the like. In addition, a strong exogenous promoter, instead of the original promoter, may be ligated to the upper end of the polynucleotide expression unit.

Furthermore, modification of the polynucleotide sequence on the chromosome may be performed by, although not specifically limited to: inducing an alteration in an expression control sequence of the polynucleotide sequence via a knockout, insertion, conservative substitution, non-conservative substitution, or a combination thereof, to further enhance the activity of the polynucleotide sequence; or replacing the polynucleotide sequence with a more active enhanced polynucleotide sequence.

In general, the introduction and enhancement of a protein activity can increase the activity or strength of the corresponding protein by at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to 1000% or 2000% relative to the activity or strength of the wild-type protein or the protein in the microorganism.

In addition, the microorganism may be a microorganism: wherein the activity of endogenous homoserine O-succinyl transferase is attenuated or inactivated compared to the endogenous activity, thereby enhancing the biosynthetic pathway of O-acetylhomoserine by blocking the pathway of O-succinylhomoserine biosynthesis from homoserine.

Attenuation and inactivation of homoserine O-succinyl transferase activity can be performed according to the above-described method.

In an exemplary embodiment of the present invention, the O-acetylhomoserine producing microorganism may be a microorganism in which: wherein the activity of an enzyme involved in the biosynthetic pathway from phosphoenolpyruvate (phosphoenolpyruvate) to homoserine is additionally introduced or enhanced to further increase the amount of homoserine (substrate for O-acetylhomoserine biosynthesis).

Specifically, the above-mentioned microorganism may be a microorganism: wherein the activity of at least one protein selected from the group consisting of phosphoenolpyruvate carboxylase (ppc, EC 4.1.1.31), aspartate aminotransferase (aspC, EC 2.6.1.1), and aspartate semialdehyde dehydrogenase (asd, EC1.2.1.11) is further introduced or enhanced.

For example, the coding sequence comprises SEQ ID NO: 20, encoding a phosphoenolpyruvate carboxylate comprising the amino acid sequence represented by SEQ ID NO: 21, and an aspC gene encoding an aspartate aminotransferase comprising the amino acid sequence represented by SEQ ID NO: 22 can be introduced into the microorganism. For example, the activities of these three different enzymes can be introduced and enhanced by: the copy number of the genes encoding these three different enzymes present in the chromosome of the host cell is all made to be at least 2, but is not limited thereto. The introduction and enhancement of the activity can be carried out as described above.

In exemplary embodiments of the present invention, the activity of citrate synthase protein is attenuated or inactivated by various methods, including the knock-out of the citrate synthase gene in an O-acetylhomoserine-producing Escherichia coli microorganism; introducing a gene encoding a modified citrate synthase protein having an attenuated activity compared to the wild type into a position of the citrate synthase gene; and an expression vector into which an antisense RNA of the citrate synthase gene is introduced. As a result, the thus constructed O-acetylhomoserine producing microorganism in which the citrate synthase protein activity is attenuated or inactivated, shows an improved O-acetylhomoserine production ability as compared with the parent microorganism (examples 1 to 4).

In another aspect, the present invention provides a method for producing O-acetylhomoserine using an O-acetylhomoserine producing microorganism having improved O-acetylhomoserine production ability. Specifically, the present invention provides a method for producing O-acetylhomoserine, comprising (a) culturing a microorganism; and (b) recovering O-acetylhomoserine produced during the culture of the microorganism.

The cultivation method of Escherichia coli having O-acetylhomoserine production ability according to the present invention can be performed according to an appropriate medium and culture conditions known in the art. The skilled person can easily adapt the cultivation process to the selected microorganism. Specifically, since the microorganism of the present invention is a microorganism in which the activity of citrate synthase (an enzyme mediating the first step of the TCA cycle) is attenuated or inactivated, the medium may include glutamate, but is not particularly limited thereto.

Examples of the culture method may include batch culture, continuous culture, and fed-batch culture, but are not limited thereto. These various methods are disclosed, for example, in "Biochemical Engineering", James M.Lee, Prentice-Hall International specifications, page 138-.

The medium used in the culture may suitably meet the requirements of the particular microorganism. Specifically, examples of microbial culture media are disclosed in "Manual of Methods for General Bacteriology", American Society for Bacteriology, Washington, DC, 1981. The medium may be a medium including a suitable carbon source, phosphorus source, inorganic compound, amino acid, and/or vitamin, etc., and the culture may be performed under aerobic conditions while adjusting temperature, pH, etc.

Examples of the carbon source may include carbohydrates such as glucose, lactose, sucrose, lactic acid, fructose, maltose, starch, and cellulose; fats, such as soybean oil, sunflower oil, castor oil, ber 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, but are not limited thereto.

Examples of the nitrogen source may include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, Corn Steep Liquor (CSL), and soybean powder; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources may be used alone or in combination, but are not limited thereto. The medium may further comprise potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as a phosphorus source. The culture medium may include metals such as magnesium sulfate and iron sulfate. In addition, amino acids, vitamins and suitable precursors may be included. These media or precursors may be added to the culture in the form of batch culture or continuous culture, but are not limited thereto.

In addition, the pH of the culture can be adjusted during the culture in a suitable manner by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. In addition, a defoaming agent such as fatty acid polyglycol ester can be used to prevent foam formation during the culture. In addition, in order to maintain aerobic conditions of the culture solution, oxygen or a gas containing oxygen (e.g., air) may be added to the culture. The culture temperature may be 20 ℃ to 45 ℃, specifically 25 ℃ to 40 ℃, but is not limited thereto. The culture may be continued until the production of O-acetylhomoserine reaches a target level, specifically for 10 hours to 160 hours, but is not limited thereto.

The method of producing O-acetylhomoserine of the present invention may further comprise recovering O-acetylhomoserine from the cultured microorganism or the culture product thereof. The recovery of the target O-acetylhomoserine can be performed by a microbial culture method according to the present invention, for example, an appropriate method known in the art, such as batch culture, continuous culture, and fed-batch culture.

Recovery may include a purification step.

O-acetylhomoserine thus recovered can produce methionine by a two-step process (Korean patent No. 10-0905381).

The two-step process includes a process for producing L-methionine and organic acids by using an enzyme having O-acetylhomoserine sulfhydrylase activity or an enzymatic reaction of a microorganism having such an enzyme, while using O-acetylhomoserine and methanethiol produced by an L-methionine precursor-producing microorganism as substrates.

More specifically, the present invention provides a method for producing L-methionine using O-acetylhomoserine accumulated by the above-described method as a substrate by enzymatic reaction of O-acetylhomoserine sulfhydrylase or the like.

In the second step, when O-acetylhomoserine is used as the L-methionine precursor, O-acetylhomoserine sulfhydrylase derived from microorganisms belonging specifically to the genera Leptospira, Chromobacterium, and Xenomyces (Hyphomonas sp.), more specifically Leptospira meyeri (Leptospira meyeri), Pseudomonas aeruginosa (Pseudomonas aurogenosa), Phomoplasma marinum (Hyphomonas Neptuninum), and Chromobacterium Violaceum (Chromobacterium Violaceae) can be used.

The reaction is as shown below.

An additional process for producing methionine is disclosed in Korean patent No. 10-0905381, the entire specification of which can be incorporated herein by reference.

Modes for carrying out the invention

The present invention is described in more detail below with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited to these examples.

Reference example: construction of O-acetylhomoserine-producing microorganism

<1-1> deletion of metB Gene derived from wild type Escherichia coli (International publication No. WO 2008/013432) an O-acetylhomoserine producing microorganism was constructed using Escherichia coli (a representative microorganism of the genus Escherichia). For this purpose, the wild-Type E.coli K12W3110(ATCC27325) obtained from the American Type Culture Collection (ATCC) was used. First, in order to block the synthetic pathway of O-succinyl-L-homoserine to cystathionine, metB gene encoding cystathionine synthase (SEQ ID NO: 10) was deleted. Specifically, the metB gene encoding cystathionine synthase was knocked out by FRT-one-step PCR knock-out method (PNAS (2000) vol 97: P6640-6645).

Specifically, using SEQ ID NOS: 30 and 31, the metB deletion cassette was constructed by PCR reaction based on pKD3 vector (PNAS (2000) vol 97: P6640-6645) as template as follows: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute, 30 cycles. The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and the 1.2kb DNA band thus obtained was purified. The recovered DNA fragment was electroporated into E.coli (K12) W3110(PNAS (2000) vol 97: P6640-6645) which had been transformed with pKD46 vector. For electroporation, pKD 46-transformed W3110 strain was cultured at 30 ℃ in LB medium containing 100. mu.g/L ampicillin and 5mM arabinose (L-arabinose) until OD₆₀₀0.6, and was used after washing twice with sterile distilled water and once with 10% glycerol. Electroporation was performed at 2500V. The recovered strain is contained inLB plates of 25. mu.g/L chloramphenicol were streaked (streak), cultured overnight at 37 ℃ and strains showing resistance were selected. The selected strain was subjected to PCR reaction under the same conditions using the same primers based on the strain as a template, and the deletion of metB gene was confirmed by observing the gene size of 1.2kb on 1.0% agarose gel. The thus confirmed strain was cultured in LB medium after being transformed again with pCP20 vector (PNAS (2000) vol 97: P6640-6645), and the final metB knock-out strain (in which the gene size was reduced to 150bp on 1.0% agarose gel by PCR performed under the same conditions) was constructed and the removal of chloramphenicol marker was confirmed. The strain thus constructed was named "W3-B".

<1-2> deletion of thrB Gene (International publication No. WO 2008/013432)

In the work to increase the amount of homoserine synthesis of O-succinylhomoserine derived from homoserine, thrB gene, homoserine kinase encoding gene, was deleted. For the deletion of thrB gene from W3-B strain constructed in example 1, FRT one-step PCR deletion method used for deletion of metB gene was used.

Using SEQ ID NOS: primers for 32 and 33, based on pKD4 vector (PNAS (2000) vol 97: P6640-6645) as template, the thrB deletion cassette was constructed by PCR as follows: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute, 30 cycles.

The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and the 1.6kb DNA band thus obtained was purified. The recovered DNA fragment was electroporated into the W3-B strain which had been transformed with pKD46 vector. The recovered strain was streaked on LB plate containing 50. mu.g/L kanamycin, cultured overnight at 37 ℃ and a strain showing resistance was selected.

The selected strains were grown using the same SEQ ID NOS: 32 and 33, directly based on the strain as a template, PCR reaction was performed under the same conditions, and the thrB gene knock-out was confirmed by selecting a strain having a gene size of 1.6kb on 1.0% agarose gel. The thus confirmed strain was again transformed with pCP20 vector and cultured in LB medium, and a final thrB knock-out strain (in which the gene size was reduced to 150bp on 1.0% agarose gel by PCR performed under the same conditions) was constructed and removal of kanamycin marker was confirmed. The strain thus constructed was named "W3-BT".

<1-3> variant metA having homoserine acetyltransferase activity (International publication No. WO 2012/087039)

In order to enhance the homoserine acetyltransferase activity of the strain obtained in reference example <1-2>, it was intended to introduce a mutant type metA gene (SEQ ID NOS: 24 and 26) encoding homoserine acetyltransferase.

First, to construct an activity-enhanced metA gene variant, the gene variants of SEQ ID NOS: 34 and 35, PCR was performed based on the chromosome of the wild type strain W3110 as a template to amplify metA gene encoding homoserine O-succinyl transferase.

Primers used in the PCR reaction were prepared based on the polynucleotide sequence of the escherichia coli chromosome NC — 000913 registered in the NIH gene bank, and SEQ ID NOS: the primers for 34 and 35 have EcoRV and HindIII restriction sites, respectively. The thus obtained PCR product and pCL1920 plasmid including Pcj1 were treated with EcoRV and HindIII, respectively, and the PCR product was cloned into pCL1920 plasmid. Coli DH 5. alpha. was transformed with the cloning plasmid, and transformed E.coli DH 5. alpha. was selected on LB plates containing 50. mu.g/mL of spectinomycin, from which the plasmid was obtained. The plasmid thus obtained was designated "pCL _ Pcj1_ metA".

Then, the 111 th amino acid of O-succinyl transferase, glycine (Gly), was substituted with glutamic acid (Glu) (G111E) using site-directed mutagenesis kit (Strata gene, USA) based on the pCL _ Pcj1_ metA plasmid constructed above as a template. The thus-constructed plasmid including the variant of G111E metA gene was named "pCL _ Pcj1_ metA (EL)".

In addition, for the substitution of amino acid 111 from glycine to glutamic acid and amino acid 112 from leucine to histidine of O-succinyl transferase, the amino acid sequences shown in SEQ ID NOS: 38 and 39. A plasmid including metA gene in which 111 th amino acid is substituted from glycine to glutamic acid and 112 th amino acid is substituted from leucine to histidine was named "pCL _ Pcj1_ metA (EH)".

Then, using pKD3 vector as template and SEQ ID NOS: primers 40 and 41, a substitution cassette for substituting meta (eh) into the strain was constructed by PCR as follows: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 2 minutes, 30 cycles. Template using pCL-Pcj1-meta (eh) as part of the substitution cassette meta (eh) and SEQ ID NOS: 42 and 43, and SEQ ID NOS: 42 and 45, to obtain respective PCR products. Three different PCR products were used and the sequences of SEQ ID NOS: 42 and 45, metA (EH) replacement cassette comprising a chloramphenicol marker moiety was constructed and electroporated into the W3-BT strain transformed with pKD46 vector constructed in reference example <1-2 >. The thus confirmed strain was again transformed with pCP20 vector and then cultured in LB medium, and the strain in which the chloramphenicol marker was removed and the metA gene was replaced with metA (EH) was named "W3-BTA".

<1-4> construction of a Strain having 2 copies of the ppc, aspC and asd genes (European patent application publication No. EP 2290051)

In order to increase the O-acetylhomoserine production capacity of the W3-BTA strain constructed in reference example <1-3>, the biosynthetic pathway was enhanced by citing the previously filed patent EP 2290051. In the same manner as in the above EP patent, the following strains each having 2 amplified copies of each gene were constructed: namely, the ppc gene encoding phosphoenolpyruvate carboxylase, which is expressed by using the nucleotide sequence shown in SEQ ID NOS: 46. 47, 48 and 49; aspC gene encoding aspartate aminotransferase, using SEQ id no: primers 50 and 51; and asd gene encoding aspartate semialdehyde dehydrogenase using SEQ ID NOS: 52. 53, 54 and 55. Specifically, the above-mentioned strain in which the biosynthetic pathway is enhanced in the production of O-acetylhomoserine was named "W3-BTA 2 PCD" (also referred to as "WCJM").

<1-5> flask culture experiment

The strains constructed in reference examples <1-3> and <1-4> were tested for O-acetylhomoserine production by Erlenmeyer flask culture.

Specifically, W3110, W3-BTA, and WCJM strains were inoculated to LB medium and cultured overnight at 33 ℃. Then, its single colony was inoculated in 3mL of LB medium, cultured at 33 ℃ for 5 hours, diluted 200-fold in a 250mL Erlenmeyer flask containing 25mL of O-acetylhomoserine production medium, cultured at 33 ℃ for 30 hours at 200rpm, and O-acetylhomoserine production was determined by HPLC analysis. The composition of the medium used is shown in Table 1 below, and the measured O-acetylhomoserine production is shown in Table 2 below.

[ Table 1] composition of culture Medium for O-acetylhomoserine production flask

[ Table 2]

As a result, it was revealed that the wild type W3110 did not produce O-acetylhomoserine at all, whereas the W3-BTA strain produced 0.9g/L O-acetylhomoserine, and the WCJM strain with enhanced biosynthetic pathway produced 1.2g/L O-acetylhomoserine.